Patent ID: 12205807

DETAILED DESCRIPTION OF DRAWINGS

FIG.1is a schematic of a cutout 3-dimensional view of a preferred embodiment of an apparatus100which 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 analyzer130through restriction126. The schematic is a generalized representation of a modification of a commercial AP ionization (API) inlet vacuum chamber120of a mass spectrometer. The vacuum chamber120may have a rotary pump124connected 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 chamber120from 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.

InFIG.1, the commercial inlet tube or aperture device with flange used with API sources has been replaced by flange device101which forms a vacuum seal with chamber120, in this case, using “O-ring”104. Other means of forming an air tight seal such as gaskets can be used. Flange101has a conduit or channel102with diameter between approximately 2 mm and 10 mm, typically on axis with the first ion transfer optics. The channel102in flange device101allows fluid communication between a higher pressure region, typically AP, and the lower pressure region within chamber120. Unless the channel102is covered to make a near air tight seal, the airflow would overload the pumping system of the analyzer. Vacuum chamber120may contain ion extraction lens, an ion guide and/or focusing elements122, 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 opening102into the ion guide/focusing elements and through a restriction126into the mass analyzer130. Restriction126is designed to provide a pressure drop between inlet chamber120and the mass analyzer130of the mass spectrometer. This pressure drop is necessary for API instruments. Flange101has situated on the face opposite chamber120, and typically at AP, two guide rails103aand103bto allow a sample plate device107to slide over the channel102in flange device101. The channel in flange device101aligns with the ion guide arranged in a manner that allows gas-phase ions and charged particles produced from a sample to traverse into the analyzer130, 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 device107containing one or more samples across channel102with minimal increase in the pressure in the analyzer. An optional valve plate105resides between the flange device101surface and the sample plate device107which slides to one of two positions. In the open position, a channel in the valve plate105of equal diameter to the inner diameter of channel102is aligned with channel102so that the lower pressure in chamber120is in fluid communication with the sample in the sample plate device107.

FIG.2further 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 device101with a valve plate106and a sample plate device107made up of a spacer plate108with multiple channels109therethrough and a sample plate110is presented inFIG.4A-C.FIG.4Ais a depiction of flange device101with channel102covered by a valve plate106, which in this case is a solid flat rectangular metal or plastic piece with a hole106atherethrough. The thickness of the valve plate106is 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 hole106aand substantially seal channel102. The valve plate106may be held in place by a cutout in the guide rails103making a slot between the guide rail and the AP face of the flange device101. In the position shown inFIG.2Aand inset expansion to the right, the valve plate seals channel102in flange device101thus separating the higher pressure region which is typically at or near AP from the lower pressure region defined by chamber120. However, sliding the valve plate left inFIG.2Aopens channel102through hole106ato the higher pressure region. The outcome depicted inFIG.2Bexpansion is undesired as channel102and hole106aare 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 plate106, the closed position shown inFIG.2Awould be used. Safeguards may be used to prevent the situation depicted onFIG.2Bfrom occurring.FIG.2Cshows an example where a spacer plate108with channels109therethrough lies over the valve plate106in the open position in which hole106alies over channel102in the flange device101. So long as there are flat surfaces in intimate contact with one another, the vacuum of the mass spectrometer is maintained provided the sample plate110is placed over spacer plate108. In this case only the gas, typically air, in channel109over channel102is drawn into the region enclosed by chamber120. Because the analytical samples residing on the sample plate align with channels109of the spacer plate, the samples never contact the valve plate106surface, or in the absence of a valve plate, the flange device101surface. In an alternative arrangement, the sample plate device107can be replaced with a sample plate device107in which the spacer plate108and flat sample plate110is 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.

FIG.3is 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 device101has attached to the surface guide rails103. In this depiction, a blank plate device105with a flat surface slides across the flat surface of flange device101and fits between the guide rails103aand103b. The purpose of the blank plate device105is to act as a valve to close channel102in flange device101to 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 plate106depicted inFIG.2lies between the flange device101and the sample plate device107and is readily replaced if necessary. Sample plate devices107can be abutted with one another or a blank valve plate105. The flat surfaces of the blank valve plate105or sample plate devices107are able to slide along the channel defined by the guide rails103aand103bwhile sealing the channel102in flange device101to maintain operational vacuum in chamber120. Likewise, a sample plate device107can replace valve plate105by sliding along the guide rails to present a flat edge surface107aagainst the flat edge surface105aof the blank valve plate105. The edge surfaces107aof sample plate device107and105aof blank valve plate105are flat so as to make a near air-tight seal when the plates are pushed together in intimate contact. In this manner, sample plate device107can be pushed against the blank valve plate105to slide the blank valve plate across channel102with minimal disturbance of the vacuum. Note that when a sample plate device covers channel102the pressure differential across the plate holds the plate against the flat surface of flange101maintaining chamber120well 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 chamber120. However, the guide rails103or other devices may be used to apply pressure to the blank valve plate105or plates device107to assure that a plate covering channel102cannot be dislodged and cause loss of instrument vacuum. Also, the viewed surfaces inFIG.2are at or near atmospheric pressure. Therefore, a sample plate device107holding sample(s) at AP can be used to replace a sample plate device107by the same mechanism described for replacing the valve plate105. Plate replacement occurs at AP with the only area exposed to sub-AP is the area of the sample plate device107or valve plate105that lies directly over the channel102in flange device101. 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 feedthrough111allows application of voltage or ground potential to the sample plate device107in order to produce a voltage gradient between the sample plate device107and, lens elements on the low pressure side of channel102. A variety of lens elements122may 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.

FIG.4depicts a partial top view schematic representation of a preferred embodiment showing automated plate movement in apparatus100using a motorized screw drive116, 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 feeder114(not shown for clarity) is the interface between a cassette (not shown in this representation) holding multiple plate devices107and a groove defined by guide rails103and the face of flange device101. In one embodiment, the plate device feeder114allows a plate device107to move into position in the plate device receiver grove and resting on guide rail103b. In this position, the sample plate device107can be moved in the groove toward channel102and between guide rails103aand143b. An automation device116having a computer controlled motor drive moves the sample plate device107to expose each sample aligned in a linear row on the sample plate sequentially over channel102. Note that channel102is at all times closed from AP or near AP by either the blank valve plate105or by the plate107. When closed by the sample plate device107, channel102is not closed by a valve plate105. Note that in this representation, the position of channel102is shown for clarity and channels109in spacer plate108are visible through the sample plate110which is represented as a glass microscopy slide.

FIG.5is a simplified schematic of a top view of a preferred representation of an ion source apparatus for automated high throughput sample analyses. The apparatus ofFIG.5allows rapid sequential exposure of samples residing on a sample plate110at or near AP to the first vacuum stage120of analyzer130when the sample is moved over channel102by sliding the sample plate device107, which incorporates sample plate110, between guide rails103. In this view, a representation of a plate device feeder114, feeder cartridge112, plate device receiver115and receiver cartridge113are combined with flange device101, guide rails103, sample plate device107, and sample plate drive116. Plate device107contains a spacer plate108sitting atop a valve plate106. Channel102is open when the hole106ain valve plate106aligns with channel102and closed when the valve plate106slides between guide rails103so that channel102and hole106aare not aligned. Under certain circumstances the valve plate106can be eliminated. In this arrangement, the channel102in flange device101can be covered or open by simply sliding the plate device107over channel102. Alternatively, a blank valve plate105can be used to cover channel102when data acquisition is not being carried out.

In the representation inFIG.5, the sample plate device107consists of a glass slide sample plate110affixed on top of the spacer plate108which has channels109. For representation purposes only, the view shows the positions of two sample plate devices107separated to show hole106ain valve plate106. So long as hole106ais not over channel102in flange device101, the pressure in the analyzer will not be affected. In operation, hole106aaligns with channel102and the two sample plate devices107abut each other and cover the hole106ain the valve plate106to substantially seal channel102from gas flow from the higher to the lower pressure regions.

The operation of the device represented inFIG.5can be visualized as follows. A sample plate device107containing samples which are typically made of a matrix and an analyte and residing on a sample plate110in positions that align with channels109in spacer plate108, the sample plate110being in intimate contact with spacer plate108to form a substantial vacuum seal, is place in the feeder assembly cartridge112, typically with other sample plate devices107containing sample to be analyzed. The bottom plate device drops into feeder assembly114when a sample plate device107sliding over valve plate106and between guide rails103clears feeder assembly114. The dropped sample plate device107rests on guide rail143band is moved towards sample plate device receiver115by the computer controlled motorized automation device116and makes intimate contact edge to edge with the next plate device in the grove between the guide rails103. A sufficient vacuum seal is created over hole106awhen the pressed together edges107aof sample plate devices107pass over hole106a. When the sample plate device107reaches the plate receiver115, it drops into the receiver cartridge113, thus freeing the next sample plate107to continue moving into the feeder receiver115. By mechanically moving only the last plate to exit the feeder cartridge112onto the guide rail103b, the resistance to movement caused by friction between the plate device107and the valve plate106, or in the case where the valve is a blank valve plate106a, the flange device101surface, results in a tight fit of the sample plate device107sides107aso that the interface of the two plates passing over hole106ain the valve plate106or the channel102in the flange device101, respectively, will not significantly disrupt the vacuum of the analyzer130.

The device represented inFIG.5can further be used to automatically acquire mass spectra of samples aligned on sample plate110in a linear fashion and in positions aligned with channels109in spacer plate108. In this mode, when the sample plate device107clears the feeder114, the next plate in feeder114drops into position onto guide rail143band the automated drive116begins moving the new sample plate toward channel102. Meanwhile, sample plate receiver115collects sample plate devices107which drop into receiver115once clear of the guide rail103b. Other configurations can be employed to achieve automated sequential exposure of samples to the vacuum of a mass spectrometer.

FIG.6is 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 beam147travel through a portion of vacuum chamber120and channel102in flange device101before striking the sample. Such an arrangement requires a transmission window into chamber120and mirrors in addition to optical focusing lens (not shown inFIG.6AorFIG.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 beam146strikes the sample from the AP side (backside). In this case, as depicted inFIG.6A, a spacer plate108with channels109therethrough lies in intimate contact with the surface of flange device101, assuming a valve plate106is not situated between the spacer plate108of plate device107and the surface of flange device101facing the AP region. The sample plate device107consisting of a spacer plate108and a sample plate110can slide over the flange device101surface or alternatively the valve plate106guided by the guide rails103. In the case of using a valve plate106, the valve plate remains stationary in the open position. So long as a sample plate device107is over channel102, the spacer plate108channel109is substantially sealed from AP and in fluid communication with the lower pressure region inside chamber120. When using a flat sample plate110for samples, indentations are not required as the samples can fit within the channels109of spacer plate108. The matrix:analyte samples, typically made up of matrix and analyte reside on the surface of the sample plate110in the position where they fit within the channels109on spacer plate108. However, indentations may be used, and in certain configurations it may include a commercial well plate. The indentations, may aid sample alignment with the channels109in the spacer plate108. The sample plate109is held stationary relative to the spacer plate so that sliding the spacer plate along the guide rails also moves the sample plate.

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 plate106as long as it is made of conductive material such as metal. In one preferred embodiment for valve plate106, Teflon is used to provide a good vacuum seal and easier movement of plate device107. 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 device111as depicted inFIG.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 plate110from the backside at angles ranging from 25 to 90 degrees, and preferable 45-90 degrees, or alternatively fiber optics can be used.

FIG.6Bdepicts 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 transmission146or reflective147geometries. The differences between this depiction and that inFIG.6Ais that the spacer plate has one large channel192instead of multiple smaller channels109. This is depicted by a top view of sample plate device107inFIG.6C. A glass or quartz sample plate111may fit on top of spacer plate108with channel192so that the sample plate110is held in place by frame194. The arrangement is especially useful for imaging where, e.g. a biological tissue slice is affixed to the transparent sample plate110and matrix solution is applied in a manner common to MALDI imaging and dried before the sample plate device107is placed over channel102in flange device101. For imaging larger sections, movement of the sample s plate device107is 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.

FIG.7is a depiction of a preferred embodiment of a means to move sample plates in 2 directions. Rails142and143are 90 degrees to one another. Valve plate181in this depiction is part of the 2 dimensional sample plate holder180so that positioning the valve plate portion181over channel102in flange device101allows removal of the 2-dimensional sample plate144having indentations145for samples.FIG.7Ais from the side facing the instrument vacuum andFIG.7Bis 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.

FIG.8is 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 device107is composed only of a blank spacer plate with indentation150normally used to hold and align a sample plate. The plate device has a channel109therethrough into which an inlet tube151is held with a vacuum tight seal represented by a screw cap152which tightens onto a ‘o’-ring153. 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 device107and near the face of the plate device which slides along the flange device101or valve plate106surface. When the inlet tube is aligned with the opening102in flange device101, the exit end of inlet capillary151is in fluid communication with the lower pressure region inside chamber120. The end in of inlet tube151does not protrude through spacer plate108so as not to contact either the flange device101surface or the surface of valve plate106when sample plate device107slides within the grove defined by guide rails103. The length of the inlet capillary on the AP side of the plate device107can be of length of a few millimeters to several meters. Interchange of the sample plate device107containing inlet tube151with any plate device107follows the same procedure as detailed above for interchanging plate devices.

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 channel102in flange device101. 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 channel102diameters up to 1 mm in diameter, typical size of channel102of 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 plate110, when not using a spacer plate108, 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 plate110has multiple indentations for multiple samples. Alternatively, the sample plate may have a spacer plate108inserted between it and the surface of flange device101in which one or more channels pass through the spacer plate108so that the sample, on a sample plate110is in fluid communication with the vacuum of the mass spectrometer when the channel102in flange device101is aligned with the channel109in spacer plate108and the matrix:analyte sample. Additionally, a valve plate106may be placed between the sample plate110, or the spacer plate108, and the flange device101to allow closure of channel102in the flange device101when no sample plate device107covers channel102.

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.

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 channel102in flange device101and in fluid communication with the vacuum of the mass spectrometer130, the remaining samples on the sample plate110are 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 analyzer130is that in each indentation on sample plate110or channel109of spacer plate108. Note that the spacer plate108may 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 plate110or the spacer plate108and the lens elements122which 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.

The ionization apparatus which employs a spacer plate108and a glass or quartz microscopy slide as sample plate110may also be used to substantially seal the channels in the spacer plate to secure the vacuum of the mass spectrometer when the sample plate device107is covering channel102in flange device101. 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.

FIG.9shows 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 channel102in flange device101. A 5 mm thick spacer plate108with 4 mm diameter channels109were 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.

FIG.10demonstrates 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 plate110from solution and allowed to air dry in a manner in which the samples aligned with the channels109in a spacer plate108. In order to acquire mass spectra of sequential samples, the sample plate device107manually slides over the channel102in the flange device101to 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.

FIG.11is shows the acquisition of five samples plus a blank using the same apparatus setup asFIG.10, but with ionization using the vMALDI method. The blank is only the CHCA matrix spotted on a glass microscopy sample plate110. 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 channels109in the spacer plate108. Transmission geometry laser ablation was employed to initiate ionization. Because the sample being ablated is exposed through channel102in flange device101to the vacuum of the mass spectrometer130when 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.

FIG.12it 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.

FIG.13is 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.

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.

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.

In the invention described herein, in a preferred embodiment, the sample plate device107is placed in the feeder cartridge112at AP for automated operation, or for manual operation directly inserted between the guide rails103aand103b. The lowest sample plate device107in the feeder114drops onto rail103band can be moved into position over the channel102of flange device101which may be covered by a sliding valve plate106with hole106a. The valve plate is then opened by moving the plate so that hole106ais aligned with channel102in the flange device101, and the sample plate device moved so the first sample is over channel102. 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 cartridge113unless 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 devices107can 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.).

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.

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 channel102until 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.

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 mm2, 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.

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.

A major advantage of the multi-ionization source that is not available with MALDI-Tof is the high resolution, accurate mass measurement, and MSn(or MSEon 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.

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.

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 chamber120with rotary pump124and restriction130, 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.

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.