Direct sub-atmospheric pressure ionization mass spectrometry: Evaporation/sublimation-driven ionization is amazing, fundamentally, and practically.

This paper covers direct sub-atmospheric pressure ionization mass spectrometry (MS). The discovery, applications, and mechanistic aspects of novel ionization processes for use in MS that are not based on the high-energy input from voltage, laser, and/or high temperature but on sublimation/evaporation within a region linking a higher to lower pressure and modulated by heat and collisions, are discussed, including how this new reality has guided a series of discoveries, instrument developments, and commercialization. A research focus, inter alia, is on how best to understand, improve, and use these novel ionization processes, which convert volatile and nonvolatile compounds from solids (sublimation) or liquids (evaporation) into gas-phase ions for analysis by MS providing reproducible, accurate, sensitive, and prompt results. Our perception on how these unprecedented versus traditional ionization processes/methods relate to each other, how they can be made to coexist on the same mass spectrometer, and an outlook on new and expanded applications (e.g., clinical, portable, fast, safe, and autonomous) is presented, and is based on ST's Opening lecture presentation at the Nordic Mass spectrometry Conference, Geilo, Norway, January 2023. Focus will be on matrix-assisted ionization (MAI) and solvent-assisted ionization (SAI) MS covering the period from 2010 to 2023; a potential paradigm shift in the making.

Instrumentation development, improvement, simplification, and miniaturization: The multifunctional plate source for use in mass spectrometry.

In remembrance of Prof. Dr Przybylski, we are presenting a vision towards his beloved mass spectrometry (MS) and its far-reaching promises outside of the academic laboratory. Sub-atmospheric pressure (AP) ionization MS is well positioned to make a step-change in direct ionization, a concept that allows sublimation/evaporation ionization and mass analyses of volatile and nonvolatile molecules from clean or dirty samples, directly, accurately, sensitively, and in a straightforward manner that has the potential to expand the field of MS into unchartered application areas. Contrary to ambient ionization MS, ionization commences in the sub-AP region of the mass spectrometer, important for practical and safety reasons, and offers inter alia, simplicity, speed, sensitivity, and robustness directly from real-world samples without cleanup. The plate source concept, presented here, provides an easy to use, rapid, and direct sample introduction from AP into the sub-AP of a mass spectrometer. Utilizing sub-AP ionization MS based on the plate source concept, small to large molecules from various environments that would be deemed too dirty for some direct MS methods are demonstrated. The new source concept can be expanded to include multiple ionization methods using the same plate source "front end" without the need to vent the mass spectrometer between the different methods, thus allowing ionization of more compounds on the same mass spectrometer for which any one ionization method may be insufficient. Examples such as fentanyl, gamma-hydroxybutyric acid, clozapine, 1-propionyllysergic acid, hydrocodone angiotensin I and II, myoglobin, and carbonic anhydrase are included.

A tutorial: Laserspray ionization and related laser-based ionization methods for use in mass spectrometry.

This Tutorial is to provide a summary of parameters useful for successful outcomes of laserspray ionization (LSI) and related methods that employ a laser to ablate a matrix:analyte sample to produce highly charged ions. In these methods the purpose of the laser is to transfer matrix-analyte clusters into the gas phase. Ions are hypothesized to be produced by a thermal process where emitted matrix:analyte gas-phase particles/clusters are charged and loss of matrix from the charged particles leads to release of the analyte ions into the gas phase. The thermal energy responsible for the charge-separation process is relatively low and not necessarily supplied by the laser; a heated inlet tube linking atmospheric pressure with the first vacuum stage of a mass spectrometer is sufficient. The inlet becomes the "ion source", and inter alia, pressure, temperature, and the matrix, which can be a solid, liquid, or combinations, become critical parameters. Injecting matrix:analyte into a heated inlet tube using laser ablation, a shockwave, or simply tapping, all produce the similar mass spectra. Applications are provided that showcase new opportunities in the field of mass spectrometry.

An overview of biological applications and fundamentals of new inlet and vacuum ionization technologies.

RATIONALE: The developments of new ionization technologies based on processes previously unknown to mass spectrometry (MS) have gained significant momentum. Herein we address the importance of understanding these unique ionization processes, demonstrate the new capabilities currently unmet by other methods, and outline their considerable analytical potential. METHODS: The inlet and vacuum ionization methods of solvent-assisted ionization (SAI), matrix-assisted ionization (MAI), and laserspray ionization can be used with commercial and dedicated ion sources producing ions from atmospheric or vacuum conditions for analyses of a variety of materials including drugs, lipids, and proteins introduced from well plates, pipet tips and plate surfaces with and without a laser using solid or solvent matrices. Mass spectrometers from various vendors are employed. RESULTS: Results are presented highlighting strengths relative to ionization methods of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization. We demonstrate the utility of multi-ionization platforms encompassing MAI, SAI, and ESI and enabling detection of what otherwise is missed, especially when directly analyzing mixtures. Unmatched robustness is achieved with dedicated vacuum MAI sources with mechanical introduction of the sample to the sub-atmospheric pressure (vacuum MAI). Simplicity and use of a wide array of matrices are attained using a conduit (inlet ionization), preferably heated, with sample introduction from atmospheric pressure. Tissue, whole blood, urine (including mouse, chicken, and human origin), bacteria strains and chemical on-probe reactions are analyzed directly and, especially in the case of vacuum ionization, without concern of carryover or instrument contamination. CONCLUSIONS: Examples are provided highlighting the exceptional analytical capabilities associated with the novel ionization processes in MS that reduce operational complexity while increasing speed and robustness, achieving mass spectra with low background for improved sensitivity, suggesting the potential of this simple ionization technology to drive MS into areas currently underserved, such as clinical and medical applications.

Resolving Isomers of Star-Branched Poly(Ethylene Glycols) by IMS-MS Using Multiply Charged Ions.

Ion mobility spectrometry (IMS) mass spectrometry (MS) centers on the ability to separate gaseous structures by size, charge, shape, and followed by mass-to-charge (m/z). For oligomeric structures, improved separation is hypothesized to be related to the ability to extend structures through repulsive forces between cations electrostatically bonded to the oligomers. Here we show the ability to separate differently branched multiply charged ions of star-branched poly(ethylene glycol) oligomers (up to 2000 Da) regardless of whether formed by electrospray ionization (ESI) charged solution droplets or from charged solid particles produced directly from a surface by matrix-assisted ionization. Detailed structural characterization of isomers of the star-branched compositions was first established using a home-built high-resolution ESI IMS-MS instrument. The doubly charged ions have well-resolved drift times, achieving separation of isomers and also allowing differentiation of star-branched versus linear oligomers. An IMS-MS "snapshot" approach allows visualization of architectural dispersity and (im)purity of samples in a straightforward manner. Analyses capabilities are shown for different cations and ionization methods using commercially available traveling wave IMS-MS instruments. Analyses directly from surfaces using the new ionization processes are, because of the multiply charging, not only associated with the benefits of improved gas-phase separations, relative to that of ions produced by matrix-assisted laser desorption/ionization, but also provide the potential for spatially resolved measurements relative to ESI and other ionization methods.

Toward understanding the ionization mechanism of matrix-assisted ionization using mass spectrometry experiment and theory.

RATIONALE: Matrix-assisted ionization (MAI) mass spectrometry does not require voltages, a laser beam, or added heat to initiate ionization, but it is strongly dependent on the choice of matrix and the vacuum conditions. High charge state distributions of nonvolatile analyte ions produced by MAI suggest that the ionization mechanism may be similar to that of electrospray ionization (ESI), but different from matrix-assisted laser desorption/ionization (MALDI). While significant information is available for MAI using mass spectrometers operating at atmospheric and intermediate pressure, little is known about the mechanism at high vacuum. METHODS: Eleven MAI matrices were studied on a high-vacuum time-of-flight (TOF) mass spectrometer using a 266 nm pulsed laser beam under otherwise typical MALDI conditions. Detailed comparisons with the commonly used MALDI matrices and theoretical prediction were made for 3-nitrobenzonitrile (3-NBN), which is the only MAI matrix that works well in high vacuum when irradiated with a laser. RESULTS: Screening of MAI matrices with good absorption at 266 nm but with various degrees of volatility and laser energies suggests that volatility and absorption at the laser wavelength may be necessary, but not sufficient, criteria to explain the formation of multiply charged analyte ions. 3-NBN produces intact, highly charged ions of nonvolatile analytes in high-vacuum TOF with the use of a laser, demonstrating that ESI-like ions can be produced in high vacuum. Theoretical calculations and mass spectra suggest that thermally induced proton transfer, which is the major ionization mechanism in MALDI, is not important with the 3-NBN matrix at 266 nm laser wavelength. 3-NBN:analyte crystal morphology is, however, important in ion generation in high vacuum. CONCLUSIONS: The 3-NBN MAI matrix produces intact, highly charged ions of nonvolatile compounds in high-vacuum TOF mass spectrometers with the aid of ablation and/or heating by laser irradiation, and shows a different ionization mechanism from that of typical MALDI matrices.

Matrix-assisted ionization mass spectrometry in targeted protein analysis - An initial evaluation.

RATIONALE: Matrix-assisted ionization (MAI) is a relatively new ionization technique for analysis by mass spectrometry (MS). The technique is simple and has been shown to be less influenced by matrix effects than e.g. electrospray ionization (ESI). These features are of interest in the targeted analysis of proteins from biological samples. METHODS: Targeted protein determination by MAI-MS was evaluated using a triple quadrupole mass analyzer equipped with a stripped nanoESI source in selected reaction monitoring (SRM) mode. The proteins were analyzed using the bottom-up approach with stable isotopic labeled peptides as internal standards (IS). The MAI matrix was 3-nitrobenzonitrile dissolved in acetonitrile. Aqueous sample and matrix solution were mixed in a 1:3 volume ratio. One microlitre of the dried matrix/analyte sample was introduced into the inlet of the mass spectrometer where ionization commences. RESULTS: SRM settings established for ESI-SRM-MS of the peptides here investigated were applicable in MAI-SRM-MS for all evaluated peptides except one that is poorly soluble in water. Addition of IS provided efficient correction at most levels (relative standard deviation (RSD) ≤28% (except lowest digest level), r2 ≥ 0.995). This was also true for the more complex biological matrices, diluted urine (1:1; RSD = 20% a synthetic peptide, NLLGLIEAK) and diluted digested serum (1:100; RSD = 7% digested cytochrome C). Biological matrix influenced the signal intensity unless sufficiently diluted. CONCLUSIONS: The results demonstrate that MAI-SRM-MS has promising potential in targeted protein determination by the bottom-up approach because of its simplicity, ease of use, and speed. However, more data is needed to confirm the results prior to application in a clinical setting.

Development of a robotics platform for automated multi-ionization mass spectrometry.

RATIONALE: Successful coupling of a multi-ionization automated platform with commercially available mass spectrometers provides improved coverage of compounds in complex mixtures through implementation of new and traditional ionization methods. The versatility of the automated platform is demonstrated through coupling with mass spectrometers from two different vendors. Standards and complex biological samples were acquired using electrospray ionization (ESI), solvent-assisted ionization (SAI) and matrix-assisted ionization (MAI). METHODS: The MS™ prototype automated platform samples from 96- or 384-well plates as well as surfaces. The platform interfaces with Thermo Fisher Scientific mass spectrometers by replacement of the IonMax source, and on Waters mass spectrometers with additional minor source inlet modifications. The sample is transferred to the ionization region using a fused-silica or metal capillary which is cleaned between acquisitions using solvents. For ESI and SAI, typically 1 µL of sample solution is drawn into the capillary tube and for ESI slowly dispensed near the inlet of the mass spectrometer with voltage placed on the delivering syringe barrel to which the tubing is attached, while for SAI the sample delivery tubing inserts into the inlet without the need for high voltage. For MAI, typically, 0.2 µL of matrix solution is drawn into the syringe before drawing 0.1 µL of the sample solution and dispensing to dry before insertion into the inlet. RESULTS: A comparison study of a mixture of angiotensin I, verapamil, crystal violet, and atrazine representative of peptides, drugs, dyes, and herbicides using SAI, MAI, and ESI shows large differences in ionization efficiency of the various components. Solutions of a mixture of erythromycin and azithromycin in wells of a 384-microtiter well plate were mass analyzed at the rate of ca 1 min per sample using MAI and ESI. In addition, we report the analysis of bacterial extracts using automated MAI and ESI methods. Finally, the ability to perform surface analysis with the automated platform is also demonstrated by directly analyzing dyes separated on a thin-layer chromatography (TLC) plate and compounds extracted from the surface of a beef liver tissue section. CONCLUSIONS: The prototype multi-ionization automated platform offers solid matrix introduction used with MAI, as well as solution introduction using either ESI or SAI. The combination of ionization methods extends the types of compounds which are efficiently ionized and is especially valuable with complex mixtures as demonstrated for bacterial extracts. While coupling of the automated multi-ionization platform to Thermo and Waters mass spectrometers is demonstrated, it should be possible to interface it with most commercial mass spectrometers.

Comparison of gaseous ubiquitin ion structures obtained from a solid and solution matrix using ion mobility spectrometry/mass spectrometry.

RATIONALE: Examining surface protein conformations, and especially achieving this with spatial resolution, is an important goal. The recently discovered ionization processes offer spatial-resolution measurements similar to matrix-assisted laser desorption/ionization (MALDI) and produce charge states similar to electrospray ionization (ESI) extending higher-mass protein applications directly from surfaces on high-performance mass spectrometers. Studying a well-interrogated protein by ion mobility spectrometry-mass spectrometry (IMS-MS) to access effects on structures using a solid vs. solvent matrix may provide insights. METHODS: Ubiquitin was studied by IMS-MS using new ionization processes with commercial and homebuilt ion sources and instruments (Waters SYNAPT G2(S)) and homebuilt 2 m drift-tube instrument; MS™ sources). Mass-to-charge and drift-time (td )-measurements are compared for ubiquitin ions obtained by inlet and vacuum ionization using laserspray ionization (LSI), matrix- (MAI) and solvent-assisted ionization (SAI), respectively, and compared with those from ESI under conditions that are most comparable. RESULTS: Using the same solution conditions with SYNAPT G2(S) instruments, td -distributions of various ubiquitin charge states from MAI, LSI, and SAI are similar to those from ESI using a variety of solvents, matrices, extraction voltages, a laser, and temperature only, showing subtle differences in more compact features within the elongated distribution of structures. However, on a homebuilt drift-tube instrument, within the elongated distribution of structures, both similar and different td -distributions are observed for ubiquitin ions obtained by MAI and ESI. MAI-generated ions are frequently narrower in their td -distributions. CONCLUSIONS: Direct comparisons between ESI and the new ionization methods operational directly from surfaces suggest that the protein in its solution structure prior to exposure to the ionization event is either captured (frozen out) at the time of crystallization, or that the protein in the solid matrix is associated with sufficient solvent to maintain the solution structure, or, alternatively, that the observed structures are those related to what occurs in the gas phase with ESI- or MAI-generated ions and not with the solution structures.

A Combination MAI and MALDI Vacuum Source Operational from Atmospheric Pressure for Fast, Robust, and Sensitive Analyses.

Previously, vacuum matrix-assisted ionization (vMAI) was employed with matrix/analyte sample introduction into the vacuum of a mass spectrometer on a probe sample introduction device. Low attomole detection was achieved, while no carryover was observed even for concentrated samples. Here, we report a new vacuum ionization source designed to duplicate the sensitivity and robustness of probe device while providing fast multisample introduction to vacuum and rapid sequential ionization. Exposure of a sample to the vacuum of the mass spectrometer provides spontaneous ionization of volatile as well as nonvolatile analytes without the need for external energy input. However, the novel source design described herein, in addition to vMAI, can employ a laser to obtain vacuum matrix-assisted laser desorption/ionization (vMALDI). In particular, ionization by vMAI or vMALDI is achieved by using the appropriate matrix. Switching between ionization modes is accomplished in a few seconds. We present results demonstrating the utility of the two ionization methods in combination to improve the molecular analyses of sample composition. In both ionization modes, multiple samples can be sequentially and rapidly acquired to increase throughput in MS. With the prototype source, samples were acquired in as little as 1 s per sample. Exchanging multisample plates can be accomplished in as little as 2 s, suggesting low-cost high-throughput automation when properly developed.

Sublimation Driven Ionization for Use in Mass Spectrometry: Mechanistic Implications.

Sublimation has been known at least since the middle ages. This process is frequently taught in schools through the use of phase diagrams. Astonishingly, such a well-known process appears to still harbor secrets. Under conditions in which compound sublimation occurs, gas-phase ions are frequently detected using mass spectrometry. This was exploited in matrix-assisted ionization in vacuum (vMAI) by adding analyte to subliming compounds used as matrices. Good vMAI matrices were those that ionize the added analyte with high sensitivity, but even matrices that fail this test often produce ions of likely matrix impurities suggesting that they may be good matrices for some compound types. We also show that binary matrices may be manipulated to provide desired properties such as fast analyses and improved sensitivity. These results imply that sublimation in some cases is more complicated than just molecules leaving a surface and that understanding the physical force responsible, and how the nonvolatile compound becomes charged, could lead to improved ionization efficiency for mass spectrometry. Here we provide insights into this process and an explanation of why this unexpected phenomenon has not previously been reported.

Expression and In Vivo Characterization of the Antimicrobial Peptide Oncocin and Variants Binding to Ribosomes.

Peptides have important biomedical applications, but poor correlation between in vitro and in vivo activities can limit their development for clinical use. The ability to generate peptides and monitor their expression with new mass spectrometric methods and biological activities in vivo would be an advantage for the discovery and improvement of peptide-based drugs. In this study, a plasmid-based system was used to express the ribosome-targeting peptide oncocin (19 amino acids, VDKPPYLPRPRPPRRIYNR) and to determine its direct antibacterial effects on Escherichia coli. Previous biochemical and structure studies showed that oncocin targets the bacterial ribosome. The oncocin peptide generated in vivo strongly inhibits bacterial growth. In vivo dimethyl sulfate footprinting of oncocin on the rRNA gives results that are consistent with those of in vitro studies but reveals additional binding interactions with E. coli ribosomes. Furthermore, expression of truncated or mutated peptides reveals which amino acids are important for antimicrobial activity. Overall, the in vivo peptide expression system can be used to investigate biological activities and interactions of peptides with their targets within the cellular environment and to separate contributions of the sequence to cellular transport. This strategy has future applications for improving the effectiveness of existing peptides and developing new peptide-based drugs.

New mass spectrometry concepts for characterization of synthetic polymers and additives.

RATIONALE: New ionization processes have been developed for biological mass spectrometry (MS) in which the matrix lifts the nonvolatile analyte into the gas phase as ions without any additional energy input. We rationalized that additional fundamental knowledge is needed to assess analytical utility for the field of synthetic polymers and additives. METHODS: Different mass spectrometers (Thermo Orbitrap (Q-)Exactive (Focus); Waters SYNAPT G2(S)) were employed. The formation of multiply charged polymer ions upon exposure of the matrix/analyte(/salt) sample to sub-atmospheric pressure directly from the solid state and surfaces facilitates the use of advanced mass spectrometers for detection of polymeric materials including consumer products (e.g., gum). RESULTS: Astonishingly, using nothing more than a small molecule matrix compound (e.g., 2-methyl-2-nitropropane-1,3-diol or 3-nitrobenzonitrile) and a salt (e.g., mono- or divalent cation(s)), such samples upon exposure to sub-atmospheric pressure transfer nonvolatile polymers and nonvolatile salts into the gas phase as multiply charged ions. These successes contradict the conventional understanding of ionization in MS, because can nonvolatile polymers be lifted in the gas phase as ions not only by as little as a volatile matrix but also by the salt required for ionizing the analyte through noncovalent metal cation adduction(s). Prototype vacuum matrix-assisted ionization (vMAI) and automated sources using a contactless approach are demonstrated for direct analyses of synthetic polymers and plasticizers, minimizing the risk of contamination using direct sample introduction into the mass spectrometer vacuum. CONCLUSIONS: Direct ionization methods from surfaces without the need of high voltage, a laser, or even applied heat are demonstrated for characterization of detailed materials using (ultra)high-resolution and accurate mass measurements enabled by the multiply charged ions extending the mass range of high-performance mass spectrometers and use of a split probe sample introduction device. Our vision is that, with further development of fundamentals and dedicated sources, both spatial- and temporal-resolution measurements are within reach if sensitivity is addressed for decreasing sample-size measurements.

1000-Fold Preconcentration of Per- and Polyfluorinated Alkyl Substances within 10 Minutes via Electrochemical Aerosol Formation.

We present a simple and efficient method for preconcentrating per- and polyfluorinated alkyl substances (PFAS) in water. Our method was inspired by the sea-spray aerosol enrichment in nature. Gas bubbles in the ocean serve to scavenge surface active material, carrying it to the air-ocean interface, where the bubbles burst and form a sea-spray aerosol. These aerosol particles are enriched in surface-active organic compounds such as free fatty acids and anionic surfactants. In our method, we in situ generate H2 microbubbles by electrochemical water reduction using a porous Ni foam electrode. These H2 bubbles pick up PFAS as they rise through the water column that contains low concentration PFAS. When these bubbles reach the water surface, they burst and produce aerosol droplets that are enriched in PFAS. Using this method, we demonstrated ∼1000-fold preconcentration for ten common PFAS in the concentration range from 1 pM to 1 nM (or ∼0.5 ng/L to 500 ng/L) in 10 min. We also developed a diffusion-limited adsorption model that is in quantitative agreement with the experimental data. In addition, we demonstrated using this method to preconcentrate PFAS in tap water, indicating its potential use for quantitative analysis of PFAS in real-world water samples.

Fundamental Studies of New Ionization Technologies and Insights from IMS-MS.

Exceptional ion mobility spectrometry mass spectrometry (IMS-MS) developments by von Helden, Jarrold, and Clemmer provided technology that gives a view of chemical/biological compositions previously not achievable. The ionization method of choice used with IMS-MS has been electrospray ionization (ESI). In this special issue contribution, we focus on fundamentals of heretofore unprecedented means for transferring volatile and nonvolatile compounds into gas-phase ions singly and multiply charged. These newer ionization processes frequently lead to different selectivity relative to ESI and, together with IMS-MS, may provide a more comprehensive view of chemical compositions directly from their original environment such as surfaces, e.g., tissue. Similarities of results using solvent- and matrix-assisted ionization are highlighted, as are differences between ESI and the inlet ionization methods, especially with mixtures such as bacterial extracts. Selectivity using different matrices is discussed, as are results which add to our fundamental knowledge of inlet ionization as well as pose additional avenues for inquiry. IMS-MS provides an opportunity for comparison studies relative to ESI and will prove valuable using the new ionization technologies for direct analyses. Our hypothesis is that some ESI-IMS-MS applications will be replaced by the new ionization processes and by understanding mechanistic aspects to aid enhanced source and method developments this will be hastened.

Novel ionization processes for use in mass spectrometry: 'Squeezing' nonvolatile analyte ions from crystals and droplets.

Together with my group and collaborators, I have been fortunate to have had a key role in the discovery of new ionization processes that we developed into new flexible, sensitive, rapid, reliable, and robust ionization technologies and methods for use in mass spectrometry (MS). Our current research is focused on how best to understand, improve, and use these novel ionization processes which convert volatile and nonvolatile compounds from solids or liquids into gas-phase ions for analysis by MS using e.g. mass-selected fragmentation and ion mobility spectrometry to provide reproducible, accurate, and improved mass and drift time resolution. In my view, the apex was the discovery of vacuum matrix-assisted ionization (vMAI) in 2012 on an intermediate pressure matrix-assisted laser desorption/ionization (MALDI) source without the use of a laser, high voltages, or any other added energy. Only exposure of the matrix:analyte to the sub-atmospheric pressure of the mass spectrometer was necessary to initiate ionization. These findings were initially rejected by three different scientific journals, with comments related to 'how can this work?', 'where do the charges come from?', and 'it is not analytically useful'. Meanwhile, we and others have demonstrated analytical utility without a complete understanding of the mechanism. In reality, MALDI and electrospray ionization are widely used in science and their mechanisms are still controversially discussed despite use and optimization of now 30 years. This Perspective covers the applications and mechanistic aspects of the novel ionization processes for use in MS that guided us in instrument developments, and provides our perspective on how they relate to traditional ionization processes.

Vacuum Matrix-Assisted Ionization Source Offering Simplicity, Sensitivity, and Exceptional Robustness in Mass Spectrometry.

Vacuum matrix-assisted ionization (vMAI) uses select matrix compounds which when exposed to the vacuum of a mass spectrometer produce gas-phase ions from associated volatile or nonvolatile analyte without external energy input. Here, a vMAI source was constructed to replace the commercial inlet of a Thermo Orbitrap mass spectrometer. This allowed for rapid introduction of the matrix/analyte sample by a probe, contrary to vacuum matrix-assisted laser desorption/ionization (MALDI) sources. The matrix/analyte sample is inserted into a region of the "S-lens" entrance, where the spontaneously formed ions can be effectively transferred to the mass analyzer. This specifically designed ion source requires no laser, high voltage, heat, or nebulizing gases. A low voltage is used to transmit the ions through the commercial "S-lens" assembly and airflow can be used to modulate the ionization event. A few picograms of the drug erythromycin, assisted by the 3-nitrobenzonitrile vMAI matrix, is sufficient to produce mass spectra for over 1 min with the MH+ ion as the base peak in each mass spectrum. There is minimal carryover when loading high concentration samples and complex mixtures, contrary to direct infusion electrospray ionization, providing the probe is thoroughly cleaned between each new sample acquisition. Analyses of biological fluids, bacterial extracts, tissue, and high concentration samples have so far shown no indication of inlet or instrument contamination with these samples. The typical ultrahigh resolution and mass accuracy of the mass spectrometer are achieved, and a path forward to potential high throughput acquisitions demonstrated. It is expected that robustness can be introduced to any mass spectrometer through implementation of such a simple source.

Unprecedented Ionization Processes in Mass Spectrometry Provide Missing Link between ESI and MALDI.

In the field of mass spectrometry, producing intact, highly-charged protein ions from surfaces is a conundrum with significant potential payoff in application areas ranging from biomedical to clinical research. Here, we report on the ability to form intact, highly-charged protein ions on high vacuum time-of-flight mass spectrometers in the linear and reflectron modes achievable using experimental conditions that allow effective matrix removal from both the sample surfaces and from the charged clusters formed by the laser ablation event. The charge states are the highest reported on high vacuum mass spectrometers, yet they remain at only around a third of the highest charge obtained using laser ablation with a suitable matrix at atmospheric pressure. Other than physical instrument modifications, the key to forming abundant and stable highly-charged ions appears to be the volatility of the matrix used. Cumulative results suggest mechanistic links between the ionization process reported here and traditional ionization methods of electrospray ionization and matrix-assisted laser desorption/ionization.

Spontaneous Charge Separation and Sublimation Processes are Ubiquitous in Nature and in Ionization Processes in Mass Spectrometry.

Ionization processes have been discovered by which small and large as well as volatile and nonvolatile compounds are converted to gas-phase ions when associated with a matrix and exposed to sub-atmospheric pressure. Here, we discuss experiments further defining these simple and unexpected processes. Charge separation is found to be a common process for small molecule chemicals, solids and liquids, passed through an inlet tube from a higher to a lower pressure region, with and without heat applied. This charge separation process produces positively- and negatively-charged particles with widely different efficiencies depending on the compound and its physical state. Circumstantial evidence is presented suggesting that in the new ionization process, charged particles carry analyte into the gas phase, and desolvation of these particles produce the bare ions similar to electrospray ionization, except that solid particles appear likely to be involved. This mechanistic proposition is in agreement with previous theoretical work related to ion emission from ice. Graphical Abstract ᅟ.

An LC/MS Method Providing Improved Sensitivity: Electrospray Ionization Inlet.

Electrospray ionization inlet (ESII) combines positive aspects of electrospray ionization (ESI) and solvent-assisted ionization (SAI). Similar to SAI, the analyte solution is directly introduced into a heated inlet tube linking atmospheric pressure and the initial vacuum stage of the mass spectrometer. However, unlike SAI, in ESII a voltage is applied to the solution through a metal union linking two sections of fused silica tubing through which solution flows into the inlet. Here, we demonstrate liquid chromatography (LC) ESII/MS on two different mass spectrometers using a mixture of drugs, a peptide standard mixture, and protein digests. This LC-ESII/MS approach has little dead volume and thus provides excellent chromatographic resolution at mobile phase flow rates from 1 to 55 µL min-1. Significant improvement in ion abundance and less chemical background ions were observed relative to ESI for all drugs and peptides tested at flow rates from 15 to 55 µL min-1. At a low inlet tube temperature, ESII has an ionization selectivity similar to that of ESI but, at higher inlet temperatures, appears to have the attributes of both ESI and SAI.