Corinne A. Lutomski

Ionization of small, large, volatile, and nonvolatile compounds with charge states nearly identical to electrospray ionization are produced from a solid matrix or solution with high sensitivity utilizing the vacuum inherent with any mass spectrometer. With the proper matrix, analytes can be analyzed from ambient conditions or by direct introduction into vacuum. The ion source is simply the inlet to the mass analyzer. The new ionization methods have been interfaced with liquid chromatography, used for imaging tissue at atmospheric pressure or in vacuum, demonstrated for high-throughput analyses, applied for fast defect analysis, and shown to be compatible with electron transfer dissociation and ion mobility spectrometry–mass spectrometry. With the proper matrix, even large proteins are converted to gas-phase multiply charged ions without application of an external source of energy other than sub-atmospheric pressure. This latter method has great potential for extending mass spectrometry to areas such as clinical analysis where cost, robustness, and simplicity are important assets.

	In matrix-assisted laser desorption–ionization (MALDI), predominantly
	singly charged gas-phase molecular ions are produced from analytes incorporated into a matrix.  However, laser ablation of a common MALDI matrix at atmospheric pressure produced ions with higher numbers of charges common with electrospray ionization (ESI). Fundamental studies directed at understanding how multiply charged ions are produced by a process that is nearly identical to MALDI, with the main difference being that ion extraction voltage is removed or lowered, led to a series of new ionization methods given the general terms 

. The laser was found to not be necessary for 

 (MAI). Ionization occurs when the analyte incorporated in a small molecule matrix is introduced into a heated inlet tube linking atmospheric pressure and the vacuum of the mass spectrometer. MAI has now been extended to operate in vacuum using laser ablation similar to MALDI, but producing ions with ESI-like charge states. Here, the laser is also not a requirement. Matrices have been discovered that efficiently lift solid-phase molecules into the gas phase as ions without any external energy source when exposed to subatmospheric pressure. The development of these novel ionization processes for use in mass spectrometry (MS) is discussed. These methods are simple to use, safe, robust, and sensitive.

A Brief Evolution of Ionization Methods for Use in Mass Spectrometry

	The inventions of ESI (1) and MALDI (2,3) in the 1980s led to important advances in science because they provided, for the first time, a means of characterizing minute quantities of nonvolatile compounds even in complex mixtures using MS. Before the advent of ESI and MALDI, methods such as field desorption, plasma desorption, fast atom bombardment, laser desorption, and thermospray ionization made inroads into converting nonvolatile compounds into gas-phase ions (4–19). ESI and MALDI quickly replaced other ionization methods used with nonvolatile compounds, each having strengths that complement the other. ESI produces multiply charged ions from compounds in solution having multiple basic sites, is compatible with liquid separation methods, and is capable of analyzing large molecules on mass-range limited mass spectrometers. MALDI replaces the solvent in ESI with a solid small-molecule matrix and produces predominatly singly charged ions upon laser ablation of the matrix. MALDI operates from surfaces and has excellent sensitivity, and singly charged ions simplify analysis of complex materials such as polymers. However, MALDI is incompatible with liquid chromatography (LC) except under certain conditions (20). Because of deficiencies in ionization of low-polarity compounds using ESI, atmospheric-pressure chemical ionization (APCI) has gained in popularity as an alternative LC–MS ionization method (21,22).

	In the past decade, numerous innovative new sampling methods have been developed under the umbrella of “ambient ionization” (23–31). The mechanisms by which ions are formed in these methods are either gas-phase ion-molecule reactions as in APCI (32) or charged droplets as in ESI (33,34). Desorption electrospray ionization (DESI) (35,36) appears to have elements of both. The ambient ionization methods minimize sample preparation (31) and are associated with direct ionization in which chromatography is not used. For complex samples, high resolving power MS or ion mobility spectrometry (IMS) enhances (37) or replaces (38–40) the chromatographic separation, but ion suppression issues are more pronounced with the direct methods. Progress has been made in coupling MS with IMS in which gas-phase ions are separated in time before MS analysis (41–43). Commercial IMS-MS instruments are now available providing rapid and high sensitivity analysis. The most prominent IMS-MS instrument uses traveling wave ion mobility spectrometry (TWIMS) technology (44). Improvements in TWIMS have enabled the separation of isomeric species (39,45,46), which is unavailable in MS alone.

	Within ambient ionization there are ESI-based approaches capable of ionizing nonvolatile compounds such as DESI, laser ablation ESI (LAESI), and matrix-assisted laser desorption ESI (MALDESI), as well as those that ionize vaporizable compounds using APCI such as atmospheric solids analysis probe (ASAP) (25) and direct analysis in real time (DART) (24). A number of ambient methods are suitable for MS imaging (47). These approaches create a molecular image from the surface of interest (48). Ambient ionization methods also show promise in the characterization of proteins, but because these approaches operate on mass spectrometers with limited mass range, they must produce multiply charged ions in sufficient abundance for imaging (49). These atmospheric-pressure imaging approaches currently suffer either from limited ionization sensitivity, low spatial resolution, or both.

	New ionization methods may be required to achieve a step-change improvement in surface characterization (50). One such new ionization method was discovered in an attempt to develop field-free transmission geometry atmospheric-pressure MALDI for tissue imaging (51,52). This new ionization method was termed 

 (LSII) (Figure 1a, left) (53). Instead of observing the expected singly charged ions of peptides applied to a glass slide in a solution of the MALDI matrix, 2,5-dihydroxybenzoic acid (2,5-DHB), multiply charged ESI-like ions were observed from the dried solid-state matrix. The initial expectation that ionization was the product of laser ablation gave way to fundamental studies that demonstrated ionization occurred when the matrix–analyte particles or molten droplets ablated from the surface entered the heated inlet tube linking atmospheric pressure and the first vacuum region of the mass spectrometer (54–57). Because identical mass spectra were obtained by physically introducing the matrix-analyte sample into the heated inlet tube rather than by laser ablation, the more general term 

 (MAII) was used (Figure 1a, center) (58). Interestingly, Leonard Nyadong working in Alan Marshall’s group with LSII on a high resolution Fourier transform MS instrument independently discovered that the laser was not necessary for ionization (59).

					Figure 1: Schematic representation of (a) inlet ionization: laserspray ionization inlet (LSII), matrix-assisted ionization inlet (MAII), and solvent-assisted ionization inlet (SAII); and (b) vacuum ionization: laserspray ionization vacuum (LSIV), matrix-assisted ionization vacuum (MAIV). Lower right: screen shots from the vacuum MALDI source capturing the matrix–analyte sample as soon as indexed to the final position (top) and after the MAIV experiment was completed by simply letting the sample sublime (bottom).

	Pagnotti and McEwen (60) discovered that a solid matrix is not a requirement for ionization to occur in a heated mass spectrometer inlet tube. Solvents also produce abundant ions having charge states nearly identical to those observed in ESI, and the sensitivity of this method, termed 

 (SAII) (Figure 1a, right), is comparable to or better than ESI at similar flow rates (60). SAII works well with microliter (61) and nanoliter (62) flow LC–MS (7 ng of bovine serum albumin tryptic digest injected on column) (62), and low femtograms for steroids (63). An example of multiplexed SAII, capable of analyzing 42 samples in about 5 min, is displayed in Figure 2 (64). SAII is a liquid introduction variant of MAII operating without a laser or a voltage.

					Figure 2: SAII-MS: (a) photograph of 96-well pipette tip holder mounted on an automated 

-stage and aligned with the commercial inlet tube to move the solution containing the sample right in front of the inlet in which the ionization event is initiated by the vacuum draw of the mass spectrometer. The source housing is removed and operation overridden for convenience entrance. (b) Total ion chronogram of 42 samples with 42 blanks in between each sample acquired within 5 min, (c) mass spectrum of clozapine (MW 326), (d) mass spectrum of leucine enkaphalin (MW 555), (e) mass spectrum of bovine insulin (MW 5730), and (f) mass spectrum of ubiquitin (MW 8560). Data were acquired using the Thermo Fisher Scientific LTQ Velos mass spectrometer. Adapted from reference 64 with permission. Copyright 2014 American Chemical Society.

	Thus, LSI was the first of a family of ionization methods in which the inlet tube becomes the ion source (Figure 1a). These methods encompass samples in solid matrix common to MALDI and in solution common to ESI. Because LSI uses laser ablation and produces highly charged ions similar to ESI, but directly from the solid state without applied voltage, it is especially useful for analysis of small areas. LSI combines the attributes of MALDI, including speed of analysis and high spatial resolution for imaging, and those of ESI, including operation from atmospheric pressure, extending the mass range of high performance mass spectrometers, improved structural information through enhanced fragmentation (such as electron transfer dissociation [ETD]), and improved IMS separation. Thus, LSI extends the mass range of compounds that can be analyzed using atmospheric-pressure laser ablation with new high performance, but mass-to-charge (

) range limited mass spectrometers. For example, using LSI, ubiquitin (MW 8561) was mass measured following laser ablation directly from the solid state using a mass spectrometer with 100,000 mass resolution, <5 ppm mass accuracy, and an upper mass limit of 4000. Sequence information was obtained from this protein using ETD fragmentation of the +11 charge state (53). LSI-ETD was also used to identify the endogenous 

-acetylated myelin basic protein fragment peptide directly from mouse brain tissue (65). Multiply charged ions help IMS-MS differentiation of isomers and LSI provides a means of producing multiply charged ions directly from surfaces (45). The spatial resolution from laser ablation in transmission geometry (backside ablation), as well as the softness of LSI, enabled the imaging of fragile gangliosides directly from mouse brain tissue (Figure 3) (66). Transmission geometry laser ablation has also been successfully coupled to other ionization methods and used to image and characterize handwriting and dusted latent fingerprints (67). Thus, the inlet ionization methods operating from atmospheric pressure provide a viable alternative to ESI and MALDI and possess unique attributes.

					Figure 3: Mass spectrometry imaging using LSII with the laser (N

) aligned in transmission geometry: (a) photograph of a mouse brain tissue section from the Allen Brain Atlas (84), (b) experimental mouse brain tissue section of 10 µm thickness precoated with matrix 2,5-dihydroxybenzoic acid and spray coated with matrix 2,5-dihydroxyacetophenone, and (c,d) LSII images of the doubly deprotonated ions of disialoganglioside GD1 at 

 917.5 and 931.5. Data were acquired on a Thermo Fisher Scientific LTQ Velos mass spectrometer at an inlet temperature of 450 °C. Adapted from reference 66 with permission.

	However, the story of the initial finding that multiply charged ESI-like ions can be generated from a solid matrix does not end with inlet ionization, which occurs within a heated inlet tube linking a higher and a lower pressure region.  Fundamental studies intended to understand the mechanism of inlet ionization (68) led to extending the LSI approach to producing highly charged analyte ions in vacuum ion sources, normally used for MALDI, without the need for a heated inlet. Because a heated inlet tube was not required, these methods were termed vacuum ionization (Figure 1b) to differentiate them from inlet ionization (Figure 1a). Matrices and instrumental conditions were found that produce charge states identical to LSII and MAII using the commercial intermediate pressure MALDI source of a Waters Synapt G2 mass spectrometer (69). Interestingly, using the same matrix–analyte sample preparation with the common MALDI matrix 2,5-dihydroxyacetophenone, either MALDI-like singly charged ions or LSI-like multiply charged ions were obtained from 

-acetylated myelin basic protein fragment peptide (Figure 4) by simply changing instrumental conditions and using lower laser fluence (70). Similar results were reported for LSII using a commercial atmospheric-pressure MALDI source (71).

-acetylated myelin basic protein fragment (MW 1833) using the same matrix: (a) soft settings, similar to commercial ESI settings (no plate extraction voltage and lower laser power); (b) harsh settings, similar to commercial MALDI settings (plate extraction voltage 20 V and high laser power). Data were acquired using the intermediate-pressure vacuum MALDI source of a Waters IMS-MS Synapt G2 mass spectrometer. Structure of matrix compound 2,5-dihydroxyacetophenone provided as insets. Adapted with permission from Springer Science and Business Media from reference 70.

	This discovery led to a problem in nomenclature because the exact same laser ablation process is used to produce either singly or multiply charged ions from a matrix commonly used in MALDI. To distinguish these processes, laser ablation under vacuum that does not require a heated inlet was termed 

 (LSIV) (Figure 1b, left) (69). It seems clear that by changing only the instrumental conditions, a different regime of ions is selected, producing ions of differing charge states. If the multiply charged ions are formed from the same process that produces these ions in LSII, then the laser may similarly not be directly involved in ionization under vacuum conditions. In this case, observation of multiply charged ions is not a MALDI process, or MALDI of multiply charged ions does not involve photochemical ionization. We suggested that for nonvolatile compounds, the ionization mechanism of MALDI, LSIV, LSII, MAII, ESI, and other methods applied to ionize nonvolatile compounds are mechanistically related (70).

	The new ionization discoveries were achieved because of the simple concept that producing charged matrix particles or droplets is common in nature and that multiply charged ions will be observed from the charged particles or droplets if the matrix evaporates or sublimes in the time frame available before ion separation and detection (70). In other words, just as in ESI, charged particles must be generated and desolvation of the charged particles must occur.  In a vacuum MALDI experiment, the laser provides the energy necessary for generating the charged particles and matrix evaporation. However, the time available before mass analysis begins in a MALDI-time-of-flight (TOF) mass spectrometer is extremely short, limiting the ability of all but small clusters to desolvate. It has been suggested that the energy available and time limitation in MALDI-TOF limit the number of charges observed on bare ions because the small clusters that are able to desolvate have few charges (70). By finding a matrix compound, 2-nitrophloroglucinol (2-NPG), that has sufficient absorbance at the laser wavelength and readily evaporates or sublimes, it was demonstrated that stable multiply charged ions can be generated in a MALDI-TOF experiment (72).

	In intermediate-pressure MALDI instruments, the mass analyzer is remote from the ionization region so that the time available for desolvation of the charged matrix particles or droplets produced by laser ablation is orders of magnitude greater than in MALDI-TOF. Desolvation is also enhanced by collisions, rf fields, and the higher pressure used in these instruments (68–70,72–74). Thus, ESI-like charge states are produced by the 2-NPG matrix as well as a number of other matrix compounds upon laser ablation. Therefore, the experiment is similar to MALDI but a combination of a more volatile matrix and tuning conditions favorable to desolvation aid in producing highly charged ions with high sensitivity. Producing ESI-like charge states from mouse tissue sections with the spatial resolution from the laser shows great promise for imaging MS experiments using intermediate-pressure ion sources (Figure 5) (74).

					Figure 5: MS imaging using LSIV with the laser (Nd:YAG) aligned in reflection geometry: (a) LSIV-IMS-MS of delipified mouse brain tissue: two-dimensional plot of drift time versus 

 insets with isotopic distributions of doubly charged 

-acetyl myelin basic protein fragment peptide ions at 

 795.8. (b) Images of endogenous peptides with 

 917.40, 831.35, and 795.81 are related to a photograph of a mouse brain tissue section (lower right) (85) with regions of high myelin content indicated by black arrows. Data were acquired using the intermediate-pressure vacuum MALDI source of a Waters IMS-MS Synapt G2 mass spectrometer. Adapted from reference 74 with permission. Copyright 2012 American Chemical Society.

	Extending the concept that a laser is not necessary to produce ions in LSII leading to the discovery of MAII, we examined a large number of small molecules seeking matrices that produce ions in an inlet with low thermal requirements. With matrices that sublime in vacuum, one should only need to produce charged matrix particles to observe bare ions. During this search, 3-nitrobenzonitrile (3-NBN) was found to spontaneously produce ions from analyte, including large proteins, when prepared similar to MALDI or LSIV matrix–analyte and introduced to vacuum conditions. It is known that 3-NBN is triboluminescent when fractured (75). 

 is caused by a discharge occurring between two fractured surfaces carrying opposite charge. Thus, if placing the 3-NBN matrix under vacuum conditions could lead to particle ejection from expanding gases such as sublimation of the matrix or evaporation of included solvent within cavities, the fracturing process could produce highly charged gas-phase matrix–analyte particles by the same process that produces the conditions for triboluminescence.

	Interestingly, 3-NBN has no acidic hydrogen atoms to donate protons to the analyte, suggesting that the observed gas-phase ions were already charged in the solid matrix. While ESI and all of the inlet and vacuum ionization methods (Figure 1b, right) produce similar charge states for gas-phase ions, MAIV must transfer ions directly from the solid state. Visualizing the process using a vacuum MALDI source (Figure 1, lower right) with a magnifying camera, one only observes that the solid matrix slowly disappears while ions are being observed.

	The method in which a matrix–analyte sample is introduced to the vacuum of a mass spectrometer to produce gas-phase ions for analysis by MS is extremely simple and highly sensitive. However, using traditional MALDI plates to introduce samples to vacuum is not efficient, as only one sample can be introduced at a time. Because the atmospheric-pressure inlet provides a small opening to the vacuum of the mass spectrometer, it is possible to produce a sealed system at the inlet to initiate ion formation (38,76). In this configuration, opening the valve that isolates atmospheric pressure from the vacuum of the mass spectrometer is all that is needed to initiate ionization. Thus, sample can be handled at atmospheric pressure and rapidly changed. However, because the atmospheric-pressure inlet is designed for gas flow-without it, ion transmission is poor.

	Providing gas flow with a semi-sealed system enhances sensitivity, and it was found that gas-permeable material such as filter paper with the matrix–analyte sample held against the commercial inlet, whether a skimmer aperture or a tube inlet with no heat, or moderate applied heat, was an efficient means of producing analyte ions using 3-NBN for MS analysis (77). A very simple means of introducing sample to vacuum is to either tap matrix–analyte into the inlet aperture similar to MAII or bring it to the inlet either wet or dry using a pipette tip. When matrix–analyte is introduced into the inlet it immediately experiences vacuum conditions and produces ions. The difference from the initial MAII method is that the inlet does not need to be an inlet tube or heated, and, in fact, an inlet that is too hot is detrimental.

	The elimination of the need for a heated inlet tube means this MAIV method can, in principle, be used with any atmospheric-pressure mass spectrometer regardless of the type of inlet that is used. Because the matrix sublimes in vacuum, no matrix contamination of the instrument is expected. Thus, this extremely simple and fast method of analyses may prove to be the least contaminating and useful for long-term analyses. While heat is not a requirement, in some cases it can be important. Introducing analyte in 3-NBN into a room temperature inlet, just as with introduction using a vacuum MALDI source, produces ions for an extended time period. For some analyses, this is important as it allows time for a variety of MS

 or IMS experiments; however, for high-throughput analyses, it is desirable for abundant ions to be produced for at most a few seconds (Figure 6). Modest heat applied to the inlet achieves this goal.

					Figure 6: MAIV-MS. (a) Photograph of a strip of filter paper spotted with four different matrix–analyte samples. The filter paper adheres to the skimmer cone by the vacuum draw of the mass spectrometer. (b) Total ion chromatogram of the sequential acquisitions;  mass spectra of (c) sphingomyelin (MW 702), (d) angiotensin I (MW 1295), (e) bovine insulin (MW 5730), and (f) ubiquitin (MW 8560). Data were acquired using a widened inner cone on the Z-spray ion source of a Waters IMS-MS Synapt G2 mass spectrometer. Adapted from reference 77 with permission. Copyright 2012 American Chemical Society.

	For 3-NBN, atmospheric-pressure inlet temperature between approximately 50 °C and 100 °C is optimal for fast analyses (77). In fact, it has been demonstrated that heat can substitute for vacuum with not only the 3-NBN matrix, but with 2,5-DHAP (78) and no doubt others, as might be expected, if expansion of subliming matrix or included solvent drives the fracturing process. Using the ASAP method for introducing sample into a Thermo Ion Max source or a Waters Z-spray source and a gentle stream of warm nitrogen gas blown over the area of the melting point tube used to hold the matrix–analyte sample, abundant ions were produced from small molecules to small proteins (Figure 7) (78).

					Figure 7: Mass spectrum of lysozyme (MW 14,300) using the atmospheric solids analysis probe for matrix (3-NBN)-analyte sample exposure in front of the inlet aperture. Data were acquired on a Thermo Fisher Scientific Orbitrap Exactive mass spectrometer with an inlet temperature of 250 °C, heated auxiliary gas (N

) of 90 °C, and 3 kV applied to the heated ESI probe. Scheme for MA-ASAP acquisition provided top right. Adapted with permission from Springer Science and Business Media from reference 78.

Applications of New Ionization Methods for Mass Spectrometry

	The MAIV method is not just applicable to running relatively pure samples, but can be coupled to IMS-MS to provide gas-phase separation and analysis of complex samples. For example, a Waters Synapt G2 system was used to analyze clozapine, levoflaxacin, and cocaine, providing efficient separation of compounds by size, shape, and charge, and high mass resolution and mass accuracy as well as MS-MS fragmentation to aid with identification (39). Other classes of compounds can be analyzed such as polymers and additives directly from a few fiber strains taken from the interior of a car (40). Furthermore, by simply spotting the matrix onto a surface, those compounds dissolved into the solvent holding the matrix can be analyzed. Only compounds from the matrix exposed area are ionized (Figure 8). Therefore, by spotting a defect and comparing the results with those from a normal area, it is possible to rapidly determine chemical composition differences in the spotted areas. Because the MAIV method only requires exposing the matrix to the analyte, including surfaces such as biological tissue, or dipping the sample such as tissue piece into the matrix solution, and introducing the matrix–analyte to the vacuum of the mass spectrometer, little user expertise is necessary. Such a simple, yet powerful method has potential in field portable mass spectrometers, and clinical and forensic (Figure 9) (76) analyses.

					Figure 8: MAIV-IMS-MS analyses of a mouse brain tissue section: (a) photograph of procedure applying with a micropipet tip the matrix (3-NBN) solution to the surface of a mouse brain tissue section adhered to a glass microscope slide; (b) 2D plot of drift time versus 

 of ions depicting separation of compound classes by charge, size, and shape of lipids (inset charge state +1), peptides, and proteins (inset charge state +4); (c) photograph of the mouse brain tissue microscopy before and after exposure to the vacuum for ionization and analysis in which the circled area highlights the tissue region that was analyzed in (b). The colored arrows are provided to guide the eye. Data were acquired using soft settings from Figure 4a on a commercial vacuum MALDI source of a Waters IMS-MS Synapt G2 mass spectrometer with the laser off. Reproduced from reference 38 with permission. Copyright to the American Society for Biochemistry and Molecular Biology.

					Figure 9: Cocaine (MW 303) and polypropylene glycol, both detected as singly charged ions, directly from a specific location (eye of the president) of a $20 bill by spotting matrix 3-NBN solution to the area of interest. (a) Mass spectrum obtained when the matrix was spotted on the eye of the bill and introduced to the vacuum; (b) MS-MS spectrum and structure of cocaine peak selected at 

 304. Data were acquired using the intermediate-pressure vacuum MALDI source of the Waters IMS-MS Synapt G2 mass spectrometer. Adapted with permission from Springer Science and Business Media from reference 76.

	Although 3-NBN is a powerful MAIV matrix, it does not efficiently ionize all compounds. In fact, basic polar compounds work extremely well with this method whereas nonpolar or acidic compounds are less efficiently ionized. The method seems to have similar attributes to ESI. Although finding a matrix that produces universal ionization is desirable (79), having matrices with selective ionization is also desirable as it allows compounds from a desired class to be analyzed with less interference from undesirable compounds. This is observed when using 3-NBN as a matrix. For example, peptides can be analyzed with very little interference from chemical background, a major interfering component in the low mass range when using MALDI.

	The matrices used in MAIV have also been used in conjunction with a DART source (80) and to obtain MS images from surfaces (81), indicating the flexibility and broadness of this new ionization method and this particular matrix. There are no “hot spot” issues with MAIV and because no laser is required, the matrix background, observed at every mass in MALDI below 

 1000 (82,83), is not observed. Because of the continuous ion production, just as in ESI, the reproducibility is improved relative to laser-based ionization technologies, but without using a laser, spatial resolution is decreased. High salt content is usually detrimental to MS analysis, although less so with MALDI than ESI, and seemingly even less so with MAIV where addition of sodium chloride had a rather modest effect with multiply charged protonated ions remaining the most abundant up to a salt concentration nearing 1 M (39).

	The newly discovered ionization process for use with MS in which analyte in a solid-state matrix is spontaneously converted to gas-phase ions on exposure to vacuum or modest heat (Figure 1 center and right), is of fundamental scientific interest and has immense analytical potential. The multiply charged ions produced by this method are well suited for high performance mass analyzers with advanced mass measurements in terms of mass resolution and accuracy, ETD structural characterization, and IMS providing inroads into the determination of structure (shape). These capabilities are usually limited to 

 <4000, and for ionization methods that produce singly charged ions, larger molecules cannot be analyzed with these instruments. Developing new matrix technology and interfaces designed for the MAIV ionization method will facilitate application to a wide array of problems. However, possibly the most promising aspect of this new ionization technology is its simplicity and cost savings as the inlet becomes the ion source. A relatively inexpensive mass analyzer with such a simple to use, nearly foolproof, and noncontaminating ionization method might find utility related to human diseases for which current methods are inadequate.

	As was demonstrated by ESI and MALDI, and, more recently, ambient ionization approaches, successful new ionization methods can have impact on science far beyond anything envisioned in their early discovery. To enhance the rate at which the newly discovered and rather astonishing ionization process contributes to measurement technology, it is of paramount importance to develop a mechanistic understanding of how molecules are transferred from the solid state to the gas phase as ions absent applied energy. This fundamental understanding will have bearing on the mechanism of ionization methods commonly used in MS, guide creation of new matrix compounds, and provide improved methods for high-throughput and spatially resolved analyses. The goal is to advance MS to near real-time molecular characterization of materials maintaining high sensitivity and providing micrometer spatial resolution so that measurements and imaging with high specificity, sensitivity, and dynamic range become possible on a broad range of materials. These goals will be realized by application of knowledge gained from fundamental research to develop a more efficient process for producing and transmitting ions into a mass analyzer from solid surfaces with ever decreasing sampling footprints.

	The authors are thankful for financial support from NSF Career Award 0955975, MSTM, ASMS Research Award, DuPont Young Professor Award, Waters Center of Innovation Award, and Eli Lilly Young Investigator Award in Analytical Chemistry (to S.T.), and Wayne State University (Schaap and Rumble Dissertation Fellowships to B.W. and Schaap Faculty Scholar to S.T.).

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