MULTI-MODE IONIZATION APPARATUS AND USES THEREOF

Abstract

An ionizing system includes a flange device for connection to a mass spectrometer or ion mobility spectrometer having the property of providing a barrier between the lower pressure region of the spectrometer and a higher pressure region substantially at atmospheric pressure, and a channel therethrough providing fluid communication between the higher and lower pressure regions. A plate device independent of the flange device which can accommodate multiple samples, such as a sample plate device, when placed over the channel in the flange device substantially seals the channel Sliding the sample plate device while in intimate contact with the flange device provides a means to sequentially and rapidly expose said samples to the opening of the channel and thus the lower pressure region. Samples are ionized when exposed to the lower pressure region in as little as one sample per second using multiple ionization methods.

Claims

1. A method of ionizing analyte in a sample for one of mass spectrometry or ion mobility spectrometry, comprising: providing a sample plate device with at least one sample well, said sample well containing analyte to be ionized; a flange device connected to an analyzer which operates at sub-atmospheric pressure, and the flange device contains at least one of a conduit therethrough providing a channel open at the first end to a higher pressure region outside of the analyzer and at the second end to the lower pressure region of the analyzer; placing the sample plate device in intimate contact with the surface of the flange device thereby forming a substantial seal to maintain the operational pressure of the analyzer, wherein the at least one sample well, when aligned on-axis with the channel is in fluid communication with the said lower pressure region of the analyzer device and in substantial alignment with first ion optics for transmitting the analyte ionized from the sample in the sample well, wherein the ionizing of the analyte in the sample occurs under sub-atmospheric pressure by one of being subjected to a lower pressure region or being subjected to a lower pressure region and ionizing energy.

2. The method of claim 1, wherein the sample comprises an analyte and a matrix.

3. The method of claim 1, wherein the sample plate device comprises one or more of a sample plate with wells, a sample plate without wells, an inlet plate with an inlet tube therethrough, and a spacer plate with channels therethrough wherein said plates have a surface which when in intimate contact with one another form a substantial vacuum tight seal.

4. The method of claim 3, wherein wells in the sample plate device comprises one of indentations in a sample plate and channels in a spacer plate.

5. The method of claim 3, wherein said at least one sample in the at least one sample well is not in contact with the flange device when the sample plate device is in intimate contact with the flange device.

6. The method of claim 4, wherein said sample well on the sample plate device is aligned with the channel in the flange device and exposed to the lower pressure region while the remaining sample wells on the sample plate device are bounded by the surface of the plate device and the surface of the flange device and are at a substantially higher pressure than the sample well aligned with the channel in the flange device.

7. The method of claim 3, wherein the inlet tube is a conduit having an inner diameter between 0.2 mm-1.5 mm with a first end at or near atmospheric pressure which is in fluid communication with a second end which when aligned with the open channel in the flange device is in fluid communication with the lower pressure region of the analyzer.

8. The method of claim 1, wherein a substantial portion of the flange surface residing in the higher pressure region is flat such that when the plate device is placed over the channel in the flange and in intimate contact with the flat flange surface provides a seal between the higher and lower pressure regions so that the plate device can slide and maintain the seal.

9. The method of claim 8, wherein the sample plate device surface, when in intimate contact with the surface of the flange device and covering the channel, seals the said channel in the e device and provides a barrier between substantially atmospheric pressure, whereof except for the portion of the sample plate device covering the channel, the sample plate device resides substantially at atmospheric pressure.

10. The method of claim 9, wherein said sample plate device is manipulated manually or by computer controlled automation to cause the sample to be sequentially aligned with the said channel in said flange device and sequentially exposed to the lower pressure region of the analyzer.

11. The method of claim 1, wherein the method of transmitting ions and charged particles produced from the ionized analyte to the analyzer uses at least one of an ion funnel, tube lens, Einzel lens, S-lens, a conduit, and a multipole ion transmission device.

12. The method of claim 11, wherein the analyzer is configured to receive at least a portion of the said gas-phase ions and charged particles.

13. The method of claim 1, wherein the analyzer is one of a mass spectrometer, ion mobility spectrometer, or a hybrid ion mobility spectrometer and mass spectrometer.

14. The method of claim 1, wherein ionizing the analyte in the sample occurs at one of sub-atmospheric pressure by one of the methods of vacuum matrix-assisted ionization, matrix-assisted laser desorption/ionization, vacuum laserspray ionization, laser desorption/ionization, surface enhanced laser desorption ionization, desorption ionization on silica, solvent-assisted ionization, and electrospray ionization inlet.

15. The method of claim 14, wherein the sample is ionized by one or more ionization methods using the same analyzer while maintaining the analyzer at operational pressure.

16. The method of claim 15, wherein the sample is ionized using the at least one of vacuum matrix-assisted ionization, laserspray ionization, and matrix-assisted laser desorption/ionization.

17. An ionization apparatus configured to establish a connection with an analyzer, comprising: a flange device constructed to attach to the analyzer and provide a barrier between the lower pressure region of the analyzer and a higher pressure region substantially at atmospheric pressure, the higher pressure region being external to the analyzer, wherein the flange device is configured with at least one of a conduit therethrough providing at least one of a channel having a first end at or near atmospheric pressure and a second end that is in fluid communication with the lower pressure region of the analyzer, wherein the at least one channel is on axis with the first ion optics element in the lower pressure region; and a sample plate device, independent of the flange device and containing at least one sample well, when in contact with the surface of the flange device and covering the first end of the channel in the flange device forms a seal to maintain pressure within the analyzer; a sample plate device that is independent of the flange device, and contains therein at least one sample well, when placed in intimate contact with the flange device surface and covering the first end of the channel in the flange device forms a substantial airtight seal in order to maintain the pressure in the analyzer.

18. The ionization apparatus of claim 17, wherein the sample plate device is constructed from at least one of metal, polymer, glass, and quartz.

19. The ionization apparatus of claim 17, wherein the sample plate device further comprises one or more of a blank valve plate, a valve plate with a channel therethrough, a sample plate with wells, a sample plate without wells, a blank plate with a channel therethrough containing an embedded inlet tube, and a spacer plate with one or more channels therethrough.

20. The ionization apparatus of claim 19, wherein the embedded inlet tube has a gas tight seal with the blank plate and has a channel therethrough with an inner diameter of between 0.2 and 1.5 mm with the first end open to atmospheric pressure and the second end in fluid communication with the lower pressure of the analyzer when aligned over the opening to the channel in the flange device.

21. The ionization apparatus of claim 18, wherein the sample plate device comprises multiple plates in a stacked arrangement and in contact with one another so that when the plate device is in contact with the surface of the flange device and covering the channel in the flange device the operational pressure of the analyzer is maintained.

22. The ionization apparatus of claim 19, wherein a blank valve plate functions to cover the channel in the flange device and maintain operational pressure in the analyzer when samples are not being ionized and can be replaced by a sample plate without substantially disrupting the pressure in the analyzer.

23. The ionization apparatus of claim 19, wherein the valve plate with a channel therethrough resides between the sample plate device and the flange device to close or open the channel in the flange device by sliding the valve plate.

24. The ionization apparatus of claim 19 wherein said sample plate is constructed from at least one of a metal plate, a plastic plate, a glass plate or quartz plate.

25. The ionization apparatus of claim 19, wherein the spacer plate has channels therethrough with diameter between 2 and 15 mm and preferably between 3 and 7 mm and in operation resides between a sample plate which covers the spacer plate channels on one side with the opposing side being in intimate contact with one of said flange device surface or a valve plate having a channel therethrough.

26. The ionization apparatus of claim 25, wherein the said channel in the spacer plate has a first end which in operation is covered by a sample plate forming a substantial airtight seal, and a second end which forms a substantial airtight seal with one of the flange device surface or the valve plate surface so that when the said spacer plate channel is aligned with the channel in the flange device, both channels are in fluid communication with the pressure in the analyzer and maintain the operational pressure of the analyzer.

27. The ionization apparatus of claim 26, wherein the sample plate attaches to the surface of the spacer plate to align samples applied to the sample plate with the first end of the channels in the spacer plate.

28. The ionization apparatus of claim 17, wherein the flange device is constructed from a polymer or metal, and contains at least one channel in the flange device having a diameter that is greater than 2 mm.

29. The ionization apparatus of claim 28, wherein the at least one channel has a diameter between 3 and 7 mm.

30. The ionization apparatus of claim 28 or 29, wherein the at least one channel has a depth less than 5 times the diameter of the channel.

31. The ionization apparatus of claim 28 or 29, wherein the at least one channel has a depth that is less than the diameter of the channel.

32. The ionization apparatus of claim 28, wherein a portion of the surface of the flange device containing the first end of the channel therethrough is flat and when in intimate contact with the flat surface of the sample plate device provides a substantial airtight seal whether the sample plate device is stationary or sliding over the flange device surface.

33. The ionization apparatus of claim 32, wherein said sample plate device holds one or more samples in a linear configuration so that movement in one direction using one of manual or automated means aligns individual samples sequentially with the channel in the flange device and thus exposed to the lower pressure within the analyzer.

34. The ionization apparatus of claim 17, wherein the ionization apparatus is configured for movement of the sample plate device over the channel in the flange device by sliding the sample plate device in one or two dimensions of axis.

35. The ionization apparatus of claim 17, wherein the sliding motion of one of said sample plate device or valve plate are controlled manually or by computer directed robotics with all devices and plates substantially at atmospheric pressure.

36. The ionization apparatus of claim 17, wherein said sample plate devices have flat edges allowing interchange with one another by sliding two plates, with flat edges in intimate contact, across the channel in the flange device without substantially disrupting the vacuum of the analyzer.

37. The ionization apparatus of claim 17, wherein the first ion optics element in the lower pressure region comprises one of an ion extraction lens, S-lens, Einzel lens, tube lens, ion funnel, conduit, or quadrupole, hexapole, and octapole ion transmission devices.

38. The ionization apparatus of claim 37, wherein the first ion optics element is heated.

39. The ionization apparatus of claim 17, wherein the analyzer is one of a mass analyzer of a mass spectrometer. an ion mobility analyzer, and an ion mobility analyzer interfaced to a mass analyzer.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0046] The foregoing aspects of the invention will be more clearly understood from the following descriptions read in conjunction with the accompanying drawings in which:

[0047] FIG. 1 is a 3-dimensional cutaway representation of a preferred embodiment of an improved vacuum ionization source for mass or ion mobility analyzers and includes a flange device, sample guide rails, ion guides, inlet chamber, and sample plate device.

[0048] FIG. 2 is a schematic side view representation of one embodiment of an improved ion source apparatus for sample introduction to the vacuum of a mass or ion mobility analyzer showing different arrangements of a sample plate device interfaced to a flange device in FIG. 4 A-C and associated expanded insets.

[0049] FIG. 3 Is a schematic top view representation of a preferred embodiment of an improved apparatus for use as an ion source of a mass spectrometer or ion mobility spectrometer including sample plate devices, flange device, plate device guides, and a device to apply voltage to the sample plate device.

[0050] FIG. 4 is a schematic top view representation of a preferred embodiment of an improved apparatus for use with an ion source of a mass spectrometer including a motorized drive for moving sample plate devices along a grove defined by guide rails.

[0051] FIG. 5 is a schematic top view representation of a preferred embodiment of an improved apparatus for automated sample introduction to the vacuum of a mass spectrometer or ion mobility spectrometer for sample ionization displaying sample plate devices, motorized drive, and sample plate device feeder and receiver cassettes.

[0052] FIG. 6 is a schematic side view representation of a preferred embodiment of an improved ion source depicting laser ablation in transmission or reflective geometry in FIG. 6A and in FIG. 6B and FIG. 6C a means to use the apparatus for surface imaging.

[0053] FIG. 7 is a schematic view of representations of a preferred embodiment of a means of sequentially exposing multiple samples from a 2-dimensional plate to the vacuum of the inlet chamber of a mass spectrometer or ion mobility spectrometer where ionization and ion transmission are initiated.

[0054] FIG. 8 is a schematic of a preferred embodiment of a sample plate holding an inlet tube to allow atmospheric pressure ionization (API) methods on the vacuum multi-mode source.

[0055] FIG. 9 shows in FIG. 9A the total ion current chronogram of the protein ubiquitin using vMAI with the improved ion source while FIG. 9B is a mass spectrum obtained from a single acquisition showing charge states of the protonated molecular ions.

[0056] FIG. 10 shows in FIG. 10A the ion current chronograms of 6 compounds in 0.4 minutes obtained using the improved ion source with vMAI and FIG. 10B shows the mass spectra obtained from 3 of the samples on an Orbitrap mass spectrometer.

[0057] FIG. 11 is a high throughput vMALDI analysis example demonstrating ionization of a blank plus 5 samples in ca. 1 second for each sample using the ion source apparatus described herein on an Orbitrap mass spectrometer.

[0058] FIG. 12 is the limited mass range mass spectrum of a fungus smeared onto a sample plate using the ion source apparatus described herein interfaced to an Orbitrap mass spectrometer using vMAI and vMALDI on the same fungus with two different matrices.

[0059] FIG. 13 is the mass spectrum of uranyl nitrate containing the most abundant uranium-238 isotope and detection of the lower abundant uranium-235 isotope (inset) using the ion source apparatus described herein on an Orbitrap mass spectrometer operated in the vMAI mode using a wet binary matrix mixture in the negative mode of detection.

DETAILED DESCRIPTION OF DRAWINGS

[0060] FIG. 1 is a schematic of a cutout 3-dimensional view of a preferred embodiment of an apparatus 100 which allows rapid or slow sequential individual exposure of samples individually to the vacuum of a mass spectrometer while all other samples remain at or near atmospheric pressure (AP). Gas-phase ions generated by vacuum matrix-assisted ionization (vMAI) are transmitted into the analyzer 130 through restriction 126. The schematic is a generalized representation of a modification of a commercial AP ionization (API) inlet vacuum chamber 120 of a mass spectrometer. The vacuum chamber 120 may have a rotary pump 124 connected thereto which is required for normal operation where an inlet aperture, typically between 400-800 microns inner diameter leaks air and/or nitrogen or other gases into chamber 120 from an API region as in electrospray ionization (ESI), AP chemical ionization (APCI), or AP matrix-assisted laser desorption/ionization (MALDI). These sources and methods are used in mass spectrometry (MS) and ion mobility spectrometry (IMS) for analyses, as well as for surface imaging MS, for large (e.g., proteins) and small molecules (e.g., lipids, drugs) within e.g., biological tissue.

[0061] In FIG. 1, the commercial inlet tube or aperture device with flange used with API sources has been replaced by flange device 101 which forms a vacuum seal with chamber 120, in this case, using “O-ring” 104. Other means of forming an air tight seal such as gaskets can be used. Flange 101 has a conduit or channel 102 with diameter between approximately 2 mm and 10 mm, typically on axis with the first ion transfer optics. The channel 102 in flange device 101 allows fluid communication between a higher pressure region, typically AP, and the lower pressure region within chamber 120. Unless the channel 102 is covered to make a near air tight seal, the airflow would overload the pumping system of the analyzer. Vacuum chamber 120 may contain ion extraction lens, an ion guide and/or focusing elements 122, which may be a single device, and in certain configurations desolvation devices such as obstructions or heater elements. Ions produced from a sample travel through opening 102 into the ion guide/focusing elements and through a restriction 126 into the mass analyzer 130. Restriction 126 is designed to provide a pressure drop between inlet chamber 120 and the mass analyzer 130 of the mass spectrometer. This pressure drop is necessary for API instruments. Flange 101 has situated on the face opposite chamber 120, and typically at AP, two guide rails 103a and 103b to allow a sample plate device 107 to slide over the channel 102 in flange device 101. The channel in flange device 101 aligns with the ion guide arranged in a manner that allows gas-phase ions and charged particles produced from a sample to traverse into the analyzer 130, which may be a mass or ion mobility analyzer. Other configurations can be envisioned, but of importance here is the ability to sequentially expose samples to the vacuum of a mass spectrometer or ion mobility spectrometer by sliding a sample plate device 107 containing one or more samples across channel 102 with minimal increase in the pressure in the analyzer. An optional valve plate 105 resides between the flange device 101 surface and the sample plate device 107 which slides to one of two positions. In the open position, a channel in the valve plate 105 of equal diameter to the inner diameter of channel 102 is aligned with channel 102 so that the lower pressure in chamber 120 is in fluid communication with the sample in the sample plate device 107.

[0062] FIG. 2 further clarifies the operations of the invention through a series of schematic representations with associated insets for additional detail. A side view of a simplified flange device 101 with a valve plate 106 and a sample plate device 107 made up of a spacer plate 108 with multiple channels 109 therethrough and a sample plate 110 is presented in FIG. 4 A-C. FIG. 4A is a depiction of flange device 101 with channel 102 covered by a valve plate 106, which in this case is a solid flat rectangular metal or plastic piece with a hole 106a therethrough. The thickness of the valve plate 106 is typically less than 5 mm and preferably between 1 and 3 mm, but can have other dimensions of thickness. The width is sufficient to cover hole 106a and substantially seal channel 102. The valve plate 106 may be held in place by a cutout in the guide rails 103 making a slot between the guide rail and the AP face of the flange device 101. In the position shown in FIG. 2A and inset expansion to the right, the valve plate seals channel 102 in flange device 101 thus separating the higher pressure region which is typically at or near AP from the lower pressure region defined by chamber 120. However, sliding the valve plate left in FIG. 2A opens channel 102 through hole 106a to the higher pressure region. The outcome depicted in FIG. 2B expansion is undesired as channel 102 and hole 106a are typically greater than 2 mm in diameter and sufficiently large to increase the pressure in the analyzer above the operational pressure. Thus, in the situation where no sample plate covers valve plate 106, the closed position shown in FIG. 2A would be used. Safeguards may be used to prevent the situation depicted on FIG. 2B from occurring. FIG. 2C shows an example where a spacer plate 108 with channels 109 therethrough lies over the valve plate 106 in the open position in which hole 106a lies over channel 102 in the flange device 101. So long as there are flat surfaces in intimate contact with one another, the vacuum of the mass spectrometer is maintained provided the sample plate 110 is placed over spacer plate 108. In this case only the gas, typically air, in channel 109 over channel 102 is drawn into the region enclosed by chamber 120. Because the analytical samples residing on the sample plate align with channels 109 of the spacer plate, the samples never contact the valve plate 106 surface, or in the absence of a valve plate, the flange device 101 surface. In an alternative arrangement, the sample plate device 107 can be replaced with a sample plate device 107 in which the spacer plate 108 and flat sample plate 110 is replaced with a sample plate having indentations or wells for samples to reside. While wells work well with vMAI, they are restrictive with vacuum MALDI (vMALDI) in that only reflective geometry laser ablation is possible unless special plates with indentations made from glass or quartz are used to allow laser beam transmission to reach the sample. By including a spacer plate with channels therethrough between the sample plate and the flange device, wells are created by the spacer plate channels.

[0063] FIG. 3 is a simplified schematic of a top view of a preferred representation of the multi-ionization apparatus with additional components. This schematic is simplified for clarity. Flange device 101 has attached to the surface guide rails 103. In this depiction, a blank plate device 105 with a flat surface slides across the flat surface of flange device 101 and fits between the guide rails 103a and 103b. The purpose of the blank plate device 105 is to act as a valve to close channel 102 in flange device 101 to prevent gas flow from the higher to the lower pressure region. In this arrangement all plate devices slide across the face of the flange device and over time may produce wear on the face of the flange device. The valve plate 106 depicted in FIG. 2 lies between the flange device 101 and the sample plate device 107 and is readily replaced if necessary. Sample plate devices 107 can be abutted with one another or a blank valve plate 105. The flat surfaces of the blank valve plate 105 or sample plate devices 107 are able to slide along the channel defined by the guide rails 103a and 103b while sealing the channel 102 in flange device 101 to maintain operational vacuum in chamber 120. Likewise, a sample plate device 107 can replace valve plate 105 by sliding along the guide rails to present a flat edge surface 107a against the flat edge surface 105a of the blank valve plate 105. The edge surfaces 107a of sample plate device 107 and 105a of blank valve plate 105 are flat so as to make a near air-tight seal when the plates are pushed together in intimate contact. In this manner, sample plate device 107 can be pushed against the blank valve plate 105 to slide the blank valve plate across channel 102 with minimal disturbance of the vacuum. Note that when a sample plate device covers channel 102 the pressure differential across the plate holds the plate against the flat surface of flange 101 maintaining chamber 120 well within the designed pressure range and less than 10 mbar and preferably less than 1 mbar pressure, although it can be designed to allow higher pressure in the region defined by chamber 120. However, the guide rails 103 or other devices may be used to apply pressure to the blank valve plate 105 or plates device 107 to assure that a plate covering channel 102 cannot be dislodged and cause loss of instrument vacuum. Also, the viewed surfaces in FIG. 2 are at or near atmospheric pressure. Therefore, a sample plate device 107 holding sample(s) at AP can be used to replace a sample plate device 107 by the same mechanism described for replacing the valve plate 105. Plate replacement occurs at AP with the only area exposed to sub-AP is the area of the sample plate device 107 or valve plate 105 that lies directly over the channel 102 in flange device 101. Note that a flat surface may be curved so long as the two surface fit together to maintain a substantial vacuum seal and in some cases slide over one another. An insulated electrically conductive feedthrough 111 allows application of voltage or ground potential to the sample plate device 107 in order to produce a voltage gradient between the sample plate device 107 and, lens elements on the low pressure side of channel 102. A variety of lens elements 122 may be used to focus and or transfer ions into the ion optics of the analyzer including but not limited to a tube lens, Einzel lens, ion funnel, and quadrupole, hexapole, or octupole ion guides.

[0064] FIG. 4 depicts a partial top view schematic representation of a preferred embodiment showing automated plate movement in apparatus 100 using a motorized screw drive 116, but other methods, as for example a belt drive may be used. Other devices familiar to those practiced in the art can also be used. In the automated mode, sample plate device feeder 114 (not shown for clarity) is the interface between a cassette (not shown in this representation) holding multiple plate devices 107 and a groove defined by guide rails 103 and the face of flange device 101. In one embodiment, the plate device feeder 114 allows a plate device 107 to move into position in the plate device receiver grove and resting on guide rail 103b. In this position, the sample plate device 107 can be moved in the groove toward channel 102 and between guide rails 103a and 143b. An automation device 116 having a computer controlled motor drive moves the sample plate device 107 to expose each sample aligned in a linear row on the sample plate sequentially over channel 102. Note that channel 102 is at all times closed from AP or near AP by either the blank valve plate 105 or by the plate 107. When closed by the sample plate device 107, channel 102 is not closed by a valve plate 105. Note that in this representation, the position of channel 102 is shown for clarity and channels 109 in spacer plate 108 are visible through the sample plate 110 which is represented as a glass microscopy slide.

[0065] FIG. 5 is a simplified schematic of a top view of a preferred representation of an ion source apparatus for automated high throughput sample analyses. The apparatus of FIG. 5 allows rapid sequential exposure of samples residing on a sample plate 110 at or near AP to the first vacuum stage 120 of analyzer 130 when the sample is moved over channel 102 by sliding the sample plate device 107, which incorporates sample plate 110, between guide rails 103. In this view, a representation of a plate device feeder 114, feeder cartridge 112, plate device receiver 115 and receiver cartridge 113 are combined with flange device 101, guide rails 103, sample plate device 107, and sample plate drive 116. Plate device 107 contains a spacer plate 108 sitting atop a valve plate 106. Channel 102 is open when the hole 106a in valve plate 106 aligns with channel 102 and closed when the valve plate 106 slides between guide rails 103 so that channel 102 and hole 106a are not aligned. Under certain circumstances the valve plate 106 can be eliminated. In this arrangement, the channel 102 in flange device 101 can be covered or open by simply sliding the plate device 107 over channel 102. Alternatively, a blank valve plate 105 can be used to cover channel 102 when data acquisition is not being carried out.

[0066] In the representation in FIG. 5, the sample plate device 107 consists of a glass slide sample plate 110 affixed on top of the spacer plate 108 which has channels 109. For representation purposes only, the view shows the positions of two sample plate devices 107 separated to show hole 106a in valve plate 106. So long as hole 106a is not over channel 102 in flange device 101, the pressure in the analyzer will not be affected. In operation, hole 106a aligns with channel 102 and the two sample plate devices 107 abut each other and cover the hole 106a in the valve plate 106 to substantially seal channel 102 from gas flow from the higher to the lower pressure regions.

[0067] The operation of the device represented in FIG. 5 can be visualized as follows. A sample plate device 107 containing samples which are typically made of a matrix and an analyte and residing on a sample plate 110 in positions that align with channels 109 in spacer plate 108, the sample plate 110 being in intimate contact with spacer plate 108 to form a substantial vacuum seal, is place in the feeder assembly cartridge 112, typically with other sample plate devices 107 containing sample to be analyzed. The bottom plate device drops into feeder assembly 114 when a sample plate device 107 sliding over valve plate 106 and between guide rails 103 clears feeder assembly 114. The dropped sample plate device 107 rests on guide rail 143b and is moved towards sample plate device receiver 115 by the computer controlled motorized automation device 116 and makes intimate contact edge to edge with the next plate device in the grove between the guide rails 103. A sufficient vacuum seal is created over hole 106a when the pressed together edges 107a of sample plate devices 107 pass over hole 106a. When the sample plate device 107 reaches the plate receiver 115, it drops into the receiver cartridge 113, thus freeing the next sample plate 107 to continue moving into the feeder receiver 115. By mechanically moving only the last plate to exit the feeder cartridge 112 onto the guide rail 103b, the resistance to movement caused by friction between the plate device 107 and the valve plate 106, or in the case where the valve is a blank valve plate 106a, the flange device 101 surface, results in a tight fit of the sample plate device 107 sides 107a so that the interface of the two plates passing over hole 106a in the valve plate 106 or the channel 102 in the flange device 101, respectively, will not significantly disrupt the vacuum of the analyzer 130.

[0068] The device represented in FIG. 5 can further be used to automatically acquire mass spectra of samples aligned on sample plate 110 in a linear fashion and in positions aligned with channels 109 in spacer plate 108. In this mode, when the sample plate device 107 clears the feeder 114, the next plate in feeder 114 drops into position onto guide rail 143b and the automated drive 116 begins moving the new sample plate toward channel 102. Meanwhile, sample plate receiver 115 collects sample plate devices 107 which drop into receiver 115 once clear of the guide rail 103b. Other configurations can be employed to achieve automated sequential exposure of samples to the vacuum of a mass spectrometer.

[0069] FIG. 6 is a representative schematics of preferred embodiments providing additional details relative to use of the invention for sample analyses using vacuum MALDI (vMALDI) and for imaging by laser ablation MALDI. Reflective geometry typically requires the laser beam 147 travel through a portion of vacuum chamber 120 and channel 102 in flange device 101 before striking the sample. Such an arrangement requires a transmission window into chamber 120 and mirrors in addition to optical focusing lens (not shown in FIG. 6A or FIG. 6B) to guide the focused laser beam to the sample, or alternatively, to use of fiber optics. For many applications of MALDI, transmission geometry laser ablation is preferred where the laser beam 146 strikes the sample from the AP side (backside). In this case, as depicted in FIG. 6A, a spacer plate 108 with channels 109 therethrough lies in intimate contact with the surface of flange device 101, assuming a valve plate 106 is not situated between the spacer plate 108 of plate device 107 and the surface of flange device 101 facing the AP region. The sample plate device 107 consisting of a spacer plate 108 and a sample plate 110 can slide over the flange device 101 surface or alternatively the valve plate 106 guided by the guide rails 103. In the case of using a valve plate 106, the valve plate remains stationary in the open position. So long as a sample plate device 107 is over channel 102, the spacer plate 108 channel 109 is substantially sealed from AP and in fluid communication with the lower pressure region inside chamber 120. When using a flat sample plate 110 for samples, indentations are not required as the samples can fit within the channels 109 of spacer plate 108. The matrix:analyte samples, typically made up of matrix and analyte reside on the surface of the sample plate 110 in the position where they fit within the channels 109 on spacer plate 108. However, indentations may be used, and in certain configurations it may include a commercial well plate. The indentations, may aid sample alignment with the channels 109 in the spacer plate 108. The sample plate 109 is held stationary relative to the spacer plate so that sliding the spacer plate along the guide rails also moves the sample plate.

[0070] While a variety of sample plate materials can be used for vMAI including, but not limited to, metal and polymer, only a plate which allows transmission of the laser beam can be used with transmission geometry vMALDI or vLSI. One possibility is use of a glass microscopy slide which does not require a conductive surface, so long as the spacer plate is conductive. However, for the positive (negative) ion mode, a microscopy slide with a coating having a positive (negative) charge is beneficial, especially for vMAI, through special coatings. With a sufficiently thin spacer plate, voltage or ground potential may be applied to valve plate 106 as long as it is made of conductive material such as metal. In one preferred embodiment for valve plate 106, Teflon is used to provide a good vacuum seal and easier movement of plate device 107. In summary, voltage or ground potential on one of valve, spacer, or sample plates is desirable for best results, especially at low gas flow (low pressure). Voltage or ground potential can be applied to the plates through electrically conductive device 111 as depicted in FIG. 3. Otherwise, a gas flow to guide ions from the sample into the ion guide/focusing region and inlet to the mass spectrometer is necessary to transmit ions and charged particles produced in the ionization process. Even with three interfaces of flat surfaces, the vacuum of the mass spectrometer is held stable with the arrangement shown. For glass or quartz sample plates, transmission geometry laser ablation can be used to obtain vacuum MALDI mass spectra. The laser beam may strike the sample through sample plate 110 from the backside at angles ranging from 25 to 90 degrees, and preferable 45-90 degrees, or alternatively fiber optics can be used.

[0071] FIG. 6B depicts a schematic representation of a preferred embodiment of a simplified method for surface imaging using the inventions described herein. The laser beam can illuminate the sample from transmission 146 or reflective 147 geometries. The differences between this depiction and that in FIG. 6A is that the spacer plate has one large channel 192 instead of multiple smaller channels 109. This is depicted by a top view of sample plate device 107 in FIG. 6C. A glass or quartz sample plate 111 may fit on top of spacer plate 108 with channel 192 so that the sample plate 110 is held in place by frame 194. The arrangement is especially useful for imaging where, e.g. a biological tissue slice is affixed to the transparent sample plate 110 and matrix solution is applied in a manner common to MALDI imaging and dried before the sample plate device 107 is placed over channel 102 in flange device 101. For imaging larger sections, movement of the sample s plate device 107 is achieved by sliding in 2 dimensions while firing the laser using optical lens elements to focus the beam on the tissue being imaged as known to those practiced in the art.

[0072] FIG. 7 is a depiction of a preferred embodiment of a means to move sample plates in 2 directions. Rails 142 and 143 are 90 degrees to one another. Valve plate 181 in this depiction is part of the 2 dimensional sample plate holder 180 so that positioning the valve plate portion 181 over channel 102 in flange device 101 allows removal of the 2-dimensional sample plate 144 having indentations 145 for samples. FIG. 7A is from the side facing the instrument vacuum and FIG. 7B is from the side at AP. The rails can be driven by computer controlled stepper motors familiar to those practiced in the art. The same arrangement can be used when imaging. Other arrangements to sequentially expose samples on a 2 dimensional plate can be envisioned.

[0073] FIG. 8 is a schematic representation of a preferred embodiment of a means of using the present invention to obtain electrospray ionization inlet, solvent-assisted ionization or matrix-assisted ionization (MAI) using an inlet tube without the necessity to vent the instrument to install an AP ion source. This invention is especially valuable for calibrating the instrument using ESI. A plate device 107 is composed only of a blank spacer plate with indentation 150 normally used to hold and align a sample plate. The plate device has a channel 109 therethrough into which an inlet tube 151 is held with a vacuum tight seal represented by a screw cap 152 which tightens onto a ‘o’-ring 153. The inlet tube with inner diameter between 0.4 mm and 1 mm is designed to be open to at or near AP at the end facing toward the viewer in the top depiction and upward in the lower depiction. The other end of the inlet tube resides in plate device 107 and near the face of the plate device which slides along the flange device 101 or valve plate 106 surface. When the inlet tube is aligned with the opening 102 in flange device 101, the exit end of inlet capillary 151 is in fluid communication with the lower pressure region inside chamber 120. The end in of inlet tube 151 does not protrude through spacer plate 108 so as not to contact either the flange device 101 surface or the surface of valve plate 106 when sample plate device 107 slides within the grove defined by guide rails 103. The length of the inlet capillary on the AP side of the plate device 107 can be of length of a few millimeters to several meters. Interchange of the sample plate device 107 containing inlet tube 151 with any plate device 107 follows the same procedure as detailed above for interchanging plate devices.

[0074] The descriptions provided is of exemplary arrangements, and one skilled in the art will be able to envision other arrangements including other 2 dimensional arrangements of samples in which the sample plate can move in the x and y directions as used with MS imaging sources and methods. The inventions described herein apply to 1- and 2-dimensional arrangements of samples because both can advantageously use flat surfaces to substantially seal the vacuum of a mass spectrometer from AP or near AP while allowing the plates to slide across channel 102 in flange device 101. In certain configurations a 3-dimensional could be analyzed directly with quite some limitations; preferably inlet ionization or traditional API methods with an extended inlet are preferred. While some API mass spectrometers have pumping capacity to operate with channel 102 diameters up to 1 mm in diameter, typical size of channel 102 of this invention is typically between 2 mm and 7 mm in diameter in order to allow maximum ion transmission and the maximum number of samples on a sample plate without any two samples being simultaneously exposed to the lower pressure of the analyzer. The sample plate 110, when not using a spacer plate 108, has at least 1 indentation or well into which the sample, typically a matrix and a analyte, is placed so that the sample does not come in direct contact with the flange surface which can result in carryover between samples. However, more typically the sample plate 110 has multiple indentations for multiple samples. Alternatively, the sample plate may have a spacer plate 108 inserted between it and the surface of flange device 101 in which one or more channels pass through the spacer plate 108 so that the sample, on a sample plate 110 is in fluid communication with the vacuum of the mass spectrometer when the channel 102 in flange device 101 is aligned with the channel 109 in spacer plate 108 and the matrix:analyte sample. Additionally, a valve plate 106 may be placed between the sample plate 110, or the spacer plate 108, and the flange device 101 to allow closure of channel 102 in the flange device 101 when no sample plate device 107 covers channel 102.

[0075] Other arrangements of the valve plate may be envisioned, including a valve built into the flange. However, the valve plates described herein are inexpensive and do not significantly increase the distance between the sample and the ion extraction lens. Another aspect of the invention is that samples may be exposed to vacuum sequentially so that only one sample is exposed to the vacuum of the mass spectrometer for transmission of ions at a time. Therefore, the pumping requirement is greatly reduced relative to inserting a sample plate through a vacuum lock into the first vacuum chamber of the mass spectrometer. However, spacer plate channels may be made sufficiently large to encompass more than a single sample or to encompass a surface for imaging.

[0076] The samples are usually prepared at AP by pipetting or using sample preparation apparatus common to MALDI, e.g. dried droplet, layered, spray coating approaches, or solvent-free matrix coating. A matrix is broadly defined as a compound providing a condition which enhances the ionization of analyte molecules. However, in laser desorption, DIOS, and SELDI, no matrix compound is necessary for ionization upon laser ablation. Except for the sample over channel 102 in flange device 101 and in fluid communication with the vacuum of the mass spectrometer 130, the remaining samples on the sample plate 110 are at or near AP. Therefore, unless gas is leaked into the system purposely to speed the ionization process or to aid in transfer of ions to the mass analyzer, the only gas introduced by each sample into the vacuum of the mass analyzer 130 is that in each indentation on sample plate 110 or channel 109 of spacer plate 108. Note that the spacer plate 108 may be of varying thickness, as for example 1 mm or 13 mm, as well as varying channel diameters such as approximately 2 mm or approximately 7 mm, or even larger and under certain circumstances smaller. While gas may be leaked into the system to hasten ionization or to transfer ions from the sample to the mass analyzer, a voltage differential may advantageously be used for ion transfer. This voltage difference is typically placed between the sample plate 110 or the spacer plate 108 and the lens elements 122 which may contain an extraction lens, but other arrangements known to those practiced in the art may be used to transport ions through an electric field.

[0077] The ionization apparatus which employs a spacer plate 108 and a glass or quartz microscopy slide as sample plate 110 may also be used to substantially seal the channels in the spacer plate to secure the vacuum of the mass spectrometer when the sample plate device 107 is covering channel 102 in flange device 101. This arrangement is advantageous in that it allows ionization by transmission geometry vMALDI or vLSI, or use of vMAI. Each ionization method may be used on sequential samples simply by employing laser ablation using a MALDI suitable matrix or a vLSI suitable matrix, or not employing the laser with a vMAI suitable matrix. The time between ionization of samples with different ionization methods, once the sample is loaded onto the sample plate may be as little as 5 seconds. Likewise, reflection geometry vMALDI, LSI and vMAI can be obtained even for adjacent sample using the same procedure so long as the laser has been set up for reflection geometry. A laser can be of low or high performance in power and speed, although for imaging applications in transmission geometry, more powerful lasers are preferred for more effective penetration through the tissue. The laser wavelengths can be ultraviolet or infrared, as known to those practiced in the art. A microscopy slide with e.g., a positive surface coating may advantageously be used for positive ion analyses. Those practiced in the art will understand other surface coatings such as gold coatings and anchor chip targets and alike constructions can work as well. With the arrangement described herein, samples on glass TLC plates can be acquired and with specially designed spacer plates can be used to obtain mass spectra from ZIP tips so long as the ZIP tip forms a snug fit with the channels in the spacer plate.

[0078] FIG. 9 shows a mass spectrum of 2.5 picomoles of the protein ubiquitin dissolved with a 2:1 binary mixture of a 3-nitrobenzonitrile (3-NBN) and alpha cyano-4-hydroxycinnamic acid (CHCA) matrix in a 2:1 acetonitrile:water solvent and applied to a glass slide and acquired using the invention described herein in the vMAI mode by simply exposing the sample to the vacuum of a Thermo Fisher Scientific Q-Exactive Focus mass spectrometer through channel 102 in flange device 101. A 5 mm thick spacer plate 108 with 4 mm diameter channels 109 were used and positive 200 V were applied to the metal spacer plate. The top graph labeled A shows the total ion current as a function of time. In this case the sample remains exposed to the vacuum of the mass spectrometer until the matrix completely sublimes. The mass spectrum acquired where the arrow is pointing is shown in the bottom graph labeled B. The peaks labeled 714.73, 779.52, 857.37, 952.64, and 1071.59 are part of a series of molecular ions of ubiquitin having 12, 11, 10, 9, and 8 protons attached, respectively, providing 12+, 11+, 10+, 9+, and 8+ charges (z). Multiplying the associated mass-to-charge number such as 857.37 times the associated number of protons (in this case 10) gives 8573.7 which provides the molecular weight (MW) of the labeled isotope peak after subtracting the mass of the 10 protons, or 8563.7 Da. The advantage of extended ionization time is that multiple experiments such as data dependent mass selected fragmentation acquisitions can be acquired.

[0079] FIG. 10 demonstrates acquiring mass spectra of six analytes associated with the 3-nitrobenzonitrile (3-NBN) by vMAI using the invention described herein. The samples were applied to a glass slide sample plate 110 from solution and allowed to air dry in a manner in which the samples aligned with the channels 109 in a spacer plate 108. In order to acquire mass spectra of sequential samples, the sample plate device 107 manually slides over the channel 102 in the flange device 101 to expose each sample sequentially to the vacuum of the mass spectrometer initiating ionization. Graph A (top) is the selected ion current chronograms for the protonated molecular positive ion for lysergic acid 2,4-dimethylazetidid (0.21 min), gramicidin S (0.27 min), erythromycin (0.37 min), 6-allyl-6-nor-LSD (0.44 min), 1-propionyl-lysergic acid diethylamide (0.52 min), and hydrochloroquine (0.58 min). In graph B (bottom), for clarity, mass spectra are only shown for retention times 0.52, 0.21, and 0.28 from top to bottom respectively. Protonated singly charged ions are observed for 1-propionyl-lysergic acid diethylamide and lysergic acid 2,4-dimethylazetidid, and the doubly charged ion of the peptide gramicidin S at m/z 571.36. No carryover is observed between samples when amounts of analyte ranging from 1.5 nanomoles to 1 picomole were loaded onto the glass slide in a matrix solution and dried before acquisition. All six samples were acquired in ca. 24 seconds or ca. 4 seconds per sample.

[0080] FIG. 11 is shows the acquisition of five samples plus a blank using the same apparatus setup as FIG. 10, but with ionization using the vMALDI method. The blank is only the CHCA matrix spotted on a glass microscopy sample plate 110. All samples were spotted from solution onto a glass microscopy slide in a linear row and allowed to dry similar to the dried droplet method used with MALDI. The spotted samples align with the channels 109 in the spacer plate 108. Transmission geometry laser ablation was employed to initiate ionization. Because the sample being ablated is exposed through channel 102 in flange device 101 to the vacuum of the mass spectrometer 130 when laser ablated, ionization is by vacuum MALDI (vMALDI). Because ionization occurs immediately upon laser ablation of the sample and ceases when the laser no longer strikes the sample, sequential analysis of samples can be faster than demonstrated with vMAI. Acquisition of the blank and 5 samples required only ca. 7 seconds or at a rate of ca. 1 second per sample as seen in the selected ion current chronogram of the protonated molecular ions in the top half of the figure. In the top graph the peak with a retention time of 0.31 minutes is of 1.5 nanomoles of 6-allyl-6-nor-LSD, the retention time 0.32 minutes is of 1.5 nanomoles of lysergic acid 2,4-dimethylazetidide, the peak at retention time 0.35 minutes is of 1.5 nanomoles of 1-propionyl-lysergic acid diethylamide, the peak at retention time 0.36 minutes is of 1 picomole of gramicidin S, and the peak with retention time 0.40 minutes is of 1 nanomole of hydroxychloroquine applied to the glass slide in a 10:1 acetonitrile:water saturated solution of the matrix CHCA. As expected for MALDI, all compounds produced singly protonated molecular ions as in shown in the bottom half of the figure for (top to bottom) hydroxychloroquine, 6-allyl-6-nor-LSD, and lysergic acid 2,4-dimethylazetidide. Despite of the short acquisition time, the ion abundances are 4.52e4, 3.21e6, and 9.92e5, respectively.

[0081] FIG. 12 it the mass spectra of a fungus sample collected via a wooden toothpick off of a strawberry and smeared onto a glass microscopy slide and treated by adding a drop of 70% formic acid and letting it dry before adding a matrix solution. To one portion of the fungus smear was added a 3:1 acetonitrile:water solution of 3-nitrobenzonitrile and to another portion was added a 3:1 acetonitrile:water solution of 2,5-dihydroxyacetophenone. Using the device of the present invention, the top spectrum represents the ions spontaneously produced with the 3-nitrobenzonitrile matrix applied to the fungus smear and exposed to sub AP of the mass spectrometer, and the bottom spectrum represents the ions produced when the 2,6-dihydroxyacetophenone matrix applied to the fungus smear was laser ablated using a nitrogen laser. For clarity, only the region between m/z 790 and 990 are shown. All charge states (z) are +1. The peaks with bolded m/z values are those that appear in the vMAI and vMALDI mass spectra and the other peaks represent ions which are unique to each method. The top mass spectrum was generated by sliding the sample platedevice so that the sample using the 3-NBN matrix was first exposed to the lower pressure region of the mass spectrometer and then the laser was switched on and the area with the 2,5-dihydroxyacetophenone matrix was exposed to the lower pressure while laser ablated. Both mass spectra were acquired in less than 30 seconds.

[0082] FIG. 13 is the mass spectrum of uranyl nitrate containing the most abundant uranium-238 isotope as well as the much lower abundance uranium-235 isotope shown in the inset. This depleted sample dissolved in an aqueous solution containing 2% nitric acid was placed together with a matrix mixture of 3-NBN and CHCA on a microscopy glass slide and analyzed immediately before the sample could dry using the ion source apparatus described herein interfaced to an Orbitrap mass spectrometer using vMAI in the negative ion mode. The detection limit for uranyl nitrate was between 1 and 10 picograms. Detection of the uranium-235 isotope was consistent when loading 1 nanogram of uranyl nitrate. Neither contamination of the source nor carryover were observed demonstrating the robustness of this source and method. Note, in the vMALDI mode of the same sample, uranyl nitrate was not detected.

[0083] The procedure by which these samples are acquired are the following. The sample solution (in water, or water and organic solvent, or an organic solvent, with or without use of additives such as acids) is applied to the sample plate followed by addition of a matrix solution, typically in an organic:water solvent mixture, as in the dried droplet method used in MALDI, or alternatively, the sample solution and matrix solution are premixed and applied to the sample plate and typically allowed to dry. In some cases, it is beneficial to expose the sample while still wet to the vacuum of the mass spectrometer. Solvents that have been used most beneficially include acetonitrile, water, methanol, and formamide, but other solvents such as dimethylsulfoxide can be advantageously used. Additives such as ammonium salts reduce the background ions, especially in vMAI.

[0084] Other methods may be successfully used. In MALDI, a matrix such as CHCA, DHAP, dihydroxybenzoic acid or other MALDI matrix and mixtures thereof or with other matrices and additives are used, whereas in vMAI, matrices such as 3-NBN, coumarin, methyl-2-methyl-3-nitrobenzoate, or other vMAI matrices and mixtures thereof with or without additives are used. Interestingly, even compounds that do not work as pure matrixes such as 3-methylnitrobenzoate, previously not reported as an MAI matrix, as well as compounds that do work as vMAI matrices such as 1,3-dicyanobenzene, or 1,3-dinitrobenzene, can be used to enhance the sensitivity or the breadth of compounds in the sample to be ionized when mixed in combination with vMAI or vMALDI matrices, to perform in either case, vMAI.

[0085] In the invention described herein, in a preferred embodiment, the sample plate device 107 is placed in the feeder cartridge 112 at AP for automated operation, or for manual operation directly inserted between the guide rails 103a and 103b. The lowest sample plate device 107 in the feeder 114 drops onto rail 103b and can be moved into position over the channel 102 of flange device 101 which may be covered by a sliding valve plate 106 with hole 106a. The valve plate is then opened by moving the plate so that hole 106a is aligned with channel 102 in the flange device 101, and the sample plate device moved so the first sample is over channel 102. If using vMAI, ionization commences at this point without need of application of any external energy to the sample. For vMALDI and vLSI, a laser must be used to ablate the sample. Once sample acquisition is complete, the valve plate is closed and the sample plate device moved into the receiver cartridge 113 unless a second sample plate device is in position. Multiple sample plates can be acquired sequentially using vMALDI, vMAI, vLSI or combinations thereof, as well as other ionization methods using laser ablation of the sample. Typically, adjacent samples can be acquired at least as fast as one/second using vMALDI and one/4 seconds using vMAI. Adjacent samples on adjacent sample device plates require as little as two additional seconds. All components of sample plate devices 107 can be cleaned and reused. Tracking of samples and correlating with date can occur using barcodes. Sample plates can be stored or immediately disposed depending on the nature of the task. Robotics, streamlining, remote control including hardware/software operation is key for safety and smooth operation independent of laboratory status such as a pandemic response and improved health outcomes (cancer detection, differentiation, classification and of type and stage; bacterial infections, multiple bacteria, specie, genus, strain level; viral infections, mutations; fungi, etc.).

[0086] In vMAI, ions are produced from the entire surface of the sample until the sample is moved out of the position over the channel to vacuum, or the matrix is depleted, whichever occurs first. For vMALDI, ions are generated only in the area receiving sufficient laser energy to ablate a portion of the sample. Thus, vMALDI samples a small area during a laser pulse. Moving the point of ablation by either moving the sample or the laser beam allows improved sensitivity by summing mass spectra and provides an improved molecular representation of the sample. In the method in which samples are in a linear row, described above, with a fixed position laser beam, laser ablation occurs in a straight line as the sample moves across the laser beam. With sufficient laser fluence a defocused beam will sample a larger area. Alternatively, the laser beam can be moved (rastered) by, for example, tilting the focusing lens. This can be accomplished in transmission or reflective geometry, and be used for surface imaging and to increase the area sampled and thus provide an improved molecular representation and increased sensitivity.

[0087] While high speed analyses are desirable for high throughput analyses, there are applications where having longer time to sum ions in for example ion mobility or to achieve multiple ion fragmentation as in data dependent acquisitions known to those practiced in the art. This is readily achieved with vMAI by simply positioning the sample over the channel 102 until the desired information is achieved before moving to the adjacent sample. In vMAI, ionization continues until the matrix has completely sublimed which is usually ample time for numerous MS, MS/MS, IMS/MS, or IMS/MS/MS experiments. Similarly, in vMALDI and vLSI, the sample or beam can be moved around the same sample to achieve prolonged ionization. Simple and fast (seconds) switching between vMAI and vMALDI allows an improved degree of chemical information obtained on the same mass spectrometer and without having to vent the mass spectrometer. This allows maximum compatibility with other techniques known to practitioners, as, for example, use of gas-phase separation technologies such as ion mobility of singly or multiply charged ions.

[0088] Moving the laser beam across a sample or moving the sample across the beam provides a method to produce a molecular image of a surface by collecting mass spectra, each of which represents a pixel in the image and transforming the data into images of any ion (more specifically, each m/z) for which there is sufficient signal. Methods of molecular imaging are available for MALDI and vLSI and can be applied here. With either transmission or reflective geometry, rastering the laser beam is only useful for imaging small areas typically less than ca. 3 mm.sup.2, and potentially single cells. One method of transmitting the laser beam for either transmission or reflective geometry is use of fiber optics which provides safety and another means of moving the position the laser beam illuminates.

[0089] However, moving the sample relative to the aperture to vacuum and the stationary laser beam allows a larger area to be imaged. As an example, a tissue slice with matrix applied can be placed on a glass slide which is then placed on a spacer plate with a channel large enough to encompasses the tissue slice. This arrangement introduces a larger gas load into the instrument, but slow movement of the channel over the channel to vacuum allows pumping the volume slowly without undue disturbance of the vacuum of the mass spectrometer. Imaging can be with the laser in transmission or reflective geometry, but transmission geometry allows easier and better focusing of the laser beam for improved spatial resolution: spatial resolution is also more effectively achieved using ultraviolet (UV) radiation relative to infrared (IR). The disadvantage of transmission geometry is the need for higher laser power to penetrate the sample holder and, e.g., the tissue and still provide sufficient energy to ablate the matrix and enable initiation ofthe analyte ionization process.

[0090] A major advantage of the multi-ionization source that is not available with MALDI-Tof is the high resolution, accurate mass measurement, and MS.sup.n (or MS.sup.E on some mass spectrometers) fragmentation which can be achieved using high performance API mass spectrometers with high resolution and mass accuracy capabilities such as the Thermo Q-Exactive Focus used in these studies. Further, for peptides and proteins, advanced fragmentation such as electron transfer dissociation (ETD) can be applied to obtain sequence information from fragment ions. MALDI does not produce the multiply charged ions necessary for successful ETD, but acquiring the same sample with vMAI only requires adding a vMAI matrix solution to the sample to obtain multiply charged ions for analysis by ETD fragmentation or collision induced fragmentation. Note, to the best of our knowledge, this is the first vMAI source operational with advanced fragmentation technology capabilities for improved sequence analyses and accurate identification (ID) of isoforms (including proteins) and chemical and post-translational modifications (PTM's). Note that a sample acquired by vMAI such that all the matrix has sublimed can be acquired by MALDI after adding only a MALDI matrix solution and dried.

[0091] The present invention also offers advantages relative to quantification. MALDI methods in general have ‘hot spots” related to the crystal formation and ablation of rather small areas of the sample. With the multi-functional ion source, it is fast and simple to switch to vMAI mode where ionization occurs from a large surface area and if desired data can be collected until the sample fully sublimes to provide improved ion statistics and a better representation of the entire sample. These improvements are especially important for extracting relative quantitative information of the full mass range for reliable analyses of sample composition without prior knowledge or assumptions using, e.g., multiple reaction monitoring (MRM) transitions of the sample composition. Best quantification is achieved with internal standards, however.

[0092] In summary, the inventions of this application entail a means of rapidly interchanging multi-sample plates and rapidly sequentially exposing the samples individually to the vacuum of the mass spectrometer such that ions or charged particles produced from the sample, either by laser ablation (vMALDI or vLSI), or by using a more volatile matrix (vMAI), traverse from the sample surface into the mass analyzer of the mass spectrometer for detection. To our knowledge, this is the first multi-mode ionization apparatus based on the principle of vacuum ionization, and methods developed using the constructed source. There are numerous advantages of the inventions described herein. Sensitivity (low attomole detection), robustness (minimized carryover and instrument contamination), speed (full scan acquisitions faster than 1 sample/second), and simplicity (sample handling from AP) have been demonstrated with this multi-ionization source. Low and high mass applications are possible and with higher mass range analyzers can be further extended for the singly charged vMALDI ions. The sample plate remains at or near AP except for the sample being analyzed. Further, the method of exposing the sample to vacuum and maintaining the vacuum of the mass spectrometer involves use of stacked plates which are able to slide one over the other to achieve the desired alignment without loss of the substantial vacuum seal. Because it is only necessary to expose a single sample to vacuum at a time, there is no need for additional pumping or a time consuming pump down chamber to load an entire plate of samples. However, it should be recognized that multiple samples may be exposed to vacuum simultaneously and then moving each sample over the channel in the flange, and for vMALDI into the laser beam. Acquiring mass spectra from sequential single samples at ca. 1 per second, and in certain configurations <1 second per sample, has been demonstrated using laser ablation and 1 sample every 3 seconds for vMAI. It is anticipated that further improvements can be made. Significantly, these are full mass spectral acquisitions. A 2-dimensional arrangement of samples on a sample plate can be used, but is unnecessary for high throughput analyses because the method herein described allows sequential loading of sample plates in about 2 seconds. In other words, there is a gap of only ca. 2 seconds between the last sample of a prior plate and the first sample of the following plate. Multiple plates with linear arrays of samples can be loaded. Further, the apparatus of the invention described herein can replace the ion source and inlet assembly of a commercial mass spectrometer in less than 1 hour plus the time to vent and pump down the mass spectrometer, and the process can be reversed for ionization by ESI or APCI, as examples. Another advantage is that this multi-functional source can be readily converted to include traditional API (e.g., ESI, AP-MALDI, as examples) and ambient ionization (e.g., DESI, paper spray, as examples) methods as well as inlet ionization. This switch does not require venting the instrument and provides a convenient means of calibrating the mass spectrometer using ESI. A further advantage of this invention is that vacuum chamber 120 with rotary pump 124 and restriction 130, necessary on API instruments, are not a requirement for the apparatus described herein. Thus, a mass spectrometer designed around this invention can be simplified with lower cost and space requirements by eliminating the inlet chamber with associated rotary pump because of the much lower gas load to the instrument using the methods described herein. Thus, this invention is not only applicable to mass spectrometers designed for API, but also to Tof mass spectrometers potentially providing more rapid analyses and use of transmission geometry laser ablation.

[0093] While specific examples are provided, it should be understood that other arrangements are possible including sample plate assemblies with larger sample indentations, or spacer plates with larger channels to allow not only surfaces for imaging, but also multiple samples when using MALDI. Circular sample plate assemblies and even 96-sample well plates of the proper design can be used. This invention also relates to use with either MALDI or vMAI and does not require that both be instituted. The apparatus of the present invention can be a standalone vMAI or standalone MALDI or LSI source. Thus, the inventions described herein should be interpreted broadly.