WO1999056304A9 - Apparatus and method for creating a continuous electrospray of a sample for mass spectrometric analysis - Google Patents

Abstract

Apparatus and method to form continuous electrospray of an analyte for mass spectrometric analaysis, wherein the analyte is stored in a resealably sealable container connected to a chamber of an ion spray body, and in fluid registry therewith, and a pressure fluid means connected to the container and in fluid connection therewith, so that pressure in the container drives the analyte to the chamber of the ion spray body where it is ionized, and then expelled through a needle connected to the chamber of the ion spray body and in fluid registry therewith, wherein the needle is downstream from the ion spray body.

Description

APPARATUS AND METHOD FOR CREATING A CONTINUOUS ELECTROSPRAY OF A SAMPLE FOR MASS SPECTROMETRIC ANALYSIS

GOVERNMENT RIGHTS CLAUSE This work was supported by Development Funds from NCI Grant P30 CA08748. The government has rights in the invention.

FIELD OF THE INVENTION The present invention relates to an apparatus and method for the formation of a continuous electrospray of an analyte.

BACKGROUND OF THE INVENTION The human genome project has redefined the role of polypeptide structural chemistry. Whereas until recently, much effort was focused on supplying data to assist in isolation and characterization of novel genes, [^U2] the next decade will bring an heightened interest in studies on protein-protein, -nucleic acid and -small molecule (drug) interactions in the cell. As a result, protein identification will most definitely become the main theme in this field.

Standard procedure has long been to search a database with limited peptide sequence, traditionally obtained by Edman-chemical analysis. Such sequences need not be of perfect quality, allowing successful analysis of subpicomole amounts in a single day.^13"61 The limiting factor to still higher sensitivities-and throughput has been liquid chromatographic (LC) isolation of pure peptides, largely because of losses incurred during collection and handling. By contrast, peptide masses can be quickly and accurately determined from unfractionated mixtures, at concentrations below 100 femtomoles / μL, by either matrix-assisted laser-desorption/ionization (MALDI) or electrospray (ESI) mass spectrometry (MS). Such information, a "peptide mass fingerprint", enables protein identification just as well.'^7"121 Unfortunately, this approach usually falls short when protein preparations are not pure or the full-length sequences are not available in any database. One option remaining then, is to obtain limited covalent structural data on single peptides by MS/MS techniques, as provided by triple quadrupole mass analyzers for instance, ^π3'¹⁴¹ and use that information to query databases directly.¹¹⁵'¹⁶¹

To that end, capillary LC- or CZE-ESI-MS offer distinct advantages in terms of sensitivity^117,181 but impose severe restrictions on analysis time for each peptide, essentially requiring full automation of MS/MS analysis. An experimental limitation that can be alleviated by the use of ultra-low-flow "infusion" devices such as nanoelectrospray

('NanoES').¹¹⁹'²⁰¹

An example of a method and apparatus for generating such an electrospray is described by Wilm, M. and Mann M. in Electrospray and Taylor-Clone theory, Dole's beam of macromolecules at last? Int. J. Mass Spectro. Ion Processes 136: 167-180 (1994), which is hereby incorporated by reference herein. Wilm et al. teach an electrospray ion source for producing ions of compounds for mass spectrometric analysis. In particular, the apparatus set forth therein employs a gold coated borosilicate glass capillary with an inner diameter of 1-3 μm, and centered at a distance of 1-2 mm from the inner orifice of a mass spectrometer. A voltage of about 600 V is applied to the capillary, with 100 V to an interface plate, and 80 V to the inner diameter. As a result, sample in the capillary is ionized and fragmented, and be subsequently analyzed with an adjacent mass spectrometer.

However, the apparatus described by Wilm et al. suffers from substantial limitations. Initially, since the capillary is coated with gold, it is expensive. More importantly, it has been observed that the gold eventually burns off the capillary with concomitant loss of conductivity, and hence ion current. Also, such gold loss results mass spectral analysis may vary over a period of time. Another potential limitation of the apparatus described by Wilm et al. concerns the inability of an operator of the electrospray apparatus to observe the analyte in the capillary. More specifically, the gold layer covering the capillary prevents the operating from determining whether a loss of ion current is due to clogging of the capillary or some other reason. Furthermore, when trying to reopen a real or perceived plug by touching the tip of the capillary to a curtain plate located between the capillary and a mass spectrometer, the impact may generate a larger than desired aperture in the tip of the capillary, resulting in severe sample loss. Another limitation involves an inability to control the flow rate of sample into the ionizing capillary. The faster the flow rate, the greater the amount of sample that will be destroyed in the production of the electrospray. Such destruction can be of critical scale when only a minute amount of sample is available.

Another limitation with such capillaries is the difficulty in preventing the trapping of air in the sample during loading of the capillary. More specifically, the typical method of loading such a capillary involves inserting a micropipette, such as the type used to deliver samples into a well of an electrophoresis gel, into the back of the capillary (opposite to the end where the electrospray is produced), and maneuvering the tip of the micropipette as close as possible to the tip of capillary where the electrospray is produced to deliver the analyte. The capillary is then installed into the electrospray apparatus. When the analyte is delivered, it is quite possible to capture air pockets within it in the capillary. These air pockets lead to interrupted and potentially unstable flow electrospray from the tip of the capillary, which can dramatically effect mass spectral data.

Yet another limitation involves an inability to retrieve sample from the capillary should it be damaged after the sample is loaded. Due to the fragility of such capillaries, it is possible to damage them after loading the sample, but prior to producing the electrospray. Under such conditions, it is impossible to retrieve the sample for use with an undamaged needle, and the sample is lost

Another example of an apparatus and method for producing an electrospray of an analyte is described in U.S. Patent 5,115,131, which teaches a similar type of apparatus. In particular, a potential is put across the electrospray capillary which ionizes and fragments the analyte contained therein. Hence, the capillary must be comprised of a conductive material, or fused silica or glass with a material such as gold, silver, or platinum deposited on the capillary by processes such as sputtering or vapor deposition. Furthermore, the '131 patent teaches placing the conductive tip of the capillary, and the mass spectrometer in a vacuum chamber. This apparatus and method suffer from limitations similar to those set forth above, and a further limitation in that the vacuum chamber must be maintained at a low pressure, preferably less than 10 millitorr during the formation of the electrospray and operation of the mass spectrometer.

Hence, what is needed is a simple, reliable apparatus for generating an electrospray that is affordable, and readily available to investigators having limited background in miniaturized ESI mass spectrometry.

What is also needed is an inexpensive apparatus for creating an electrospray of analyte for mass spectrometric analysis which is amenable to continuous production of electrospray, adaptation to automation so that numerous samples can be consecutively analyzed, the rate of formation of electrospray can be automatically controlled.

The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.

SUMMARY OF THE INVENTION

There is provided in accordance with the present invention an improved apparatus that can create a continuous electrospray of an analyte for mass spectrometric analysis that does not possess the shortcomings of other electrospray devices explained above, and further offers the advantages of reliable, simple to operate, adaptable to full automation.

Accordingly, the present invention extends to an apparatus to form a continuous electrospray of an analyte for mass spectrometric analysis, comprising:

a fluid pressure source;

a resealably sealable container for holding the analyte, wherein the container is adapted for fluid registration with the pressure source;

an ion spray needle made of a non-electrically conductive material, wherein the needle comprises a tip having an orifice and a distal end opposite to the tip, wherein the distal end of the needle is in fluid registration with the resealably sealable container;

an electrical potential applied to the analyte upstream from the needle tip, wherein the electrical potential ionizes the analyte,

such that when the fluid pressure source increases pressure in the container, the analyte exits the container and is ionized prior to entering the needle, which discharges an electrospray of the ionized analyte.

Furthermore, the present invention extends to the apparatus as described above, wherein the fluid pressure source comprises a source of inert gas that is in fluid registration with the resealably sealable container. Examples of inert gases having applicatons herein include, but certainly are not limited to N, He, Ne, Ar, Kr, Xe, and Rn. Furthermore, the pressure source comprises a pressure regulator, vent and pressure gauge which are in fluid registration with the source of inert gas and the resealably sealable container, which regulate and monitor the pressure of the inert gas entering the resealably sealable container. The vent is located downstream of the pressure regulator and upstream of the resealably sealable container.

In addition, an apparatus of the invention can comprise an ion spray body having a chamber which is in fluid communication with the resealably sealable container and the needle, such that the ion spray body is downstream from the resealably sealable container, and upstream from the needle, such that the analyte passes through the chamber, and the ion spray body is made of an electrically conductive material that is chemically inert to the analyte. In a particular embodiment, the ion spray body is comprised of titanium, and its chamber has a diameter of approximately 0.15 mm.

Naturally, the electrical potential applied to the analyte upstream of the ion spray needle can be obtained from a voltage source, such as a power supply, electrically connected to the ion spray body, such that the electrical potential is applied to the analyte while in the chamber.

Furthermore, the present invention extends to an apparatus as described above, wherein the ion spray needle is comprised of fused silica, and thus, is transparent. In a particular embodiment, the ion spray needle has a length of approximately 3 cm, and the orifice has an inner diameter of not more than 10, and particularly not more than 5 micrometers.

What's more, numerous analytes can be electrosprayed with an apparatus of the invnention. Particular examples of such analytes include, but certainly are not limited to polar solutions, a protein, a peptide, a nucleic acid, a fatty acid, a carbohydrate, a synthetic polymer, or a mixture thereof.

Furthermore, the present invention extends to a method for creating a continuous electrospray of an analyte for mass spectrometric analysis, comprising:

providing a resealably sealable container containing the analyte to be analyzed;

providing an ion spray needle comprising a tip and a wide end, wherein the wide end is in fluid registration with the resealably sealable container, and the needle is comprises of an electrically non-conductive material;

creating a flow of the analyte from the resealably sealable container to the needle; and

applying an electrical potential to the analyte up stream of the needle which ionizes the analyte, and the ionized analyte is discharged from the needle tip as an electrospray.

The present invention extends to the method described above, further comprising the step of providing an ion spray body made of an electrically conductive material that is chemically inert to the analyte, wherein the ion spray body comprises a chamber that is in fluid registration with the wide end of the needle and the resealably sealable container, such that the analyte flows through the chamber.

In addition, the step of the method of the invention involving applying the electrical potential to the analyte comprising electrically connecting a voltage source to the ion spray body such that the electrical potential is applied to the analyte within the chamber of the ion spray body, and is ionized.

Naturally, the present invention extends to the method described above, wherein the voltage source comprises a power supply that is electrically connected to the ion spray body. However, as explained throughout the instant specification and claims, the electrical potential can applied to the analyte anywhere upstream of the ion spray needle tip.

Thus, the present invention extends to the method described above, wherein the step of applying and generating an electrical potential for ionizing the sample upstream of the needle tip comprises inserting an electrode in the resealably sealable container and connecting the electrodes to a voltage source. Furthermore, as explained above, in a particular embodiment of the method of the invention, the ion spray needle is made of fused silica, has a length of approximately 3 cm, and the tip has an inner diameter of not more than 10, and particularly not more than 5 micrometers.

In addition, the present invention extends to a method as described above, wherein the step of creating a flow of the analyte comprises connecting a fluid pressure source to the resealably sealable container such that the fluid pressure source is in fluid registration with the container.

Also, the present invention extends to a method described above, wherein fluid pressure source comprises a source of inert gas in fluid registration with the resealably sealable container. Examples of inert gases having applications herein include, but certainly are not limited to, N, He, Ne, Ar, Kr, Xe, and Rn.

What's more, the fluid pressure source can comprise a pressure regulator, a pressure vent downstream of the pressure regultor, and a pressure gauge which are in fluid registration with the fluid pressure source and the resealably sealable container.

Examples of analyte that can be electrosprayed in a method of the invention include, but certainly are not limited to a protein, a peptide, a nucleic acid, a fatty acid, a carbohydrate, a synthetic polymer, or a mixture thereof.

Furthermore, the present invention extends to an automated electrospray apparatus comprising: a tee having a first port, a second port, and a third port such that fluid can pass through the tee;

an ion spray needle having a tip with an orifice, and a wide end opposite to the tip, wherein the ion spray needle is comprised of an electrically non-conductive material, and the wide end passes through the first and third ports of the tee, such that a pressure tight seal is formed between the needle and the first port of the tee; a pressure source which is fluid registration the second port of the tee;

an insertion line having a first end in fluid registration with a third port of tee, and a second end forming a beveled and tapered end, such that the wide end of the needle passes through the insertion line and extends beyond the beveled and tapered point;

a sample rack having a plurality of sockets in which a plurality of resealably sealable containers are affixed to the sample rack, such that one resealably sealable container is placed in one socket;

a translation stage upon which the sample rack is mounted, wherein the translation stage translates the rack such that the beveled and tapered end, and the wide end of the needle enter a first resealably sealable container containing an analyte such that the wide end contacts the analyte, and fluid from the pressure source travels through the insertion line into the first container, which increases the pressure within the first container and forces the analyte from the container into the needle;

an electrical potential which is applied to the first analyte upstream of the needle, which ionizes analyte from the first container upstream of the needle tip, such that the ionized analyte is discharged as electrospray from the needle tip; and

the translation stage translates the sample rack such that the beveled and tapered end, and the wide end of the needle are removed from the first container afer discharge of the electrospray, and enter a second resealably sealable container such that the wide end of the needle contacts analyte within the second container.

In addition, the present invention extends to an automated electrospray apparatus as described above, further comprising an ion spray body having a chamber, wherein the chamber is in fluid registration with the tee, and in fluid registration with the wide end of the ion spray needle, and the analyte enters the ion spray body from the tee. In a particular embodiment, wherein the electrical potential is applied to the analyte while in the chamber of the ion spray body, the ion spray body is comprised of an electrically conducting material, including, but not limited to an electrically conducting polymer or a metal, such as titanium.

Furthermore, as explained above, the electrical potential can be applied to the analyte anywhere upstream of the ion spray needle. Thus, in an embodiment of automated electrospray apparatus of the invention comprising an ion spray body made of an electrically conducting material, a voltage source can electrically connected to the ion spray body, such that the electrical potential is conducted to the ion spray body, and ionizes the analyte while the analyte is in the chamber of the ion spray body.

Naturally, in an automated electrospray apparatus of the invention, ion spray needle is transparent, and preferably, composed of fused silica.

In addition the translation stage of an automated electrospray apparatus of the invention is described infra.

What's more, the present invention extends to an automated electrospray apparatus as described above, wherein the insertion line is comprised of an electrically conductive material, such as an electrically conductive polymer or a metal. Thus, when a voltage source, such as a power supply is electrically connected to the insertion line, an electrical potential is applied to the analyte while in the insertion line.

In yet another embodiment of an automated electrospray apparatus, an electrode is located within the resealably sealable container, wherein the electrode is electrically connected to a voltage source, and the electrode ionizes the analyte while in the container.

Naturally, the present invention extends to an automated electrospray apparatus as described above, wherein the pressure source comprises comprises a source of inert gas that is in fluid registration with the second port of the tee. Examples of inert gases having applications herein include, but certainly are not limited to N, He, Ne, Ar, Kr, Xe, and Rn. What's more, the fluid pressure source further comprises a pressure regulator, a pressure vent, and pressure gauge which are in fluid registration with the source of inert gas and the tee, which regulate and monitor the pressure of the inert gas entering the resealably sealable container, wherein the pressure vent is downstream of the pressure regulator and upstream of the tee.

In another embodiment, the present invention extends to an apparatus that can form a continuous electrospray of an analyte for mass spectrometric analysis. One such apparatus comprises a fluid pressure source, a resealably sealable analyte container adapted for fluid registration with the pressure source, an ion spray body having a chamber, wherein the chamber is in fluid registration with the resealably sealable container, an ion spray needle made of an electrically nonconductive material, wherein the needle comprises a tip having an orifice and a distal end opposite to the tip. The distal end of the needle is in fluid registration with the chamber of the ion spray body such that analyte can travel from the container, through the chamber, and then into the needle. The apparatus also comprises an electrical potential that is applied to the analyte upstream of the needle tip, which ionizes the sample. Thus, when the pressure source increases the pressure in the container, the analyte is pushed from the container into the ion spray needle. Since the analyte is ionized upstream of the needle tip, the analyte enters the needle in an ionized form, and is discharged from the tip in an electrospray.

Furthermore, the present invention extends to an apparatus that can form a continuous electrospray of an analyte for mass spectrometric analysis, as set forth above, in which the fluid pressure source comprises a source of inert gas, such as a tank of inert gas, that is in fluid registration with the resealably sealable analyte container. Numerous inert gases have applications in the present invention, including, but not limited to N, He, Ne, Ar, Kr, Xe, and Rn. In a preferred embodiment, the source of inert gas comprises a tank of helium gas.

In addition, the pressure source of the apparatus of the present invention can further comprise a pressure regulator, a vent down stream of the regular and a pressure gauge which are fluid registration with the source of inert gas and the resealably sealable analyte container for regulating and monitoring the pressure of inert gas entering the resealably sealable container. Thus, the pressure of the inert gas (for example, helium) entering the resealably sealable container holding the analyte can be controlled, i.e. increased or decreased. As a result, the flow rate of production of electrospray of the analyte can be controlled. In a preferred embodiment of the present invention, a pressure line connects the gas regulator in fluid registration with the container. Similar lines can connect the gas regulator in fluid registration with the source of inert gas. This pressure/vent line can be composed of any material that does not chemically react with the analyte, such as fused silica. The pressure/vent line has an internal diameter which can range from about 150 to 800 micrometers. In a particular embodiment, the pressure/vent line has a diameter of about 800 micrometers.

As explained above, the electrical potential can be applied to the analyte anywhere upstream of the ion spray needle tip. In a particular embodiment of the invention, the electrical potential is applied to the ion spray body, such that analyte within the chamber of the ion spray body is ionized. Naturally, in such an embodiment, the ion spray body is comprised an electrically conductive material that is chemically inert to the analyte being analyzed. A voltage source, such as a power supply is then electrically connected to the ion spray body. As a result, analyte in the chamber of the ion spray body is ionized, and discharged via the ion spray needle as an electrospray. The ion spray body can be comprised of any electrically conductive material, such as a metal or an electrically conductive polymer, provided the material is not soluble in the analyte, or chemically reactive with the analyte. In a preferred embodiment of the present invention, the ion spray body is composed of titanium. More preferably, the ion spray body has a length of approximately 0.5 mm with a chamber diameter of not greater than 0.15 mm.

The present invention further extends to an apparatus that can form a continuous electrospray of an analyte for mass spectrometric analysis, as described herein, wherein the ion spray needle is comprised of an electrically non-conductive material, such as glass or fused silica, i.e., that it be transparent in the electromagnetic spectrum of visible light. In a particular embodiment of the present invention, the ion spray needle comprises fused silica, has a length of approximately 3 cm, and tip inner diameter of not more than 10 micrometers, and particularly not more than 5 micrometers.

In addition, the present invention extends to an apparatus that can form a continuous electrospray of an analyte for mass spectrometric analysis, and be suitable for an analyte that is soluble in a polar solution. Examples of analytes which can form a continuous electrospray pursuant to the present invention include, but are not limited to proteins, peptides, nucleic acids, fatty acids, carbohydrates, synthetic polymers, or a mixture thereof to name only a few.

In another embodiment, the present invention extends to an apparatus for regulating the rate of formation of continuous electrospray of an analyte for mass spectrometric analysis, comprising a fluid pressure source, a resealably sealable analyte container adapted for fluid registration with the fluid pressure source, a regulator, a vent upstream of the container and downstream of the regulator, and a gauge for regulating which are in fluid registration with the pressure source and the resealably sealable container. The regulator, vent and gauge respectively regulate and monitor the pressure of the fluid entering the resealably sealable analyte. The apparatus also comprises an ion spray body having a chamber, wherein the chamber is adapted for fluid registry with the resealably sealable analyte container, and an ion spray needle having a tip with an orifice and a wide end opposite to the tip, wherein the wide end is in fluid registration with the ion spray body. Thus, the regulator and gauge monitor the rate at which the analyte passes from the resealably sealable container, through the chamber of the ion spray body, and through the needle. Also, the present invention comprises an electrical potential which is applied to the analyte upstream of the ion spray needle tip, which ionizes the sample prior to its entering into the needle, such that the needle discharges the ionized analyte in the form of an electrospray.

Furthermore, the present invention relates to an apparatus for regulating the rate of formation of continuous electrospray of an analyte for mass spectrometric analysis, as described above, wherein the fluid pressure source comprises a source of inert gas that is in fluid registration with the resealably sealable analyte container. As explained above, inert gases such as N, He, Ne, Ar, Kr, Xe, and Rn, to name only a few, have ready applications in the present invention.

Furthermore, as explained above, ionization of the analyte occurs upstream of the ion spray needle tip in the apparatus of the invention. In one embodiment of the invention, ionization of the analyte occurs in the chamber of the ion spray body. Thus, the present invention extends to an apparatus as described above, wherein the ion spray body is made of an electrically conductive material, such as a metal or an electrically conductive polymer that is chemically inert to the analyte being analyzed. Furthermore, a voltage source such as a power supply is electrically connected to the ion spray body. Since the ion spray body conducts an electric current, the potential is applied to the analyte while in the chamber of the ion spray body. In a particular embodiment, the ion spray body is made of titanium.

Naturally, the ion spray needle is comprised of an electrically non-conductive material, and preferrably is transparent. An example of such a material having applications herein is fused silica. In a particular embodiment, the ion spray needle has a length of approximately 3 cm, and the tip has an inner diameter of at most one micrometer, and particularly at most 5 micrometers. Preferably, the analyte analyzed are soluble in a polar solution. Such analytes can be a protein, a peptide, a nucleic acid, a fatty acid, a carbohydrate, a synthetic polymer, or a mixture thereof.

Moreover, the present invention extends to an apparatus to form a continuous electrospray of an analyte for mass spectrometric analysis, as described infra, further comprising a structure means upon which the resealably sealable analyte container and the ion spray body can be affixed. In a preferred embodiment, the structure means comprises a sample holder made of an inert material, non electrically conductive material, such as "DELRIN".

The present invention also provides methods for creating a continuous electrospray of an analyte for mass spectrometric analysis. One such method comprises providing a resealably sealable analyte container containing an analyte, and connecting an ion spray body having a chamber to the container such that the chamber is adapted for fluid registry with the container so that the analyte can enter the chamber. An ion spray needle made of an electrically non-conductive material, and having a tip with an orifice and a wide end opposite to the tip is also in fluid registration with the chamber of the ion spray body connected to the chamber and is in fluid registry with the chamber. An electrical potential is generated and applied to the analyte upstream of the ion spray needle tip which ionizes the analyte. The method also comprises creating a flow of analyte from the resealably sealable container, through the chamber of the ion spray body and through the needle. Since the analyte is ionized upstream of the ion spray needle tip, ionized analyte is continuously expelled from the tip of the ion spray needle. In one such embodiment, the ion spray body is made of an electrically conductive material, such as a metal or electrically conductive polymer, that is chemically inert to the analyte. Preferably, the ion spray body is made of titanium. More preferably, the ion spray body is approximately 0.5 mm long, and the chamber has a diameter of not more than 0.15 mm.

In another embodiment of this type, the step of creating and generating the electrical potential which ionizes the analyte upstream of the needle tip comprises connecting a power supply to the ion spray body. What's more, preferably, the ion spray needle is made of fused silica. In a particular embodiment, the ion spray needle has a length of approximately 3 cm, and the tip has an inner diameter of not more than 10 micrometers, and particularly, not more than 5 micrometers.

In yet another embodiment of this type, the step of creating a flow of the analyte comprises connecting a fluid pressure source to the resealably sealable analyte container such that the fluid pressure source is in fluid registry with the container. Preferably, the fluid pressure means comprises a source of inert gas which is in fluid registration with the resealably sealable analyte container. Numerous inert gases have applications herein, including but not limited to N, He, Ne, Ar, Kr, Xe, and Rn.

In a related embodiment of this type, the method further includes the step of regulating and monitoring the flow of the analyte from the resealably sealable analyte container to the chamber of the ion spray body. Preferably, the regulating step comprises connecting a pressure regulator and vent to the source of inert gas, and to the resealably sealable container such that the pressure regulator and vent are in fluid registry with the source and the container and the vent is downstream of the regulator. More preferably the monitoring step comprises connecting a pressure gauge to the pressure regulator such that the pressure gauge is in fluid registry with the pressure regulator. Preferred analytes for such methodology are soluble in a polar solution. More preferably, the analyte is a protein, a peptide, a nucleic acid, a fatty acid, a carbohydrate, a synthetic polymer, an ion, or a mixture thereof.

Accordingly, it is a principal object of the invention to provide an apparatus and method to form a continuous electrospray of an analyte from a resealably sealable container removed from the point of discharge of the electrospray. Hence, electrospray can be continuously formed until the analyte in the container is totally depleted or alternatively, subsequent resealably sealable containers, when used in an autosampling configuration, are depleted.

It is another object of the present invention to provide an apparatus and method to form a continuous electrospray of an analyte in which the rate of formation of electrospray can be readily regulated, and is not independent of the control of the operator of the mass spectrometer.

It is yet another object of the present invention to provide an apparatus and method to form a continuous electrospray of an analyte in which ionization occurs in the chamber of an ion spray body, which is upstream of the ion spray needle tip. Hence, it is not necessary to use a needle or capillary which is electrically conductive, or which is coated with an electrically conductive material. Hence, the ion spray needle of present invention can be made of inexpensive materials, such as glass or fused silica, which is transparent so that the operator can view the analyte within the needle to determine whether any air pockets are trapped within the needle which will distort mass spectral results.

It is yet another object of the present invention to provide an apparatus and method to form a continuous electrospray of an analyte wherein it is not necessary to load the analyte into the needle prior to installing the needle into the apparatus. It is possible that such installation will damage the needle, resulting in inaccurate mass spectral results, or the loss of sample. Rather, the present invention comprises an apparatus in which the needle is left installed and undisturbed during the loading of the analyte into the apparatus. In particular, the analyte is placed into a resealably sealable container, and not directly into the needle.

It is yet another object of the present invention to provide an apparatus and method to form a continuous electrospray of analyte for mass spectrometric analysis, wherein the apparatus is readily adaptable to automation. In particular, the present invention can be adapted to automating the rate at which electrospray is formed, and the positioning of the needle relative to the input of a mass spectrometer. Other variables which can be automated in the present invention are the potential delivered to ionize an analyte in the chamber of the ion spray body, and the analysis of consecutive analytes, to name only a few. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematical view of an embodiment of the invention, wherein ionization of the liquid analyte occurs in the ion spray body of the apparatus of the present invention.

FIG. 2 is a schematical view of a fluid pressure source of the present invention.

FIG. 3 is MS data acquired during continuous operation of the JaFIS ion source using 30 μL of 50 fmoles β-galactosidase tryptic peptides per μL. FIG 4A shows the total ion current over the full 32 hours of the experiment; indicating an average spray rate of 16 nL/min. Figures 4 B, C and D are spectra averaged over 5 min (12 scans) and taken at time points around 20 min, 15 hours and 26 hours, respectively; consistency over time, with respect to overall ion patterns as well as peak intensities is clearly illustrated (selected ions are labeled to serve as reference points). Details about operation of the API300 triple quadrupole mass spectrometer are given under 'experimental' in Example I.

FIG 4. Stable intensities of selected ions during 32 hours continuous operation of JaFIS-MS. Arbitrarily selected ions with m/z of 532, 681 and 872 represent three different tryptic peptides from β-galactosidase ( — 50 fmoles/μL each), and are derived from the experiment depicted in Fig. 4 (ions are labeled in FIG. 5B-D). Relative intensities are averaged from 12 scans (5 minutes total) and have been plotted here at 1 hour intervals; relative standard deviations were in the 7 to 18% range.

FIG. 5 MS data acquired during semi-continuous operation of the JaFIS ion source analyzing three different samples consecutively. Panel A shows TIC over almost 5 hours of total analysis time. The sequence of events is depicted by horizontal bars (numbered 1 to 7), representing a linear time line: (1), (4) and (7), JaFIS-MS of 50 fmol/μL tryptic BSA peptides, 25 fmol/μL tryptic β-galactosidase peptides, and 50 fmol/μL tryptic G6PD peptides, respectively; (2) and (5), sample exchanges; (3) and (6), transition ion current resulting from sample exchanges. Panels B, C and D depict spectra averaged over 5 min (12 scans) to illustrate the lack of cross-contamination between the peptide mixtures being analyzed. Details about operation of the API300 triple-quadrupole mass spectrometer are given under 'Experimental' in Example I. FIG. 6. JaFIS-MS/MS spectrum of 25 fmol/μL of a β-galactosidase tryptic peptide. Fragmentation of the doubly charged ion at m/z 872, selected from the Ql spectrum as shown in Fig. 4C (same ion is also labeled in Fig. 4B-D), is presented. A limited y"-ion series (y"6 to 11) was determined manually, and used to assign a partial sequence (ct~T-V-E-I/L-T-nt); which, in turn, was taken to create a SequenceTag and to query a sequence database. After delineation of this particular peptide in the entire β-galactosidase sequence, additional y"-ions (y" 1 to 5) were calculated and retroactively assigned to less prominent peaks in the spectrum. Details about operation of the API300 triple-quadrupole mass spectrometer are given under 'Experimental' in Example I.

FIG. 7 is a schematical cross sectional view of an apparatus of the invention wherein ionization of the analyte liquid occurs in the ion spray body, and further, the apparatus includes an automated sampler.

FIG. 8 is a schematical cross sectional view of an apparatus of the invention comprising an automated sampler, wherein the sample is ionized by titanium insertion line 37.

FIG. 9 is a schematical cross sectional view of an apparatus of the Invention wherein the sample is ionized in the resealably sealable container via a platinum electrode.

FIG. 9a is a schematical cross sectional view of a sampling nozzle (50).

FIG. 10 is a graphical representation of the effect of voltage applied on signal strength. ♦ , D, Δ represent peptide ions of molecular weight of 593.0, 681.2, and 871.8 respectively, and x represents the average of the three. The sample pressure of 6 psi The ion source was configured for a flow of 4 nL/min using the following parameters:

ISN orifice ID: 0.8 μm sample pressure: 8 psi axial distance from aperture: 1 mm.

FIG. 11 is a graphical representation of the effect of axial distance from aperture on ion signal strength. ♦ , D, Δ represent peptide ions of molecular weight of 593.0, 681.2, and 871.8 respectively, and x represents the average of the three ions. The sample pressure was 6 psi. The ion source was configured for a flow rate of 4 nL/min using the following parameters:

ISN orifice ID: 0.8 μm sample pressure: 8 psi ISP: 600 volts.

FIG: 12 is a graphical representation of the effect of flow rate on signal strength. ♦ , D, Δ represent peptide ions of molecular weight of 593.0, 681.2, and 871.8 respectively, and x represents the average of the three ions.

FIG. 13 is a graphical representation of the effect of ion spray potential on signal strength while holding the field strength contsant at 250 V/mm. ♦ , D, Δ represent peptide ions of molecular weight of 593.0, 681.2, and 871.8 respectively, and x represents the average of the three. The sample pressure was 6 psi. The ion source was configured for a flow of 4 nL/min using the following parameters:

ISN orifice ID: 0.8 μm sample pressure: 8 psi axial distance from aperture: 1 mm.

FIG. 14 is a graphical representation of the effect of flow rate on ion transfer. ♦ , D, Δ represent peptide ions of molecular weight of 593.0, 681.2, and 871.8 respectively, and x represents the average. Ion transfer is defined as ions detected/ ions available or a period of time.

FIG. 15 is a graphical representation of a reaction kinetics experiment. Panel a illustrates a TIC (total ion chromatogram) covering 30 minutes. Panel b depicts 5 minutes of averaged data over a mass to charge range of 700 to 1100 collected from 50 μl of 250 fm/μl of the control solution of Example I in 8% acetonitrile and 0.1 % formic acid, running at 40 nL/min. At approximately 12 minutes into the run, the resealably sealable container was vented and 10 μl of a slurry of Porous R2 beads, suspended in 50% methanol was introduced into the resealably sealable container at a flow rate of 50 μl/minute. Panel c depicts 5 minutes of average data over a mass to charge range of 700-1100 collected the control/slurry mixture. Although the addition of the 10 microliter volume reduces the analyte concentration, the data illustrates a signal strength of great than 20%.

FIG. 16 depicts MS data acquired during automated operation of the apparatus of the invention as set forth in FIG. 7 configured for a flow rate of 25 nL/min, wherein four separate analytes were sequentially analyzed. Panel a shows a TIC collected over 5 hours. All analytes were digested with trypsin and diluted to a final concentration of 100 fm/μl in 33% acetonitrile, 0.1 % formic acid. The sequence of analysis was as follows: 0-20 minutes carbonic anhydrase; 20-55 minutes BSA; 60-95 minutes β-galactosidase; 100-135 minutes lysozyme; 140-175 β-galactosidase; 180-215 minutes lysozyme; 220-255 minutes carbonic anhydrase; 260-300 BSA. Panels b-i illustrate 5 minutes of average data ten minutes after the changeover from one analyte to another analyte.

DETAILED DESCRIPTION OF THE INVENTION The present invention is based upon the Applicants' discovery that surprisingly, it is not necessary to load an analyte into the needle prior to installing the needle in the aparatus, as is done in methods previously described. Rather, a resealably sealable container remote from the place of electrospray production can be used. Moreover, the volume that can be analyzed is not limited by the volume of the needle, as in previously described systems, but rather can be much greater, and is limited only by the volume of the container. In this way unlike previously described methods, the container can be easily installed, removed or replaced without disturbing the needle, which is aimed at the mass spectrometer. Thus, the resealably sealable container allows for analysis of multiple samples unlike previously described techniques of creating nanoelectrospray.

Consequently, Applicants have discovered that inexpensive, electrically nonconductive materials, such as glass or fused silica, can be used as ion spray needles for delivering electrospray to a mass spectrometer. This unexpected discovery results in substantial benefits as compared to previously used techniques of creating electrospray. For example, as explained above, ion spray needles used herein are very inexpensive. Furthermore, since such ion spray needles are transparent, one can readily observe whether air is trapped in them, which would result in inaccurate mass spectral analysis of the analyte. In addition, there is no worry regarding the degradation of the needles, as opposed to capillaries coated with a conductive material, such as gold, which burns off during the life of the needle.

Moreover, this discovery has led to the discovery of an apparatus for producing an electrospray in which the rate of production of electrospray can be regulated and controlled. In particular, Applicants have developed an ingenious apparatus for regulating the rate of production which employs a fluid pressure source in fluid registry with the resealably sealable container. Hence, increasing the pressure in the container results in an increase in flow rate of the analyte to the needle. Furthermore, since ionization of the analyte occurs upstream of the needle tip, the present invention permits an increase in electrospray formation. In contrast, a decrease in the pressure in the container results in a decrease in flow rate of analyte to the needle, which in turn results in a decrease in the formation of electrospray. Thus, the present invention permits one on ordinary skill in the art to control the rate of formation of electrospray.

Accordingly, the present invention extends to an apparatus to form a continuous electrospray of an analyte for mass spectrometric analysis, comprising a fluid pressure means, a resealably sealable analyte container adapted for fluid registration with the pressure means, an ion spray body having a chamber adapted for fluid registry with the resealably sealable analyte container so that the analyte can enter the chamber, a means for generating and applying an electrical potential cooperatively associated with the ion spray body to ionize analyte in the chamber, and a spray delivery means connected to the chamber and in fluid registry with the chamber, for discharging an electrospray of the ionized analyte.

As used herein, "fluid registration" and "fluid registry" refer to the connection of separate parts of the Invention in a manner that permits fluid to flow from one part to the other part.

A schematic view of an embodiment of the present invention is set forth in FIG. 1. As explained above, resealably sealable container (7) for holding analyte is adapted for fluid registration with a pressure source, which is set forth schematically in FIG. 2. Any container which can be opened and resealed, and which can hold a solution, has applications as a resealably sealable container in the present invention. In a preferred embodiment, resealably sealable container (7) comprises a 0.2 mL PCR tube (United Scientific Products, San Leando. CA. Part No. PCR-02), which is inserted into nylon collar (8), which, in turn, fits into the threaded socket of a "DELRIN" sample holder (5). Furthermore, prior to inserting nylon collar (8) into threaded socket of sample holder (5), an O-ring (6) is fitted into the top of the threaded socket of sample holder (5). Hence, when container (7) is inserted in nylon collar (8), and nylon collar (8) is screwed into the threaded socket of sample holder (5), compressing )-ring (6) between container (7) and the samle holder (5), container (7) is sealed. Furthermore, when nylon collar (8) is unscrewed from the threaded socket of sample holder (5), container (7) is unsealed. Hence, container (7) is resealable.

Moreover, as set forth above, resealably sealable container (7) is in fluid registration with the chamber of the ion spray body of the invention. Referring again to FIG. 1 , when container (7) is sealed as described above, analyte within container (7) comes in contact with pick-up line (9), which traverses sample holder (5), and is sealed in place. In the preferred embodiment, pick-up line (9) is sealed in place using a 1/32"- >0.4 mm polyimide reducing ferrule (14) (Valco Part No.FS.4-5) and a 1/32" titanium long nut (31) (Valco Instruments, Houston, TX. Part No. LZN.5-10) which is screwed into sample holder (5) on a side opposite to that of container (7). Pick-up line (9) then passes out of sample holder (5) and is connected to the chamber (not shown) of ion spray body (10) (Valco Part No. MU.5XCTI) via a short titanium nut (30) (Valco Part No. ZN.5-10). In the preferred emboidment, pick-up line (9) passes through nut (31) and is connected to the chamber (not shown) of ion spray body (10) via a short 1/32" titanium nut (30) and reducing ferrule similar to (14) (not shown). Thus, the chamber of the ion spray body (10) is in fluid registration with container (7), and analyte can travel from container (7), through pick-up line (9) to the chamber of ion spray body (10). Pick-up line (9) can be made from numerous types of materials, provided the materials do not chemically react with the analyte. Examples of such materials include "TEFLON," glass or fused silica. In a preferred embodiment, pick-up line (9) comprises a fused silica capillary with an outer diameter of approximately 365 μm.

As explained above, the fluid pressure source, which regulates the rate of formation of electrospray in the present invention, is also in fluid registration with container (7). In particular, the fluid pressure source of the present invention comprises a source of insert gas (not shown), such as N, He, Ne, Ar, Kr, Xe, or Rn, to name only a few. In particular embodiment, the inert gas is helium.

Furthermore, referring again to FIG. 1, the fluid pressure source also comprises pressure regulator (2) for regulating and monitoring the pressure of inert gas entering the container. Pressure regulator (2) is in fluid registration with the source of inert gas (not shown) and container (7). In order to form such fluid registration, a pressure/vent line (4) is connected to pressure regulator (2) and container (7). Through pressure/vent line (4), inert gas is delivered to resealably resealable container (7). Thus, the inert gas increases the pressure in container (7), which then causes the sample to travel up pick-up line (9). Generally, the pressure/vent line (4) is sealed in fluid registration with container (7) through a bore in the sample holder (5). In the preferred embodiment, the pressure/vent line (4) is sealed in place using a 1/16" flanged tube end fitting (32)(Valco Part No. CF-1) screwed into sample holder (5) on side opposite that facing the mass spectrometer. Furthermore, in this embodiment of the present invention, a second vent line (26) is sealed into sample holder (5) and traverses sample holder (5) in a manner similar to pickup line (9), except that the vent line (26) does not come in physical contact with the analyte. On the side of the sample holder (5) opposite to the container (7), vent line passes first through a 1/32"- >0.4 mm polyimide reducing ferrule similar to (14) and second through a 1/32" titanium long nut similar to (31), which is screwed into the sample holder (5) to maintain the position of vent line (26). The opposite end of the vent line (26) is then connected to a vent valve (not shown). Hence, when container (7) is place into nylon collar (8), as described above, and screwed into threaded socket of sample holder (5), container (7) is in fluid registration with pressure regulator (2) and purge valve (1) via pressure/vent line (4) and with vent valve (not shown) via vent line (26). Pressure may be applied to container (7) via pressure regulator (2) and reduced or removed by opening either purge valve (1) or vent valve (not shown). Numerous materials can be used in the production of pressure/vent line (4) and vent line (26), provided they do not chemically react with analyte in container (7). Examples of such materials include, but are not limited to, glass, "TEFLON", or fused silica, to name only a few. In a preferred embodiment, pressure/vent line (4) is comprised of "TEFLON" with an outer diameter of 1/16" and vent line (26) is comprised of fused silica with and outer diameter of approximately 365 μm. In addition, in an embodiment of the present invention, the fluid pressure source futher comprises pressure gauge (3) which is connected to pressure regulator (2), and is in fluid registry therewith. Pressure guage (3) permits one of ordinary skill in the art to monitor the pressure of inert gas entering resealably sealable container (7). In a particular embodiment, the pressure gauge (3) is a 0-60 psi gauge. Furthermore, in order to vent inert gas from the pressure source to avoid an excessive buildup of pressure, the pressure source further comprises purge valve (1), which is connected to pressure gauge (3) and is in fluid registration therewith.

FIG. 2 further sets forth a schematical view of the fluid pressure source of the present invention, which regulates the formation of electrospray of the analyte. In particular, pressure regulator (2) (Porter Instrument, Hatfield, PA. Part No. 8310ANVS60) is connected to pressure gauge (3) (McMaster-Carr Supply, New Brunswick, NJ. Part No. 4000K51960) and in fluid registry therewith. Such fluid registration connection is provided by male run tee, 1/4" tubing - 1/8" NPT (15) (Swagelock, Solon, OH. Part No.

B-400-3-TMT) and 1/4" outer diameter polypropylene tubing (21), so that the pressure of gas from the source of inert gas (not shown) which enters pressure regulator (2) can be measured. Furthermore, purge valve (1) (Swagelock Part No. B-4P-4) is also connected to pressure regulator (2) and pressure gauge (3) and in fluid registry therewith. Hence, inert gas can readily be bled from the system.

As explained above, inert gas travels from the source of inert gas to the pressure source, and enters pressure regulator (2), which is in fluid registry with the inert gas source. Referring again to FIG. 2, the source of inert gas, preferrably helium, is in fluid registration with quick-connect body, 1/4" tubing (18) (Swagelock, Part No.

B-QC4-B-400). Quick-connect body (18) in turn is in fluid registration with bulkhead double-end shutoff (DESO) quick-connect stem, 1/4" tubing (17) (Swagelock Part No. B-QC4-D 1-400), which in turn is in fluid registration with an inlet of a first 1/4" outer diameter polypropylene tubing (16). The outlet of first polypropylene tubing (16) is in fluid registration with a first end of a first male elbow, 1/4" tubing - 1/8" NPT (19) (Swagelock Part No. B-400-2-2), while the second end of first male elbow (19) is in fluid registration with pressure regulator (2). As a result of these connections, the source of inert gas, such as a tank of helium gas, is in fluid registration with pressure regulator (2) and in fluid registry therewith.

Furthermore, pressure regulator (2) is in fluid registration with the inlet of a second male elbow, 1/4" tubing - 1/8" NPT (22). The second end of the male elbow (22) is in fluid registration with a second polypropylene tube (23). The outlet of the second polyproylene tube(23) is in fluid registration with a bulkhead quick-connect body (24) (Swagelock Part No. B-QC4-B 1-400). A DESO quick-connect stem, 1/8" tubing (25) (Swagelock Part No. B-QC4-D-200) in turn is in fluid registration with bulkhead body quick-connect (24). Pressure/vent line (4) is connected to container (7) (not shown) and quick-connect DESO stem (25). Hence pressure regulator (2) is also in fluid registration with container (7) so that inert gas from the source of inert gas, such as a tank of gas, can travel to pressure regulator (2), have its pressure measured with pressure gauge (3), and continue through pressure/vent line (4) to container (7) (not shown). Purge valve (1) is in fluid registration with container (7) as well so that inert gas may be bled from the container (7) through the pressure/vent line (4) and out the purge valve (1) Pressure regulator (2) and purge valve (1) therefore, can control the pressure of inert gas entering container (7), the rate of formation of electrospray of the analyte.

Furthermore, pursuant to FIG. l, and as explained above, ion spray needle (11) is in fluid registration with the chamber of ion spray body (10). Needle (11) can be made of a variety of materials, provided it is not electrically conductive and does not react with the analyte. Also, it is preferred that needle (11) be transparent so that an operator can see whether air pockets are trapped in needle (11), or needle (11) is clogged. Examples of such materials comprise fused silica, "TEFLON", or glass. In a preferred embodiment, needle (11) comprises fused silica, and has a wide or distal end connected to the chamber of ion spray body (10), and a narrow needle tip from which electrospray is discharged. The inner diameter of the tip is dependent upon the application. In a particular embodiment, the inner diameter (ID) of the tip is not more than 10 micrometers, and particularly not more than 5 micrometers. Particular examples of applicable IDs are set forth infra.

Referring again to FIG. l, needle (11) is inserted through titanium internal nut (28), and into 1/32" -< 0.4 mm polyimide reducing ferrule (27), which is located behind 1/32" internal nut (28). Internal nut (28) is then screwed in the chamber of ion spray body (10), and then to needle (11). As explained above, ionization of the analyte can occur anywhere upstream of the tip of needle (11), including within container (7). In the embodiment of the invention set forth in FIG. 1, ionization occurs in the chamber of ion spray body (10). In particular, voltage source (13), such as a power supply, is electrically connected to 8-32 x 1" titanium round head machine screw (29) which passes through sample holder (5). Ion spray body (10) is connected to screw (29) so that an electrical current is conducted via screw (29) to ion spray body (10). In this embodiment, ion spray body is comprised of an electrically conductive material, such as an electrically conductive polymer, or a metal. In a particular embodiment, ion spray body (10) is made of titanium. Thus, the voltage which is conducted to ion spray body (10) ionizes analyte in the chamber of ion spray body (10), which is then discharged from needle (11) in an ionized form. It is important to note however, that successful operation of the apparatus of the invention does not require ionization of the analyte liquid in the chamber of the ion spray body. Rather, as described infra, other embodiments permit ionization of the analyte upstream of the ion spray needle tip, i.e., closer to container (7).- After being ionized in the chamber of ion spray body (10), the ionized sample is discharged through needle (11).

FIG. 7 is a schematical cross sectional view of an embodiment of the invention wherein the electrospray apparatus of the invention interfaced with a multi-sampler. As explained above, according to the present invention, analyte liquid is ionized upstream of the needle tip. Thus, there is no need to provide the needle a coating of conductive material, such as gold for example, which is burned off during the use of the needle. Moreover, unlike traditional electrospray devices, the present invention does not require analyte liquid be loaded into the needle prior to installing the needle into the apparatus. Thus, the present invention is extremely well suited to auto sampling. Referring again to FIG. 7, pick-up line (9), having a length of approximately 10 cm, is threaded through a 1/16" tee assembly (35) with a 0.020" bore (Upchurch Scientific Part No. U-248) and a pressure tight seal is formed between pick-up line (9) and the tee assembly (35) using a 1/16" <0.4 mm polyimide reducing ferrule (33) (Valco Part No. FS1.5-5) and standard 1/16" titanium nut (34). As a result, pick-up line (9) is in fluid registration with tee assembly (35). Pick-up line (9) passes through the center of tee assembly (35) and exits through a 5 cm length of 1/16" OD 0.020" ID titanium insertion line (37), which is also connected to the tee (35) and is in fluid registration therewith. The distal end of titanium insertion line (37), i.e., the end opposite to the end connected to tee (35), forms a beveled and tapered end (not shown). Pick-up line (9) passes through stainlessinsertion (37) and extends beyond the beveled and tapered end. Titanium insertion line (37) is also held in place in tee assembly (35) with a standard 1/16" titanium nut similar to nut (34) and ferrule (36) (Valco Part No. ZF1-10). Pressure line (4) connects the pressure source (not shown) to the remaining port of the tee in a similar fashion. In a preferred embodiment, pressure line (4) is comprised of 1/16" outer diameter (OD), 800 μm inner diameter (ID) "TEFLON" tubing. Furthermore, sample rack (41) is provided which comprises a plurality of internally threaded sockets. Resealably sealable containers (7) in this embodiment are constructed using the same PCR tubes as described in FIG.l, wherein these tubes are inserted through a nylon collar (6) threaded throughout its entire exterior length. A pressure tight seal is maintained by threading cap (39) containing a 1 mm thick PTFE/silicone septa (38) within cap (39) onto collar (6). Once cap (39) with septa (38) is secured to container (7), container (7) is capable of withstanding up to 40psi of head pressure. Nylon collar (6) is then screwed into a threaded socket of sample rack (41), which affixes container (7) to sample rack (41).

Referring again to FIG. 7, analyte liquids are initially placed in multiple containers (7), wherein each analyte liquid is placed in a separate container. Then, each of the containers are inserted into nylon collar (6), and capped with cap (39) having therein septa (38). Collars (6) are then screwed into theaded sockets in sample rack (41). After the containers are secured into sample rack (41), rack (41) is positioned via computer controlled xyz translation stage (42) (Sutter Instruments, Novato CA. Model MP-285), which presents samples to titanium line (37). Each container (7) is presented separately to insertion line (37). After presentation, stage (42) translates rack (41), and thus the presented container (7) towards insertion (37). Beveled and tapered end of insertion line (37) pierces cap (39) and septa (38), and enters container (7). Naturally, the end of pick-up line (9) which extends beyond the beveled and tapered end of insertion line (37) also enters container (7) and makes contact with the analyte liquid. Inert gas is then flowed from the pressure source, through the tee, and insertion line (37) into container (7). As a result of this inert gas flow, the pressure increases in container (7) such that the analyte liquid is driven from container (7), through pickup line (9) to a chamber within ion spray body (10). An electrical current from voltage source (13) is conducted through screw (29) and ion spray body (10). The analyte liquid is then ionized in the chamber of ion spray body (10), and then discharged through needle (11) for analysis with an analytical instrument coupled to the ionspray device, such as a mass spectrometer.

After the analyte liquid has been electrosprayed, stage (40) translates the sample rack (41) such that container (7) is translated away from insertion line (37) so that its tapered end is clear of cap (39). Stage (40) then translates sample rack (41) such that a second container (7) is presented to insertion line (37) and translated toward the insertion line (37) such that its tapered end pierces second cap (39) and septa (38), repeating the process described above. Since the electrospray apparatus can spray continuously, the number of samples which can be sprayed with the sample needle is limited only by the number of resealably sealable containers (7) that can be held in the sample rack (41).

FIG. 8 sets forth another embodiment of the invention comprising an autosampling capability. This embodiment of the invention is similar to the embodiment of the invention schematically set forth in FIG. 7. However, in this embodiment, voltage source (13) is electrically connected to titanium insertion line (37). Thus, when the insertion line (37) pierces the cap (39) and septa (38) it is in fluid registration with the analyte in the container (7), and ionization of the analyte occurs in the container (7). This allows a longer needle (11) to extend in a continuous line through a microvolume connector (44) (Valco Part No. MU1XTSI), a length of 1/16" outer diameter/ 0.02" inner diameter titanium tubing (43) (Valco Part No. T5C20-10), the tee (35), and insertion line (37) into fluid registration with the analyte in the container (7). Thus, when the increased pressure in container (7) forces analyte liquid into the needle (11) within insertion line (37), the analyte liquid is already ionized. The position of the needle (11) in the microvolume connector (44) is maintained by a 1/16" - >0.4 mm polyimide reducing ferrule (45) (Valco Part No. FS1.4-5) and 1/16" titanium nut (46) (Valco Part No. ZN1-10).

FIG. 9 schematically shows an embodiment of the invention wherein the sample is ionized in resealably sealable container (7) prior to entering pick-up line (9). One of ordinary skill in the art can readily appreciate this embodiment has ready applications in single, or multi- sampling. In particular, sample holder (5) has a first bore therethrough which runs from a first side of holder (5) to the threaded socket of sample holder (5) into which nylon sampling nozzle (50) is screwed intosample holder (5). Platinum electrode (47) is connected to the first side of sample holder (5) using a standard titanium 1/32" nut (48) and ferrule (49) (Valco Part No. ZN.5-10, ZF.5S6-10). Platinum electrode (47) is then passed through the first bore and the threaded socket, and extends beyond the threaded socket. On a second side of holder (5), i.e., the side opposite to which platinum electrode (47) is connected to holder (5), there is a threaded port for receiving a 1/32" bulkhead internal union (53) (Valco Part No. ZBU1M). A second bore located within sample holder (5) runs from the threaded port to the threaded socket. The diameter of the second bore is about 0.5 cm. A short length (approximately 2 cm) of 1/8" OD/ 1.5 mm ID "TEFLON" tubing (52) is laid within the second bore so that is runs from the threaded socket to the threaded port in which bulkhead union (53) is screwed into sample holder (5). Tubing (52) extends down through the second bore, and permits a container (7) connected to sampling nozzle (50) to be in fluid communication with bulkhead union (53). A 1/32" >0.4 mm ID polyimide reducing ferrule (27) is connected to bulkhead union (53) forming a pressure tight seal, and permitting liquid analyte to travel through bulkhead union (53) and ferrule (27). A 1/32" titanium nut (28) is then connected to ferrule (27). Nut (28) has a bore therethrough so that needle (11) can be installed into nut (28), and have a pressure tight seal. As a result, needle (11) is in fluid registration with the threaded socket. When presenting a reasealably sealable container (7) to the socket, initially, an o-ring (6) is inserted into the socket. Then, annular externally threaded nozzle (50) is screwed into the socket, trapping o-ring (6) between sample holder (5) and nozzle (50). FIG.9(a) sets forth a schematical cross sectional view of nozzle (50). In particular, as can be seen in FIG. 9(a), nozzle (50) comprises an exterally threaded portion (54) and a nozzle portion (55). An annular groove (56) is located between nozzle portion (55) and externally threaded portion (54).

Referring again to FIG. 9, o-ring (6) is placed within the threaded socket , and then nozzle

(50) is screwed into the socket of sample holder (5). As a result, o-ring (6) is trapped between sample holder (5) and nozzle (50), forming a pressure tight seal. A second o-ring

(51) is placed around nozzle portion (55) of nozzle (50) so that second o-ring (51) fits snugly around annular groove (56) (not shown), thus forming a pressure tight seal among resealably sealable container (7) containing an analyte liquid, pressure line (4), and pick-up line (9) when container (7) is placed upon nozzle (50). The pressure source used in this embodiment is similar to that described above, and schematically shown in FIG 2. Naturally, platinum electrode (47) passes through the first bore, o-ring (6), nozzle (50), and second o-ring (51) such that it can extend into container (7), and make contact with analyte liquid when container (7) is placed upon second o-ring (51).

Voltage source (13) is electrically connected to platinum electrode (47). Thus, when voltage is permitted to run from voltage source (13) to platinum electrode (47), and when resealably sealable container having an analyte liquid is placed over o-ring (51), an electrical potential is formed within container (7), which ionizes the analyte liquid. When the pressure is increased in resealably sealable container (7), as described above, the ionized analyte liquid passes through tubing (52), ferrule (27), bulkhead union (53), and is discharged from needle (11). O-rings (6) and (51) can be made of any material that is chemically resistant to analyte and of sufficient durability to withstand repeated compressions and releases. An example of such a material includes, but is not limited to 'NITON". Naturally, numerous materials can be used to make sample holder (5), tubing (52), and nozzle (50), provided the materials do not conduct an electric current, and are chemically inert such that they do not react with the liquid analyte. Examples of such materials having applications herein include, but certainly are not limited to "TEFLON" and "DELRIN". Furthermore, using routine experimentation, one of ordinary skill in art can readily configure the embodiment of the invention set forth in FIG. 9 for use in autosampling techniques.

The present invention may be better understood by reference to the following non-limiting Examples, which are provided as exemplary of the invention. The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.

EXAMPLE I Nanoelectrospray (nanoES) tandem mass spectrometry of complex peptide mixtures has become a certified and highly reliable technique for the identification of proteins. The typical low flow rates of nanoES, its extended analysis times for small samples, high ion transmission and its overall ease-of-use provide important practical advantages for polypeptide covalent microanalysis. A novel and useful nanoelectrospray ion source has been constructed and described herein, which is highly durable and user-friendly, and allows for full auto-sampling operation. The injection adaptable Eine Ion Spray (JaFIS) source can be operated at flow rates of 10-100 nL per minute and with sensitivities in the 25 femtomoles peptide per microliter range. The ion spray needles las indefinitely, allowing for standards and multiple samples to be analyzed consecutively under similar conditions. In this configuration, quality controlled needles can also be saved and reused, providing for more consistent and reproducible day-to-day operating conditions. JaFIS-ES also permits sample recovery should any failure occur during analysis.

EXPERIMENTAL Materials and reagents β-galactosidase (E. coli), yeast glucose-6-phosphate dehydrogenase (G6PD), bovine serum albumin (BSA), and ammonium bicarbonate were purchased from Sigma (St. Louis, MO); acetonitrile was from Burdick & Jackson (Muskegon, MI); formic acid and Ponceau S were from Fluka (Ronkonkoma, NY); Zwittergent 3-16 was from Calbiochem (San Diego, CA); all chemicals and equipment for casting and running polyacrylamide gels, and for electroblotting onto nitrocellulose, were from Bio-Rad (Richmond, CA). Trypsin was "modified sequencing grade" from Promega (Madison, WI). Poros 50 R2 beads were obtained from PerSeptive BioSystems (Framingham, MA).

A first 'control' peptide mixture was generated by resuspending β-galactosidase to 5 μg/μL in 100 mM ammonium bicarbonate and digesting with trypsin (E/S: 1/20; w/w) for 3 h at 37° C. After incubation, the resulting mixture was diluted with 30% acetonitrile/0. 1 % formic acid to a 1 pmol/μL stock, and frozen in 35 μL aliquots. Final concentration of the control was adjusted by diluting the freshly thawed stock solution twenty-fold (in 30% acetonitrile/0. 1 % formic acid) just before use.

G6PD and BSA were resuspended in Laemmli sample buffer and subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by electroblotting and in-situ proteolysis as described.¹²⁴¹ Briefly, the protein bands were visualized with Ponceau S and excised, and the proteins were digested with trypsin in an ammonium bicarbonate/ Zwittergent 3-16 buffer. An aliquot of each digest mixture was transferred to an Eppendorf gel loading tip packed with 2 μL bed volume of Poros 50 R2 beads,¹²⁴'²⁵¹ washed with 10 μL 0.1 % formic acid, and eluted in 4 μL 30% acetonitrile/0. 1 % formic acid.

Mass Spectrometry

All experiments were performed with an API 300 triple-quadrupole mass spectrometer (PE Sciex, Thornhill, Canada). The micro-ion source was replaced with JaFIS (see below). The API 300 instrument does not allow for user control of the curtain plate voltage, so a Y-connection was inserted between the ring and the curtain plate to set them to the same potential. All associated covers and interlocks relating to the orifice were either removed or disabled. No nebulizing gas or sheath flow was employed. Ultra high purity nitrogen was used as a curtain gas at a flow rate of 0.2 to 0.6 L/min. Needle voltage ranged from 600 to 1350 volts depending on the application. The voltages for the orifice and the curtain plate were set at 5 and 150, respectively. Positioning of the needle tip relative to the orifice was accomplished through the instrument control software (ICS) and varied depending on the ionization potential and flow rate.

The instrument was calibrated using the polypropylene glycol standards provided by the manufacturer. Ql scans were collected using a 0.2 amu step size, and a 5 msec dwell time over a mass range from 500 to 1500 amu. For statistical analysis, 12 scans were averaged (5 minutes). Ql resolution was set such that the charge state of singly, doubly, and triply charged ions could be ascertained. For operation in the MS/MS mode, Ql was set to transmit the complete isotopic envelope of the parent. All spectra were averaged with a 0.2 Dalton step size and a 5 msec dwell time for 5 minutes over the mass range of the singly charged m/z. Q3 resolution was set such that the charge state of the fragment ions could be distinguished. Collision energies, as well as CAD gas pressures, were optimized individually for each peptide as to obtain the best MS/MS spectra.

Construction of the JaFIS source

A schematic view of the JaFIS assembly is given in Figure 1.

The needle (ISN) is a 3 cm long, pointed piece of FSC which has been narrowed down at one end to an inner diameter of, depending on the application, not more than 10 micrometers, and particularly, not more than 5 micrometers, using a P-2000 laser puller (Sutter Instruments, Novato CA); the tip was inspected and calibrated using an Optiphot-2 microscope (Nikon Inc., Melville, NY) with a dry 100X objective and measuring reticle. The ISN was connected to the ISB as illustrated in FIG. 1.

The pressure source is schematically shown in FIG.2, and described above.

JaFIS was positioned through the use of a robot comprising three stepper-motor driven translation stages (Newport Corp., Irvine, CA.) which translate in the x, y, and z directions, controlled by a motion controller/driver (Newport) and a Macintosh Powerbook G3 (Apple Computers, Cupertino, CA) portable computer with a PCMCIA GPIB card (National Instruments, Austin, TX). The ICS was developed using LabView (National Instruments). The ICS software has three preset positions: 'Change ISN' ; 'Extend' ; 'View' ; and a user-defined position, 'Spray'. Monitoring of the ISN position and ionization characteristics, and of the liquid level in the sample reservoir is done through a number of high resolution, magnifying CCD cameras (Chugai Boyeki, Japan; and Panasonic/ Matsushita, Japan), strategically positioned and mounted to a vibration-proof table. The video output is displayed on two 9 inch and one 13 inch monitor (Panasonic).

Run control

The collar around the resealably sealable container containing 5 μL of the control peptide mixture (50 femtomoles of β-galactosidase tryptic digest per μL), was threaded into the sample holder and the pressure within the container was slowly increased (typically to 8 psi) until the migration of the meniscus of the sample all the way through the ISN could be viewed. Any air trapped in the pickup line or the ISN (ions spray needle) must be displaced before a stable spray ion current can be established. Since JaFIS was developed for flow rates between 1-250 nL/min, and with a sample to tip dead volume of approximately 50 nL, the time required to displace the air varies from 1-10 minutes depending on the flow. Once trapped air pockets are displaced, a bubble formed at the tip of the ISN, and the needle tip was moved to a safe location approximately 1-4 mm in front of the orifice ('Extend' position). The voltage was then applied, and the position of the needle tip and voltage were optimized empirically by viewing the total ion current (TIC) and spectra. Once optimized, the position of the needle tip was saved, and 5 minutes of Ql data was averaged and compared against a reference spectrum. Several peaks from the averaged data were then arrow listed and the background-corrected intensities were exported to a FileMaker Pro (Claris Corporation, Santa Clara, CA) database and analysis performed. If the results met quality control criteria, MS/MS analysis was done on two predetermined doubly charged ions, and the process repeated. When all the QC requirements had been met, the voltage was disabled and the needle tip was moved to a preset 'Sample Change' position. The resealably sealable container was then vented and replaced with a resealably sealable container containing 0.5-10 μL of the analyte solution of interest. The needle tip was then returned to the saved 'Spray' location and the voltage and pressure were reapplied. The TIC was monitored through the Acquire Data feature in the LC2Tune ICS (PE Sciex). When five minutes of acceptable continuous ion current from the analyte had been achieved, mass analysis was started.

Mass spectra interpretation and database searching

Software has been developed on the basis of AppleScript™ (Apple Computers), FileMaker Pro and BioMultiview™ (PE-Sciex). After averaging 12 scans (5 minutes), all ions of interest were arrow listed, extracted and transferred to a FileMaker Pro database. The database compares all ions to the MALDI spectra (if available) for that particular sample, and the resulting doubly charged ions which match the predicted mlz values from the MALDI were then selected for MS/MS experiments. For database identification, the mass fragments with mlz values higher than the precursor ion were inspected to identify a y" ion series. A short amino acid sequence, the bracketing mlz values and the molecular mass of the entire peptide were then taken to generate a 'sequence tag'¹¹⁵¹ for subsequent query of a non-redundant sequence database (NRDB, from Dr. C. Sander, European Bioinformatics Institute, Hinxton, UK), which presently contains more than 280,000 entries, by using PeptideSearch version 3.0.2; no restrictions on protein molecular weight, pi or species of origin were used in the database searches.

RESULTS AND DISCUSSION

As previously explained, traditional nanoelectrospray tips are sputter-coated with gold to provide ion spray potential to the analysis solution.¹²⁰'²⁶'²⁷¹ It has been observed both here and elsewhere that the gold eventually burns off the tip with concomitant loss of ion current.'²⁶'²⁸¹ However, as the results set forth above clearly show, the needles used in an apparatus of the invention do not use such conductive materials, and in fact are nonconducting. Thus, since the ion spray potential in JaFIS is applied directly to the sample liquid, the gold coating is no longer necessary and spray time is limited only by the sample volume.

Analysis of multiple samples The extended ISN life time, combined with the ability to quickly and easily change samples, suggested that multiple samples could be analyzed consecutively through JaFIS with little cross-contamination. In one such "multi-sample" experiment, 50 fmol/μL BSA tryptic peptide mixture was sprayed for 30 min from a previously optimized source. This sample was then replaced with a new reservoir containing 3.5 μL of 25 fmol/μL β-galactosidase tryptic digest, which was sprayed for 2 Vτ hours (the average analysis time of an unknown); and then again replaced with 3.5 μL of 50 fmol/μL G6PD tryptic digest solution. The TIC was monitored until continuous ion current was fully restored and the Ql scans indicated the perfect signature spectra of the G6PD tryptic map. Figure 5 gives an overview of these experiments. As depicted in FIG. 5(a), TIC was lost as voltage was removed and one sample reservoir was replaced with the next. But when the voltage and spray position were restored, the TIC returned. It became unstable for about 15 min as the BSA peptide mixture remaining in the pickup line was consumed and the air trapped between the BSA and β-galactosidase digest solutions passed through the ISN. This did not occur during the transition from β-galactosidase to G6PD. Figures 5(B), (C) and (D) show representative spectra to illustrate the experiment described above. Note that in Fig. 5(C), only 5 minutes after the first identifiable ion signals from the β-galactosidase digest had appeared, there was no cross-contamination from any of the BSA peptides visible. Under the low flow ( ~ 20 nL/min) operating conditions that JaFIS is typically used, the amount of sample lost while waiting for the cross-contamination to disappear can be considered negligible.

In order to verify if MS/MS spectra of specific peptides out of any mixture could be obtained during lengthy, multi-sample MS analyses, the doubly charged ion of m/z 871.8 (labeled '872' in Figure 5C; 25 fmoles β-galactosidase peptides per μL) was analyzed. As shown in Fig. 6, a partial y" ion series was easily distinguished above background. The fragment ions were then arrow-listed and a script was activated that queried the database with a sequence tag (peptide M_r 1742 + 1; [814.0]TVE[I/L]T[1357.5]) using PeptideSearch software; β-galactosidase (represented numerous times in the database) was unequivocally identified.

User Interface

JaFIS was designed with a major emphasis on ease of use, so that laboratory personnel only moderately experienced in mass spectrometry could readily operate JaFIS and obtain reproducible results. The automated positioning feature of this particular ion source enables the user to quickly switch samples. Indeed, the ICS software permits rapid movement of the source between positions and ensures reproducibility once these conditions have been optimized. Moreover, the JaFIS source travels between any of the fixed positions in less than 4 seconds with an error of ±2 μm. This is important since preliminary data indicates that the distance from the ISN to the orifice is critical in maximizing ion transmission through the first quadruple at a given ion spray potential. As demonstrated in Fig. 5(a), the sample can be exchanged and data collection resumed in less than two minutes; needles can be replaced just as rapidly by loosening the bushing on the ISB and switching needles. Again, this is rather important, for it allows the use of rigorously pretested ISN needles, a major factor in ensuring maximal success rate of analysis.

The use of the FileMaker Pro database to correlate MALDI and ESI MS data allows for rapid selection of ions likely to provide the best MS/MS data. The database also provides the operator with recommendations for configuring the mass spectrometer's CAD gas pressure and collision energy on the selected ions for MS/MS, thus reducing the time required to perform each analysis.

Fault Tolerance The JaFIS source was found to be tolerant of casualties that can easily result in sample loss in conventional nanoelectrospray tips. The absence of the gold coating on the ISN allows the user to view the inside of the tip when ion current becomes unstable. This heightened visibility prevents wrongly ascribing a loss in signal to a plugged needle, rather than air. It was found that no corrective action is required for an air pocket, and that it typically clears within minutes. When a clogged tip or other damage to the ISN is identified, the resealably sealable container is vented, voltage is removed, and the needle tip moved to the "Change Sample" position for ISN replacement. Since the only analyte lost in such a maneuver would be the analyte within the needle, such mishaps typically result in a loss of less than less than 2% of the 3 μL sample volume. In those cases when the entire sample volume is needed to be recovered, the ISN can be readily replaced with a pressure line which forces the sample back through the ISB and pickup line into the resealably sealable container.

By comparison, when a loss in ion current occurs with conventional nanoelectrospray sources, the user is unable to clearly ascertain the cause of the problem and must assume clogging. When trying to reopen a real or perceived plug by touching the tip to the curtain plate, the user sometimes generates a larger than desired aperture, resulting in severe sample loss.

CONCLUSION

The JaFIS ion source was designed and developed to make the process of MS/MS protein identification available to investigators with limited background in miniaturized ESI mass spectrometry; an out-of-the-box functional system. With sensitivity in the 25-50 fmol/μL range and flow rates between 1-250 nL/min, JaFIS is ideally suited for analysis of unfractionated protein digest mixtures at the 100-200 femtomole (FM) level. Furthermore, the data presented herein clearly demonstrates the feasibility of JaFIS as a multi-sample, continuous flow nanoelectrospray ionization system. Durability and ease-of-use compare favorably with previously described low-flow ionization sources. Because of the extended ISN lifetime, long spray periods that require minimal user intervention, and accurate positioning through the ICS, JaFIS undoubtedly provides for high sample throughput scenarios and is adaptable to full automation. Furthermore, facile sample recoverability means enhance success rates necessary for routine protein identification. Furthermore, the apparatus of the present invention is readily adaptable to autosampling, and computer-control of optimal pressure within the resealably sealable container in order to automatically regulate the rate at which electrospray is formed, and to accurate automatic positioning of the ion spray needle relative to the intake of the mass spectrometer through feedback from reference peptide ion current.

EXAMPLE II

Analysis of performance of ionspray Device of the Invention In this example, an investigation was conducted to determine the effects of head pressure, position and ion spray potential on flow rate and ion spray stability of an electrospray apparatus of the invention.

Needle Construction:

Fused silica capillaries used to produced needles herein were purchased in bulk from Polymicro Technologies (Phoenix AR). In order to minimize the dead volume associated with the needles, only raw material having an internal diameter of 25 μm, 50 μm, and 75 μm was used. The OD was held constant at 365 microns.

The fused silica was then cut to six inch lengths and the center one centimeter of the polyamide coating was removed using CE Solve, a polyamide stripper. A P2000 laser puller (Sutter Instrument Co. Novato CA) was then used to pull the capillaries using a series of four line programs that reproducibly provided needles having internal diameters of about 0.4 to about lOμm in 0.4μm increments.

Once the capillary had been pulled, it was separated int two equal halves forming two needles. The needles were then visually inspected and calibrated, and then stored in plastic boxes in groups of six until used. Before installation, the distal end of tip, i.e., the end connected to the ion spray body, was cut to a length of 3 cm. The associated dead volume and tip geometry for each needle is illustrated in Table 1.

Table 1

Pressure, Position, and Ion Spray Potential Of Needles

Larger ID needles (greater than 5 microns) were used in this investigation. The most common interfaces to tandem mass spectrometers which are likely to provide structural information for assisting in the identification of biologically relevant proteins include commercially available liquid chromatography instruments (LC), and electrically overcoated single analysis nano-electrospray ionization sources. The nominal flow rates for these devices range from about 200 nL to about 1 μl for LC-MS, and from about 20 to 60 nL/min for commercially available single analysis nano-electrospray.

Initially, a measuring station comprising a high magnification, high resolution CCD camera was mounted above the needle tip. The ion spray body was affixed to an x-y-z manual translation stage. An external power supply, which served as the voltage source, was connected to the ion spray body as schematically shown in Figure 1. A curtain plate identical to one in an API300 triple quadrupole mass spectrometer described above, was mounted perpendicular to the needle. A counter electrode comprising a 6 cm square, 1 /8-inch thick piece of titanium was placed behind the curtain plate, and a pico- ammeter (Kiethy Instruments) was used to measure current between the needle tip and the counter electrode. The current generated at each pressure, position of the needle tip, and ion spray potential combination was recorded. To measure the flow rate, a second CCD camera was aimed at the resealably sealable container containing the analyte liquid, wherein the container was affixed to the sample holder as described above. A video display was interfaced with the second CCD camera, and an acetate sheet was taped to the video display. The acetate sheet was calibrated to measure the volume of sample removed from the resealably sealable container in approximately 200 nL volumetric increments.

In all experiments, approximately 5 μL of a control solution comprising tryptically digested, E. coli β-galactosidase at a concentration of 100 fm/μL was initially placed in the resealably sealable container. The digest mixture was diluted from a concentrated stock in 33% CH_jCN, 0.5% HCOOH. Once the control solution was loaded into the resealably sealable container and the container was affixed to the sample holder, the pressure was incrementally increased, and the position of the needle and the ion spray potential was adjusted to maximize the current to the counter electrode. For statistical relevance, once optimized, a data point was only taken after 30 minutes of continuous spray. Spray stability was considered acceptable by monitoring the current to the counter electrode, and ensuring a deviation no greater than 15 % during the conduction of the experiment. The data generated from the measuring station was used as starting point for mass spectrometric determination. The robot set forth in Example I was mounted in front of a modified API300 triple quadrupole mass spectrometer. Monitoring of needle position, ionization characteristics, and sample volume was accomplished through a number of high resolution, magnifying CCD cameras (Chugai Boyeki, Japan; and Panasonic/Matsushita, Japan) strategically positioned and mounted to a vibration proof table (Newport). The video output was displayed on two 9 inch and one thirteen inch monitors (Panasonic). In this configuration, micron resolution was permitted with respect to positioning as well as rapid and facile movement of the needle between stations. More specifically the translation speed of the needle in the x, y, or z directions is > 2 cm/sec.

In these experiments, once the current was optimized, approximately five minutes of averaged signal for each data point was recorded. Furthermore, in all experiments, the ion current was monitored to ensure spray stability. Table 2 illustrates the mass to charge ratios (m/z), charge state and amino acid sequence of three randomly selected peptides ions from the control mixture which were used for the statistical analyses.

Table 2

For each data set, needle tip orifice ID, pressure, position, and ion spray potential, each parameter was empirically optimized. Initially, such optimization occurred by holding the needle tip orifice ID and pressure constant while adjusting ion spray potential and position of the needle. On the off-line station, corona discharge was found too be extremely detrimental to needle performance. Viewing the needle tip at higher flows indicated that under the high field strength necessary to produce corona discharge, the increased electric field strength caused cavitation. The field appeared to pull the analyte liquid out of the needle tip orifice and created gas pockets within the needle, behind the meniscus formed at the needle tip. This anomaly led too exceedingly unstable flow and as such was deemed undesirable. In order to eliminate this possibility, the needle was translated to the "See Tip" position. At this position, the needle tip was approximately 6 cm away from the bottom left portion of the curtain plate. Also, while in this position, the needle tip was directly in front of the viewing CCD camera. With the voltage off, a droplet of analyte liquid (the control) formed on the needle tip's surface area. When the correct voltage was applied however, a bubble formed at the needle tip which detached therefrom. Also, when the needle tip was set to a position which optimized spraying, the user could visualize ions and noise hitting the detector via the acquisition software. At that point, the potential can be toggled to see the creation of and detachment of the droplet, ensuring a complete electrical-liquid interface. The tip can also be translated along the flow path for visual inspection. This added functionality also allows for detailed inspection of the needle tip for particulate buildup, as well as formation of gas pocket in the tip. If a gas pocket does form, its progress through the flow path of the needle can be observed. In the case of particulate matter in the flow path of the needle, the user can toggle the voltage and look for droplet formation. In the event that the needle tip orifice is completely obstructed, the needle tip can be translated to the "Change ISN" position, the pressure vented from the resealably sealable container, and thus from the needle. The voltage can be removed, and the needle can be readily replaced with an unclogged needle. The ability to change needles mid-run results in only nominal sample loss (not greater than 60 nL).

With a needle tip having an orifice size of about 0.8 μm installed in the apparatus and given a pressure of about 6 psig, a potential of about 1500 volts was applied to the ion spray body. Then the flow path of the needle was inspected, and the electrical-liquid interface was confirmed. Subsequently, the needle tip was translated to the "Extend" position, and the optimization of the electrospray was started. The "Extend" position is about 2 cm from the aperture of a mass spectrometer interfaced with the electrospray apparatus of the invention. This point is referred herein as the starting point for all ion transfer and sensitivity experiments set forth in this example. While the needle was in the "Change ISN" position, 5μL of the control mixture was loaded into a resealably sealable container, which was then connected to the sample holder as described above. An 0.8μm ID needle was then installed into the apparatus. The needle was then translated to the "See Tip" position. About 1500 volts was then applied to the ion spray body and the container was pressurized to 6 psig. The flow path was inspected and the electrical-liquid interface confirmed. The needle was then translated to the "Extend" position and acquisition started. From the measuring station, it had been empirically determined that the highest current to the counter electrode was recorded when the needle tip was approximately 1 mm from the curtain plate. At this distance, the potential of the electrospray was about 750 volts. The measured flow under these conditions was 4 nL/min. The needle tip was then translated from the "extend" position towards the aperture in 1 mm increments, wherein the voltage was reduced by 150 V in each incremental translation. Once the needle tip was positioned 1 mm from the aperture, the ion current was monitored until a stable, continuous ion current was recorded. Once the parameters for a stable electrospray were recorded, the position of the needle was held constant and the ion spray potential was varied in 50-volt increments every five minutes. Each experiment was repeated at least five times with three different needles. Data taken in these experiments is graphically set forth in FIG.10. If the standard deviation of the data set generated was not within 20%, all experiments were repeated. Each data point represented in FIG. 10 is the average of the three experiments. All of these experiments were performed using the variables set forth in Table 3.

Table 3 Parameter Windows for generation of 80% of optimal Ion Transfer and Sensitivity from

User Defined Flow Rates

*gas used for pressurization is helium.

The combination of the variables needle tip orifice ID, fluid pressure, and ion spray potential, to obtain a flow rate of 4 nL/ min as set forth in Table 3, was selected for illustration of the optimization of the parameters set forth in Table 3, and thus to illustrate the optimization of sensitivity and ion transfer.

Knowing the optimal ion spray potential for all needle orifice ID as set forth in Table 3, pressure and distance combinations, subsequent experiments were designed to measure the effect of position of the needle tip on ion transfer and signal strength. Using the robot of Example I, the needle was translated to the "spray" position, and data was acquired. Once the electrospray had stabilized, the needle was translated forward by 500 μm and then back at 500μm increments relative to the ion curtain until the ion spray could no longer be maintained. The voltage was held constant during these experiments. These experiments were repeated using a variety of different needles, and no data set was considered valid unless within a 20% relative standard deviation. Figure 11 sets forth these results.

Also, these experiments were substantially repeated except that both the voltage and the needle tip's axial distance (z) from the curtain were held constant at each flow rate's respective optimum (Table 3). In the case of the data illustrated in Figures 10 & 11, the optimums were 600 volts the needle was peφendicular with the plane of the aperture, and concentric with the plane of the aperture, and at an axial distance of 1 mm from aperture.

The optimal position in both the x and y coordinate was then investigated. In each experiment, the needle was moved in both directions off the center, i.e., relative to a plane of symmetry through the entire length of the needle and the aperture. Translation of the needle was made in 50 μm increments until the maximum signal was obtained. A compilation of all the data relating to optimization of ion transfer and sensitivity involving positioning, ion spray potential, pressure, and tip orifice ID is illustrated in Table 3. Field strength was the next variable to be examined. In particular, it was desired to determine whether electrospray could be sustained with a constant field strength at distances from the aperture greater than the optimal distances set forth in Table 3.

A detailed examination of the data generated in the experiments set forth above indicates a relationship exists among flow, ISP and distance of the needle tip from the aperture of a mass spectrometer. For example, at a flow of 4 nL/min, the ISP was 600 volts and the axial distance of the needle tip from the aperture of the mass spectrometer was 1mm. However, at a flow of 25 nL/min, the ISP was 1100 volts and the axial distance of the needle tip from the aperture was 3 mm. The electric field strength in these experiments was calculated to be the potential difference between the aperture and the needle tip divided by the distance. In both instances, the calculated field strength was found to 250 v/mm. This calculated field strength was then compared with the other optimas outlined in Table 3. In order to validate results set forth in FIG. 11, the control solution was loaded into a resealably sealable container which was affixed to the sample holder as explained above, and the apparatus was configured so that the flow rate of the control solution from the container to the needle was 4 nL/min. The ISP and the position of the needle tip relative to the aperture were set in optimal levels set forth in Table 3 to maximize the signal strength. Next, the needle tip was translated away from the aperture of the mass spectrometer in 1 mm increments, and in each incremental translation, the ISP was raised by 250 volts. FIG. 12 graphically depicts these results. Thus, this data clearly shows that effective electrospray can be sustained with a constant field strength at greater distances from the mass spectrometer. More specifically, in the experiments where the ISP potential was held constant at its optimum as shown in FIG. 11, the average signal strength when the needle tip is 3 millimeters from the aperture was measured to be 2X10⁵ counts/second, with no detectable signal when the needle tip is 4 mm away from the aperature. In comparison, with the same source configuration, and a constant field strength, a similar average signal of 2X10⁵ counts/second can be sustained when the needle tip is 5 mm away from the aperture of the mass spectrometer.

As a result of these experiments, it was determined that as flow rate of the analyte liquid in the apparatus of the invention is increased, all parameters set forth in Table 3 become less critical to sustaining an electrospray. Furthermore, since Table 3 clearly illustrates a thorough understanding of the variables involved in generating a stable electrospray at various flow rates, a skilled artisan can readily automate the opitimization of these variables using readily known and available algorithms like "fuzzy logic." Thus the apparatus of the invention can be readily used in an automated fashion in applications such as proteomics, i.e., the determination of the protein structure, modication of proteins via phosphorylation, etc.

Flow Rates, Ion Transfer and Sensitivity

The experiments set forth in this section were designed to determine the effect of flow rate on ion transfer and sensitivity. In order to measure the effect of flow rate on sensitivity, 5 μL of the control solution was serially loaded into the apparatus of the Invention schematically shown in FIG. 1, for each of the variables set forth in Table 3. Once the electrical liquid interface was determined to be continuous, the needle tip was translated to the optimum position, and the correct ionization potential for that flow was applied to the sample as illustrated in Table 3. A stable flow was attained, and five minutes of averaged MS data was collected. Again, each experiment was repeated at least five times with three different needles. The results are represented in FIG. 12, wherein each data point is the average of the fifteen experiments. For statistical analysis, the low and high were removed and if the standard deviation of any single data set was not within 20% of the mean the complete sample set was repeated.

The data presented in Figure 12 clearly illustrates that lower flow results in higher sensitivity. More specifically, at a flow rate of 4 nL/min, the signal strength is approximately four times greater than signal strength obtained using a conventional conductivity coated nano-electrospray needles (20-40 nL/min), with a minimal six-fold increase in analysis time. Similarly, signal strengths of the present invention at a flow rate of 4 nL/min are approximately 3 times that of commercially available micro-ion-spray devices (100-200 nL/min), with a forty-fold increase in analysis time. The increased analysis times permit the successful identification of all proteins in a non-equimolar heterogeneous mixture, and readily permits the identification of sites of post-translational modification of proteins, including phosphorylation and glycosylation.

In order validate these unexpected and suφrising results, subsequent experiments were preformed using the apparatus of the invention set forth in FIG. 1 , wherein 3 μL of the control solution was loaded into apparatus as schematically shown in FIG. 1 and explained above. Flow rates similar to those used in the Experiments described above were also used. From results obtained, transfer efficiencies of 1 in 480 at 25 nL/min and 1 in 4200 at 250 nL/min were calculated. These results compare favorably to the previously reported values⁵ . To further investigate transfer efficiency the flow experiments were repeated and the efficiencies calculated and plotted, and set forth in Figure 13 . This figure graphically demonstrates the results of these experiments.

In the embodiment of the Invention set forth in FIG. 1, the present invention permits a spray time of a sample that is limited only by sample volume. Figure 3 clearly illustrate the longevity, stability, and reproducibility, of the spray. More specifically, Figure 3a illustrates a Total Ion Chromatogram (TIC) collected over 32 hours from a single sample source configured for a flow rate of ~20 nL/min. In this experiment, the analyte solution comprised about 30 μL of a two fold dilution of the control solution, thus having a concentration of 50 fm/μL. Figure 3b, c, and d illustrate that the spectra varied little in relative intensity, despite being separated by over 600 min of continuous spraying. Three ions, with m/z of 593.0, 681.2, and 871.8, were selected at random and their relative intensities compared. As shown in FIG. 4, the percent relative standard deviation for these ions was calculated to be 14.7, 5.5, and 7.2, respectively, which indicates an extremely stable electrospray was formed.

Example III Automated multiple Sampling with apparatus to form a continuous electrospray of an analyje

Automatic Sampling The preferred embodiment of the micro-sampling JaFIS is illustrated in FIG. 7. Simply, the micro-sampler allows for automated serial injection of an array of low microliter sample volumes. The tip-to-tube dead volume of the JaFIS-microsampler is < 50 nL, when used with needles manufactured from 25 μm ID fused silica capillaries (FSC). With a nominal flow rate of 4 nL/min, the interval between analysis of samples is about 12.5 minutes. However, this time period can be varied by selecting different needle tip orifice internal diameters and flow rates. More specifically, when interfaced with a quadrupole time of flight mass spectrometer, a user may take advantage of the increased sensitivity afforded by the non-scanning mass analyzer and increase the flow rate. In this configuration, the decrease in signal strength due to the higher flow is offset by the increase in sensitivity provided by the non-scanning mass analyzer.

In microsampling, individual samples are placed in individual resealably sealable containers, which in this case were commercially available PCR tubes. With the cap removed, the container is inserted through a threaded annular nylon collar as described above. The thread size and count of the collar is such that a commercially available autosampler cap with an approximately 1 mm thick septa can be screwed on to provide a pressure tight seal. The bottom 1 /4-inch of the collar is then threaded into the sample rack to hold the assembly in place during insertion and removal of the beveled and tapered end of the titanium insertion line, as described above. In this embodiment, the septa is constructed of a silicone PTFE bi-layer. When assembled and inserted into the sample rack, the resealably sealable container is capable of withstanding up to 40 psig of pressure.

Briefly, samples were pipetted or directly eluted into the containers. The caps of the containers with a septa were then used to seal the containers as described above. Then, the containers were screwed into the sample rack, as schematically shown in FIG. 7. The rack was affixed to a Sutter Instruments, Novato CA. Model MP285 xyz translation stage mounted to a PESciex (Toronto Canada) Quadrupole type mass spectrometer or a Micromass (Manchester, UK) mass spectrometer, and an optical rail. The rail had two preset mechanical stops; "load" and "sample". For mounting of the rack in this configuration, the rail was manually translated to the load position. For use on a Finnigan (San Jose CA) LCQ ion trap, the stage was inverted and mounted to the bottom of the instrument's rail. To load, the stage was swivelled from the "sample" position to the "load" position, and the rack was mounted to the xyz translation stage. Though functional in this configuration, the limited translation distances of the MP285 ( ~ 2.54cm) allowed only for sampling from 4 separate resealably sealable containers. However, one of ordinary skill in the art can readily modify the microsampler to use stepper motors with worm gear spindles and driving gears for movement to permit the use of circular or rectangular sample racks, which increase the number of samples that can be analyzed. Once the samples were loaded and the sampler was manually translated to the sample position as schematically shown in FIG. 7, the robot translated the stage to the appropriate sample position and the rack was raised. As explained above, the xyz translation stage was controlled by a microprocessor that can be readily programed to perform as set forth above by one of ordinary skill in the art. The 1 mm of 25 μm ID FSC pickup line which extended beyond the beveled and tapered end of the titanium insertion came into contact, and initially distorted the silicone of the septa. As a result, the beveled and tapered end could readily pierce the septa and enter the container. Once the septa had been pierced, fluid, such as an inert gas, flowed from the pressure source into the container, so that the pressure within the container increased. The rack was then raised so that the pickup line in the container could make contact with the analyte liquid volume. The translation distance for the raising of the sample rack was calibrated at setup. The 1 mm extension of the FSC pickup line facilitated two needs in this process. Initially, it ensured that the beveled and tapered end of the titanium insertion did not pierce the bottom of the resealably sealable container. More importantly, once the pickup line made contact with the analyte liquid and pierced its meniscus, the complete volume of analyte could be sprayed even if the pickup line did not make contact with the bottom of the container. Thus, in this configuration, the microsampler is capable of serial analysis of sample volumes as low as two microliters.

Figure 16 illustrates a TIC collected over 300 minutes from an integrated JaFIS- microsampling device of the invention on a PESciex API300 triple quadrupole mass spectrometer. In particular, FIG. 16 depicts MS data acquired during automated operation of the apparatus of the invention as set forth in FIG. 7 configured for a flow rate of 25 nL/min. Four separate analytes were sequentially analyzed. Panel a shows a TIC collected over 5 hours. All analytes were digested with trypsin and diluted to a final concentration of 100 fm/μl in 33% acetonitrile, 0.1 % formic acid. The sequence of analysis was as follows: 0-20 minutes carbonic anhydrase; 20-55 minutes BSA; 60-95 minutes β-galactosidase; 100- 135 minutes lysozyme; 140-175 β-galactosidase; 180-215 minutes lysozyme; 220-255 minutes carbonic anhydrase; 260-300 BSA. Panels b-i illustrate 5 minutes of average data ten minutes after the changeover from one analyte to another analyte. In order illustrate the reproducibility of positioning of the sample rack, the analytes were analyzed twice, and in different sequence.

The results set forth herein clearly indicate that the apparatus of the invention can produce a continuous electrospray, and further can readily be interfaced with an autosampler. As a result, the present invention clearly permits high throughput of numerous samples with a high sensitivity, which heretofore has not been possible using conventional electrospray devices.

The present invention is not to be limited in scope by the specific embodiments describe herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description.

Various publications are cited herein, the disclosures of which are incoφorated by reference in their entireties.

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It is to be understood that the invention is not limited to the illustrations described and shown herein, which are deemed to be merely illustrative of the best modes of carrying out the invention, and which are susceptible of modification of form, size, arrangement of parts and details of operation. The invention rather is intended to encompass all such modifications which are within its spirit and scope as defined by the claims.

Claims

WHAT IS CLAIMED TS:

1. An apparatus to form a continuous electrospray of an analyte for mass spectrometric analysis, comprising:

a fluid pressure source;

a resealably sealable container for holding the analyte, wherein the container is adapted for fluid registration with said pressure source;

an ion spray needle made of a non-electrically conductive material, wherein said needle comprises a tip having an orifice and a distal end opposite to said tip, wherein said distal end of said needle is in fluid registration with said resealably sealable container;

an electrical potential applied to said analyte upstream from said needle tip, wherein said electrical potential ionizes said analyte,

such that when said fluid pressure source increases pressure in said container, said analyte exits said container and is ionized prior to entering said needle, which discharges an electrospray of said ionized analyte.

2. The apparatus of Claim 1, wherein said fluid pressure source comprises a source of inert gas that is in fluid registration with said resealably sealable container.

3. The apparatus of Claim 2, wherein said inert gas is selected from the group consisting of N, He, Ne, Ar, Kr, Xe, and Rn.

4. The apparatus of Claim 5, wherein said pressure source further comprises a pressure regulator and pressure gauge which are in fluid registration with said source of inert gas and said resealably sealable container, which regulate and monitor the pressure of said inert gas entering said resealably sealable container.

6. The apparatus of Claim 1, further comprising an ion spray body having a chamber which is in fluid communication with said resealably sealable container and said needle, such that said ion spraybody is downstream from said resealably sealable container, and upstream from said needle, such that said analyte passes through said chamber, and said ion spray body is made of an electrically conductive material that is chemically inert to said analyte.

7. The apparatus of Claim 6, wherein said ion spray body is made of titanium, is approximately 0.5 mm long, and said chamber has a diameter of approximately 0.15 mm.

8. The apparatus of Claim 6, further comprising a power supply electrically connected connected to said ion spray body, such that said electrical potential is applied to said analyte while in said chamber.

9. The apparatus of Claim 1, wherein said needle comprises fused silica.

10. The apparatus of Claim 9, wherein said needle is transparent.

11. The apparatus of Claim 10, wherein said ion spray needle has a length of approximately 3 cm, and said orifice has an inner diameter of not more than 10, and particularly not more than 5 micrometers.

12. The apparatus of Claim 1, wherein said analyte is soluble in a polar solution.

13. The apparatus of Claim 1, wherein said analyte comprises a protein, a peptide, a nucleic acid, a fatty acid, a carbohydrate, a synthetic polymer, or a mixture thereof.

26. A method for creating a continuous electrospray of an analyte for mass spectrometric analysis, comprising:

providing a resealably sealable container containing the analyte to be analyzed; providing an ion spray needle comprising a tip and a wide end, wherein said wide end is in fluid registration with said resealably sealable container, and said needle is comprises of an electrically non-conductive material;

creating a flow of the analyte from the resealably sealable container to the needle; and

applying an electrical potential to the analyte up stream of the needle which ionizes the analyte, and said ionized analyte is discharged from said needle tip as an electrospray.

27. The method of Claim 26, further comprising the step of providing an ion spray body made of an electrically conductive material that is chemically inert to the analyte, wherein said ion spray body comprises a chamber that is in fluid registration with the wide end of the needle and the resealably sealable container, such that the analyte flows through the chamber.

28. The method of Claim 27, wherein the step of applying the electrical potential to the analyte comprising electrically connecting a voltage source to the ion spray body such that the electrical potential is applied to the analyte within the chamber of the ion spray body, and is ionized.

29. The method of Claim 28, wherein the voltage source comprises a power supply that is electrically connected to the ion spray body.

30. The method of Claim 27, wherein the ion spray body is made of titanium, is approximately 0.5 mm long, and the chamber has a diameter of approximately 0.15 mm.

31. The method of Claim 26, wherein the step of applying and generating an electrical potential for ionizing the sample upstream of the needle tip comprises inserting an electrode in the resealably sealable container and connecting the electrodes to a voltage source.

32. The method of Claim 26, wherein the ion spray needle is made of fused silica, has a length of approximately 3 cm, and said tip has an inner diameter of not more than 10, and particularly not more than 5 micrometers.

33. The method of Claim 26, wherein the step of creating a flow of the analyte comprises connecting a fluid pressure source to the resealably sealable container such that the fluid pressure source is in fluid registration with the container.

34. The method of Claim 33, wherein the fluid pressure source comprises a source of inert gas in fluid registration with the resealably sealable container.

35 The method of Claim 34 wherein the inert gas is selected from the group consisting of N, He, Ne, Ar, Kr, Xe, and Rn.

36. The method of Claim 33, further comprising the step of regulating and monitoring the flow of the analyte from the resealably sealable container to the ion spray needle.

37. The method of Claim 36, wherein the regulating step comprises providing a pressure regulator and a pressure monitor which are in fluid registration with the fluid pressure source and the resealably sealable container such that the pressure regulator.

38. The method of Claim 26, wherein the analyte is soluble in a polar solution.

39. The method of Claim 38, wherein the analyte comprises a protein, a peptide, a nucleic acid, a fatty acid, a carbohydrate, a synthetic polymer, or a mixture thereof.

40. An automated electrospray apparatus comprising: a tee having a first port, a second port, and a third port such that fluid can pass through said tee;

an ion spray needle having a tip with an orifice, and a wide end opposite to said tip, wherein said ion spray needle is comprised of an electrically non-conductive material, and said wide end passes through said first and third ports of said tee, such that a pressure tight seal is formed between said needle and said first port of said tee; a pressure source which is fluid registration said second port of said tee;

an insertion line having a first end in fluid registration with a third port of tee, and a second end forming a beveled and tapered end, such that said wide end of said needle passes through said insertion line and extends beyond said beveled and tapered point;

a sample rack having a plurality of sockets in which a plurality of resealably sealable containers are affixed to said sample rack, such that one resealably sealable container is placed in one socket;

a translation stage upon which said sample rack is mounted, wherein said translation stage translates said rack such that said beveled and tapered end, and said wide end of said needle enter a first resealably sealable container containing an analyte such that said wide end contacts said analyte, and fluid from said pressure source travels through said insertion line into said first container, which increases the pressure within said first container and forces said analyte from said container into said needle;

an electrical potential which is applied to said first analyte upstream of said needle, which ionizes analyte from said first container upstream of said needle tip, such that said ionized analyte is discharged as electrospray from said needle tip; and

said translation stage translates said sample rack such that said beveled and tapered end, and said wide end of said needle are removed from said first container afer discharge of said electrospray, and enter a second resealably sealable container such that said wide end of said needle contacts analyte within said second container.

41. The apparatus of Claim 40, further comprising an ion spray body having a chamber, wherein said chamber is in fluid registration with said tee, and in fluid registration with said wide end of said ion spray needle, and said analyte enters said ion spray body from said tee.

42. The apparatus of Claim 41, wherein said ion spray body is comprised of an electrically conducting material.

43. The apparatus of Claim 42, wherein said electrically conducting material comprises an electrically conducting polymer or a metal.

44. The apparatus of Claim 43, wherein the ion spray body comprises stainless steel.

45. The apparatus of Claim 44, further comprising a voltage source which is electrically connected to said ion spray body, such that said electrical potential is conducted to said ion spray body, and ionizes said analyte while said analyte is in said chamber of said ion spray body.

46. The apparatus of Claim 40, wherein said ion spray needle is transparent.

47. The apparatus of Claim 46, wherein said ion spray needle comprises fused silica.

48. The apparatus of Claim 40, wherein said translation stage comprises a computer controlled xyz translation stage.

49. The apparatus of Claim 40, wherein said insertion line is comprised of an electrically conductive material.

50. The apparatus of Claim 49, wherein said insertion line is comprised of an electrically conductive polymer or a metal.

51. The apparatus of Claim 50, wherein said insertion line is comprised of stainless steel.

52. The apparatus of Claim 40, further comprising a voltage source that is electrically connected to said insertion line, such that said electrical potential is applied to said analyte and ionizes said analyte while said analyte is in said insertion line.

53. The apparatus of Claim 40, further comprising an electrode within said container, wherein said electrode is electrically connected to a voltage source, and said electrode ionizes said analyte while in said container.

54. The apparatus of Claim 53, wherein the voltage source comprises a power supply.

55. The apparatus of Claim 40, wherein said pressure source comprises comprises a source of inert gas that is in fluid registration with said second port of said tee.

56. The apparatus of Claim 55, wherein said inert gas is selected from the group consisting of N, He, Ne, Ar, Kr, Xe, and Rn.

57. The apparatus of Claim 55, wherein said pressure source further comprises a pressure regulator, a pressure vent, and pressure gauge which are in fluid registration with said source of inert gas and said tee, which regulate and monitor the pressure of said inert gas entering said resealably sealable container, wherein said pressure vent is downstream of said pressure regulator and upstream of said tee.