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.

Matrix-assisted ionization (MAI)-mass spectrometry (MS) eliminates the need for high voltage, a heat source, lasers, and compressed gases in the ionization process and uses minimal solvents in sample preparation, thus making MAI ideal for field-portable mass spectrometers. The broad applicability of MAI is demonstrated by simple, rapid, and robust positive and negative detection mode analyses of low and high mass compounds including some pesticides, dyes, drugs, lipids, and proteins (186 Da to 8.5 kDa) from various materials including urine, biological tissue sections, paper, and plant material on a low pumping capacity, single-quadrupole mass spectrometer. Different sample introduction methods are applicable, including the use of a pipet tip or glass melting point tube, allowing integration of sample preparation with sample introduction for increased analytical utility and ease of operation, even when sampling directly from surfaces.

The systematic study of the temperature and pressure dependence of matrix-assisted ionization (MAI) led us to the discovery of the seemingly impossible, initially explained by some reviewers as either sleight of hand or the misinterpretation by an overzealous young scientist of results reported many years before and having little utility. The “magic” that we were attempting to report was that with matrix assistance, molecules, at least as large as bovine serum albumin (66 kDa), are lifted into the gas phase as multiply charged ions simply by exposure of the matrix:analyte sample to the vacuum of a mass spectrometer. Applied heat, a laser, or voltages are not necessary to achieve charge states and ion abundances only previously observed with electrospray ionization (ESI). The fundamentals of how solid phase volatile or nonvolatile compounds are converted to gas-phase ions without added energy currently involves speculation providing a great opportunity to rethink mechanistic understanding of ionization processes used in mass spectrometry. Improved understanding of the mechanism(s) of these processes and their connection to ESI and matrix-assisted laser desorption/ionization may provide opportunities to further develop new ionization strategies for traditional and yet unforeseen applications of mass spectrometry. This Critical Insights article covers developments leading to the discovery of a seemingly magic ionization process that is simple to use, fast, sensitive, robust, and can be directly applied to surface characterization using portable or high performance mass spectrometers.

The power of ion mobility spectrometry–mass spectrometry (IMS-MS) as an analytical technology for differentiating macromolecular architecture is demonstrated. The presence of architectural dispersity within a sample is probed by sequentially measuring both the drift time and the mass-to-charge ratio for every component within a polymer sample. The utility of this technology is demonstrated by investigating three poly(ethylene glycol) (PEG) architectures with closely related average molecular weights of about 9000 Da: a linear PEG, an unevenly branched miktoarm star PEG, and evenly branched homoarm star PEGs. The three architectures were readily distinguished when analyzed separately as “pure” architectures or when analyzed as mixtures. IMS-MS results are contrasted with matrix-assisted laser desorption/ionization-MS and viscometry measurements.

Matrix assisted ionization vacuum (MAIV) rapidly generates gas-phase analyte ions from subliming solid-phase matrix:analyte crystals for analysis by mass spectrometry (MS). Ionization from the solid-phase allows the use of a variety of surfaces for introducing matrix:analyte samples to the vacuum of a mass spectrometer, including common laboratory materials, such as disposable pipet tips, filter paper, tooth picks, and nylon mesh. MAIV is shown here to be capable of analyses as fast as 3 s per sample with achievable sensitivities in the low femtomole range. MAIV-MS coupled with ion mobility spectrometry (IMS)-MS and tandem mass spectrometry (MS/MS) is shown to be especially powerful for analysis and characterization of a wide range of molecules ranging from small molecules such as drugs and metabolites (∼300 Da) to intact proteins (25.6 kDa). Automated sample introduction is demonstrated on two different commercial mass spectrometers using a programmable XYZ stage. A MAIV high-throughput nontargeted MSE approach is also demonstrated utilizing IMS for rapid characterization of small molecules and peptides from standard solutions, as well as drug spiked human urine.

Matrix-assisted ionization (MAI) mass spectrometry (MS) is a simple and sensitive method for analysis of low- and high-mass compounds, requiring only that the analyte in a suitable matrix be exposed to the inlet aperture of an atmospheric pressure ionization mass spectrometer. Here, we evaluate the reproducibility of MAI and its potential for quantification using six drug standards. Factors influencing reproducibility include the matrix compound used, temperature, and the method of sample introduction. The relative standard deviation (RSD) using MAI for a mixture of morphine, codeine, oxymorphone, oxycodone, clozapine, and buspirone and their deuterated internal standards using the matrix 3-nitrobenzonitrile is less than 10% with either a Waters SYNAPT G2 or a Thermo LTQ Velos mass spectrometer. The RSD values obtained using MAI are comparable to those using ESI or MALDI on these instruments. The day-to-day reproducibility of MAI determined for five consecutive days with internal standards was better than 20% using manual sample introduction. The reproducibility improved to better than 5% using a mechanically assisted sample introduction method. Hydrocodone, present in a sample of undiluted infant urine, was quantified with MAI using the standard addition method.

•Forty new compounds are presented that spontaneously convert analyte molecules to gas phase ions.•Sublimation and possibly triboluminescence appear to be necessary properties of MAIV matrices.•Compounds that are liquids at room temperature are converted to MAIV matrices by freezing.•Analyte ion abundance vs. inlet temperature and pH is studied for 25 solid MAIV matrices.•Pressure and temperature are variables that control the time over which anayte ions are observed.The initial discovery that a heated inlet tube of a mass spectrometer is an ionization source producing ions from volatile, nonvolatile, small, and large molecules with charge states similar to electrospray ionization has been advanced to ionization requiring only the vacuum inherent with a mass spectrometer and a suitable matrix. This spontaneous ionization method was first applicable with the matrix 3-nitrobenzonitrile. Here we report that over 40 compounds have now been discovered that spontaneously convert molecules to gas-phase ions when exposed to sub-atmospheric pressure, some with remarkable sensitivity (10 fmol of protein insulin). The commonality of all matrices is the ability to sublime, preferably near room temperature, through exposure to vacuum, and the ability to create charge separation under these conditions. The effect of vacuum, airflow, temperature (−80 to +150 °C) and pH (1–9) on the effectiveness of these newly discovered matrices to ionize peptides and proteins is presented. Compounds with and without acidic hydrogen atoms act as matrices and ionize specific compound classes. The new matrices extend applications from peptides, proteins and drugs to compound classes without basic functionality such as lipids and synthetic polymers in the negative and positive modes. Mass resolution and ion mobility spectrometry aspects are also discussed.

The analytical utility of a new and simple to use ionization method, matrix-assisted ionization (MAI), coupled with ion mobility spectrometry (IMS) and mass spectrometry (MS) is used to characterize a 2-armed europium(III)-containing poly(ethylene glycol) (Eu-PEG) complex directly from a crude sample. MAI was used with the matrix 1,2-dicyanobenzene, which affords low chemical background relative to matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). MAI provides high ion abundance of desired products in comparison to ESI and MALDI. Inductively coupled plasma-MS measurements were used to estimate a maximum of 10% of the crude sample by mass was the 2-arm Eu-PEG complex, supporting evidence of selective ionization of Eu-PEG complexes using the new MAI matrix, 1,2-dicyanobenzene. Multiply charged ions formed in MAI enhance the IMS gas-phase separation, especially relative to the singly charged ions observed with MALDI. Individual components are cleanly separated and readily identified, allowing characterization of the 2-arm Eu-PEG conjugate from a mixture of the 1-arm Eu-PEG complex and unreacted starting materials. Size-exclusion chromatography, liquid chromatography at critical conditions, MALDI-MS, ESI-MS, and ESI-IMS-MS had difficulties with this analysis, or failed.

This represents the first report of laserspray ionization vacuum (LSIV) with operation directly from atmospheric pressure for use in mass spectrometry. Two different types of electrospray ionization source inlets were converted to LSIV sources by equipping the entrance of the atmospheric pressure inlet aperture with a customized cone that is sealed with a removable glass plate holding the matrix/analyte sample. A laser aligned in transmission geometry (at 180° relative to the inlet) ablates the matrix/analyte sample deposited on the vacuum side of the glass slide. Laser ablation from vacuum requires lower inlet temperature relative to laser ablation at atmospheric pressure. However, higher inlet temperature is required for high-mass analytes, for example, α-chymotrypsinogen (25.6 kDa). Labile compounds such as gangliosides and cardiolipins are detected in the negative ion mode directly from mouse brain tissue as intact doubly deprotonated ions. Multiple charging enhances the ion mobility spectrometry separation of ions derived from complex tissue samples.

Introducing water or methanol containing a low concentration of volatile or nonvolatile analyte into an inlet tube cooled with dry ice linking atmospheric pressure and the first vacuum stage of a mass spectrometer produces gas-phase ions even of small proteins that can be detected by mass spectrometry. Collision-induced dissociation experiments conducted in the first vacuum region of the mass spectrometer suggest analyte ions being protected by a solvent cage. The charges may be produced by processes similar to those proposed for charge separation under freezing conditions in thunderclouds. By this process, the surface of an ice pellet is charged positive and the interior negative so that removal of surface results in charge separation. A reversal of surface charge is expected for a heated droplet surface, and this is observed by heating rather than cooling the inlet tube. These observations are consistent with charged supercooled droplets or ice particles as intermediates in the production of analyte ions under freezing conditions.

In this work we developed a multiplexed analysis platform providing a simple high-throughput means to characterize solutions. Automated analyses, requiring less than 5 s per sample without carryover and 1 s per sample, accepting minor cross contamination, was achieved using multiplexed solvent assisted ionization inlet (SAII) mass spectrometry (MS). The method involves sequentially moving rows of pipet tips containing sample solutions in close proximity to the inlet aperture of a heated mass spectrometer inlet tube. The solution is pulled from the container into the mass spectrometer inlet by the pressure differential at the mass spectrometer inlet aperture. This sample introduction method for direct injection of solutions is fast, easily implemented, and widely applicable, as is shown by applications ranging from small molecules to proteins as large as carbonic anhydrase (molecular weight ca. 29 000). MS/MS fragmentation is applicable for sample characterization. An x,y-stage and common imaging software are incorporated to map the location of components in the sample wells of a microtiter plate. Location within an x,y-array of different sample solutions and the relative concentration of the sample are displayed using ion intensity maps.

Mass spectrometry has emerged as a powerful tool for the bioanalytical sciences because of its ability to characterize small and large biomolecules in vanishingly small amounts. A recurring motif in mass spectrometry aims to decipher the chemical composition of biological samples at the molecular level, requiring drastic improvements in the ability to interrogate well defined and highly spatially resolved areas of a sample surface. With the growth of novel ionization methods, numerous advances have been made in sampling biological tissue surfaces. Here, current advancements in ambient, inlet, and vacuum ionization methods are discussed with respect to the potential improvements in the goal of achieving high spatial resolution and/or fast surface analysis. Of similar importance is the need for improvements in applicable characterization strategies using high performance fragmentation technologies such as electron transfer dissociation and electron capture dissociation directly from surfaces, and gas-phase separation through ion mobility spectrometry and high resolution mass spectrometry.

Matrix assisted ionization vacuum (MAIV) is a new ionization method that does not require high voltages, a laser beam, or applied heat and depends only the proper matrix, 3-nitrobenzonitrile (3-NBN), and the vacuum of the mass spectrometer to initiate ionization. Analyte ions of volatile as well as nonvolatile compounds are formed by simply exposing the matrix–analyte to the vacuum of a mass spectrometer. The reduced pressure at the inlet of an atmospheric pressure ionization mass spectrometer suffices to produce analyte ions, but unlike the previously reported matrix assisted ionization inlet method, with MAIV, heating the inlet is not necessary. Singly and multiply charged ions are formed similar to electrospray ionization but from a surface. Mass spectrometers in which a heated inlet tube is not available can be used for ionization using the 3-NBN matrix. We demonstrate rapid, high-sensitivity analyses of drugs, peptides, and proteins in the low femtomole range. The potential for high-throughput analyses is shown using multiwell plates and paper strips.

An astonishingly simple new method to produce gas-phase ions of small molecules as well as proteins from the solid state under cold vacuum conditions is described. This matrix assisted ionization vacuum (MAIV) mass spectrometry (MS) method produces multiply charged ions similar to those that typify electrospray ionization (ESI) and uses sample preparation methods that are nearly identical to matrix-assisted laser desorption/ionization (MALDI). Unlike these established methods, MAIV does not require a laser or voltage for ionization, and unlike the recently introduced matrix assisted ionization inlet method, does not require added heat. MAIV-MS requires only introduction of a crystalline mixture of the analyte incorporated with a suitable small molecule matrix compound such as 3-nitrobenzonitrile directly to the vacuum of the mass spectrometer. Vacuum intermediate pressure MALDI sources and modified ESI sources successfully produce ions for analysis by MS with this method. As in ESI-MS, ion formation is continuous and, without a laser, little chemical background is observed. MAIV, operating from a surface offers the possibility of significantly improved sensitivity relative to atmospheric pressure ionization because ions are produced in the vacuum region of the mass spectrometer eliminating losses associated with ion transfer from atmospheric pressure to vacuum. Mechanistic aspects and potential applications for this new ionization method are discussed.

Recently discovered ionization methods for use in mass spectrometry (MS), are widely applicable to biological materials, robust, and easy to automate. Among these, matrix assisted ionization vacuum (MAIV) is astonishing in that ionization of low and high-mass compounds are converted to gas-phase ions with charge states similar to electrospray ionization simply by exposing a matrix:analyte mixture to the vacuum of a mass spectrometer. Using the matrix compound, 3-nitrobenzonitrile, abundant ions are produced at room temperature without the need of high voltage or a laser. Here we discuss chemical analyses advances using MAIV combined with ion mobility spectrometry (IMS) real time separation, high resolution MS, and mass selected and non-mass selected MS/MS providing rapid analyte characterization. Drugs, their metabolites, lipids, peptides, and proteins can be ionized simultaneously from a variety of different biological matrixes such as urine, plasma, whole blood, and tissue. These complex mixtures are best characterized using a separation step, which is obtained nearly instantaneously with IMS, and together with direct ionization and MS or MS/MS provides a fast analysis method that has considerable potential for non-targeted clinical analyses.

Mechanistic arguments relative to matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) address observations that predominately singly charged ions are detected. However, recently a matrix assisted laser ablation method, laserspray ionization (LSI), was introduced that can use the same sample preparation and laser as MALDI, but produce highly charged ions from proteins. In MALDI, ions are generated from neutral molecules by the photon energy provided to a matrix, while in LSI ions are produced inside a heated inlet tube linking atmospheric pressure and the first vacuum region of the mass spectrometer. Some LSI matrices also produce highly charged ions with MALDI ion sources operated at intermediate pressure or high vacuum. The operational similarity of LSI to MALDI, and the large difference in charge states observed by these methods, provides information of fundamental importance to proposed ionization mechanisms for LSI and MALDI. Here, we present data suggesting that the prompt and delayed ionization reported for vacuum MALDI are both fast processes relative to producing highly charged ions by LSI. The energy supplied to produce these charged clusters/droplets as well as their size and time available for desolvation are determining factors in the charge states of the ions observed. Further, charged droplets/clusters may be a common link for ionization of nonvolatile compounds by a variety of MS ionization methods, including MALDI and LSI.

Matrix assisted inlet ionization (MAII) is a method in which a matrix:analyte mixture produces mass spectra nearly identical to electrospray ionization without the application of a voltage or the use of a laser as is required in laserspray ionization (LSI), a subset of MAII. In MAII, the sample is introduced by, for example, tapping particles of dried matrix:analyte into the inlet of the mass spectrometer and, therefore, permits the study of conditions pertinent to the formation of multiply charged ions without the need of absorption at a laser wavelength. Crucial for the production of highly charged ions are desolvation conditions to remove matrix molecules from charged matrix:analyte clusters. Important factors affecting desolvation include heat, vacuum, collisions with gases and surfaces, and even radio frequency fields. Other parameters affecting multiply charged ion production is sample preparation, including pH and solvent composition. Here, findings from over 100 compounds found to produce multiply charged analyte ions using MAII with the inlet tube set at 450 °C are presented. Of the compounds tested, many have –OH or –NH2 functionality, but several have neither (e.g., anthracene), nor aromaticity or conjugation. Binary matrices are shown to be applicable for LSI and solvent-free sample preparation can be applied to solubility restricted compounds, and matrix compounds too volatile to allow drying from common solvents. Our findings suggest that the physical properties of the matrix such as its morphology after evaporation of the solvent, its propensity to evaporate/sublime, and its acidity are more important than its structure and functional groups.

Liquid chromatography (LC) solvent assisted inlet ionization (SAII) mass spectrometry (MS) was previously reported to give good chromatographic resolution and MS detection injecting 66 ng of a BSA tryptic digest. In analogy to nano-electrospray ionization (nESI), we extend SAII LC/MS to nano-SAII (nSAII) operating at nL min–1 flow rates and demonstrate good quality ion chromatograms and mass spectra from injection of as little as 0.7 ng of BSA digest onto a capillary LC column. Data dependent fragmentation is demonstrated for injection of 7 ng of a BSA digest. This method has advantages over nESI in ease of use and low cost as it requires no voltage and is operational without the necessity of connectors or fragile nESI emitters, although similar constricted tips can be helpful in nSAII to stabilize the signal at low nanoliter flow. At a flow rate of 0.8 μL min–1, the only requirement for nSAII is that the exit-end of the capillary LC column be adjusted near the aperture of the heated inlet of the mass spectrometer.

The first examples of highly charged ions observed under intermediate pressure (IP) vacuum conditions are reported using laser ablation of matrix/analyte mixtures. The method and results are similar to those obtained at atmospheric pressure (AP) using laserspray ionization (LSI) and/or matrix assisted inlet ionization (MAII). Electrospray ionization (ESI), LSI, and MAII are methods operating at AP and have been shown, with or without the use of a voltage or a laser, to produce highly charged ions with very similar ion abundance and charge states. A commercial matrix-assisted laser desorption/ionization ion mobility spectrometry (IMS) mass spectrometry (MS) instrument (SYNAPT G2) was used for the IP developments. The necessary conditions for producing highly charged ions of peptides and small proteins at IP appear to be a pressure drop region and the use of suitable matrixes and laser fluence. Ionization to produce these highly charged ions under the low pressure conditions of IP does not require specific heating or a special inlet ion transfer region. However, under the current setup, ubiquitin is the highest molecular weight protein observed. These findings are in accord with the need to provide thermal energy in the pressure drop region, similar to LSI and MAII, to improve sensitivity and extend the types of compounds that produce highly charged ions. The practical utility of IP-LSI in combination with IMS-MS is demonstrated for the analysis of model mixtures composed of a lipid, peptides, and a protein. Further, endogenous multiply charged peptides are observed directly from delipified mouse brain tissue with drift time distributions that are nearly identical in appearance to those obtained from a synthesized neuropeptide standard analyzed by either LSI- or ESI-IMS-MS at AP. Efficient solvent-free gas-phase separation enabled by the IMS dimension separates the multiply charged peptides from lipids that remained on the delipified tissue. Lipid and peptide families are exceptionally well separated because of the ability of IP-LSI to produce multiple charging.

Inlet ionization is a new approach for ionizing both small and large molecules in solids or liquid solvents with high sensitivity. The utility of solvent based inlet ionization mass spectrometry (MS) as a method for analysis of volatile and nonvolatile compounds eluting from a liquid chromatography (LC) column is demonstrated. This new LC/MS approach uses reverse phase solvent systems common to electrospray ionization MS. The first LC/MS analyses using this novel approach produced sharp chromatographic peaks and good quality full mass range mass spectra for over 25 peptides from injection of only 1 pmol of a tryptic digest of bovine serum albumin using an eluent flow rate of 55 μL min–1. Similarly, full acquisition LC/MS/MS of the MH+ ion of the drug clozapine, using the same solvent flow rate, produced a signal-to-noise ratio of 54 for the major fragment ion with injection of only 1 μL of a 2 ppb solution. LC/MS results were acquired on two different manufacturer’s mass spectrometers using a Waters Corporation NanoAcquity liquid chromatograph.

A new matrix compound, 2-nitrophloroglucinol, is reported which not only produces highly charged ions similar to electrospray ionization (ESI) under atmospheric pressure (AP) and intermediate pressure (IP) laserspray ionization (LSI) conditions but also the most highly charged ions so far observed for small proteins in mass spectrometry (MS) under high vacuum (HV) conditions. This new matrix extends the compounds that can successfully be employed as matrixes with LSI, as demonstrated on an LTQ Velos (Thermo) at AP, a matrix-assisted laser desorption/ionization (MALDI)-ion mobility spectrometry (IMS) time-of-flight (TOF) SYNAPT G2 (Waters) at IP, and MALDI-TOF Ultraflex, UltrafleXtreme, and Autoflex Speed (Bruker) mass spectrometers at HV. Measurements show that stable multiple charged molecular ions of proteins are formed under all pressure conditions indicating softer ionization than MALDI, which suffers a high degree of metastable fragmentation when multiply charged ions are produced. An important analytical advantage of this new LSI matrix are the potential for high sensitivity equivalent or better than AP-LSI and vacuum MALDI and the potential for enhanced mass selected fragmentation of the abundant highly charged protein ions. A second new LSI matrix, 4,6-dinitropyrogallol, produces abundant multiply charged ions at AP but not under HV conditions. The differences in these similar compounds ability to produce multiply charged ions under HV conditions is believed to be related to their relative ability to evaporate from charged matrix/analyte clusters.

An extra dimension of polymer analysis: ion mobility spectrometry-mass spectrometry (IMS-MS) separates ions according to their size in the gas phase, allowing differentiation of linear and cyclic polymeric isomers. This analytical technique is a rapid and sensitive method for assessing cyclic polymer purity in one step. As highly pure cyclic polymers are crucial for adequately assessing architecture dependent properties, IMS–MS offers great promise in the characterization of this unique class of polymers.

Laserspray ionization (LSI) is a new approach to producing multiply charged ions from solids on surfaces by laser ablation of matrixes commonly used in matrix-assisted laser desorption/ionization (MALDI). We show that the only necessity of the laser for producing multiply charged ions is to deliver particles or droplets of the matrix/analyte mixture to an ionization zone which is simply a heated inlet to the vacuum of the mass spectrometer. Several other methods for delivering sample are demonstrated to produce nearly equivalent results. One example shows the use of an air gun replacing the laser and producing mass spectra of proteins by shooting pellets into a metal plate which has matrix/analyte applied to the opposite side and near the ion entrance inlet to the mass spectrometer. Multiply charged ions of proteins are produced in the absence of any electric field or laser and with only the need of a heated ion entrance capillary or skimmer. The commonality of the matrix with MALDI and the mild conditions necessary for formation of ions brings into question the mechanism of formation of multiply charged ions and the importance of matrix structure in this process.

The ability to analyze complex (macro) molecules is of fundamental importance for understanding chemical, physical, and biological processes. Complexity may arise from small differences in structure, large dynamic range, as well as a vast range in solubility or ionization, imposing daunting tasks in areas as different as lipidomics and proteomics. Here, we describe a rapid matrix application that permits the deposition of matrix-assisted laser desorption/ionization (MALDI) matrix solvent-free. This solvent-free one-step automatic matrix deposition is achieved through vigorous movements of beads pressing the matrix material through a metal mesh. The mesh (20 μm) produces homogeneous coverage of <12 μm crystals (DHB, CHCA matrixes) in 1 min, as determined by optical microscopy, permitting fast uniform coverage of analyte and possible high-spatial resolution surface analysis. Homogenous tissue coverage of <5 μm sized crystals is achieved using a 3 μm mesh. Solvent-free MALDI analysis on a time-of-flight (TOF) mass analyzer of mouse brain tissue homogenously covered with CHCA matrix subsequently provides a homogeneous response in ion signal intensity. Total solvent-free analysis (TSA) by mass spectrometry (MS) of tissue sections is carried out by applying the MALDI matrix solvent-free for subsequent ionization and gas phase separation for decongestion of complexity in the absence of any solvent using ion mobility spectrometry (IMS) followed by MS detection. Isobaric compositions were well-delineated using TSA by MS.

The ability of laserspray ionization (LSI) to produce multiply charged ions by laser ablation from the solid state, directly from a surface, and at atmospheric pressure allows protein analysis on an ion mobility spectrometry (IMS)−mass spectrometry (MS) instrument (SYNAPT G2) having a mass-to-charge limit of 8000. The matrix, 2,5-dihydroxyacetophenone, lowers the thermal requirements for desolvation of matrix/analyte clusters to produce the highly charged LSI ions under gentle conditions to retain structural integrity of the proteins. Examples include cytochrome C and lysozyme. The solvent-free IMS gas-phase separation is used to baseline separate in the drift time dimension the isomeric solubility restricted β-amyloid (1−42) from the reversed (42−1). The LSI process is shown to be sufficiently soft to preserve structural integrity and permit separation according to the different shapes. These results suggest that LSI-IMS-MS potentially combines speed of analysis and imaging capability common to matrix-assisted laser desorption/ionization and multiple charging with the potential for structural analysis common to electrospray ionization.

A simple device is described for desolvation of highly charged matrix/analyte clusters produced by laser ablation leading to multiply charged ions that are analyzed by ion mobility spectrometry-mass spectrometry. Thus, for example, highly charged ions of ubiquitin and lysozyme are cleanly separated in the gas phase according to size and mass (shape and molecular weight) as well as charge using Tri-Wave ion mobility technology coupled to mass spectrometry. This contribution confirms the mechanistic argument that desolvation is necessary to produce multiply charged matrix-assisted laser desorption/ionization (MALDI) ions and points to how these ions can be routinely formed on any atmospheric pressure mass spectrometer.Laserspray ionization (LSI) has elements of MALDI and ESI. Laser ablation of a MALDI sample in transmission geometry produces charged droplets similar to ESI.Figure optionsDownload full-size imageDownload high-quality image (139 K)Download as PowerPoint slide

Increasingly comprehensive questions related to the biosynthesis of lipids relevant to understanding new signaling pathways have created daunting tasks for their chemical analysis. Here, ion mobility spectrometry (IMS) and mass spectrometry (MS) techniques combined with electrospray ionization have been used to examine mixtures of closely related lipid structures. The drift time distributions of sphingomyelins show baseline separations for ethylene chain length differences (Δ ∼ 1.2 ms) and partial separations in single unsaturation differences (Δ ∼ 0.3 ms) revealing that the most compact structures are observed with shorter chains and increasing unsaturation. Drift time distributions of different ionizations frequently fall into families with the same drift times (isodrifts) indicating that the ion attached to the lipid has little structural influence. The present data show that phospholipids, especially phosphatidylinositol, aggregate to form inverted micelles. Phospholipids (phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and phosphatidylinositol) are effectively separated according to their polar head groups. This method also provides information about the mixture composition of the chemically different lipids N-palmitoyl glycine, N-arachidonoyl ethanolamide, and phosphatidylcholine existing over an array of charge states and sizes (inverted micelles) depending on mixture concentration. Multidimensional IMS3-MS introduces an additional dimension to fragmentation analysis by separating the fragmented ions into groups related to size, shape and charge and allows determination of sn-1 and sn-2 substitution as is shown for phosphatidylglycerols. This contribution provides evidence for extending the targeted approach to global lipidomics analysis using the high-efficiency gas-phase separation afforded by multidimensional IMS-MS.

The synthesis of increasingly complex polymers has created daunting, sometimes insurmountable problems for their chemical analysis. The importance is magnified by outsourcing of production and their use in consumer products, including medical devices and food storage, and therefore requires a new generation of technology for quality assurance. Here, we report capturing subtle differences at the molecular level in complex polymer mixtures nearly instantaneously using a prototype multidimensional ion mobility spectrometry/mass spectrometry spectrometry instrument. Bulk activation/fragmentation strategies reported here provide signatures of structural characteristics that permit effortless recognition of minor differences in blends and copolymers, even as structural isomers and from a quantitative perspective. The data displayed as a pictorial snapshot provide a visual pattern that is sufficiently distinctive that computer-aided pattern recognition can be used to address process control and regulatory issues.

•Matrix-assisted ionization (MAI) from surfaces with and without laser ablation.•Membrane-associated proteins ionized from buffer solutions for mass spectrometry (MS) analysis.•Rapid protein analysis from high salt content samples using MAI at (ultra) high resolution.•Comparison of MAI, ESI, and MALDI for analyzing cholera toxin directly from buffer.•MAI analysis of hexameric pertussis toxin from buffer showing protein subunits.Matrix-assisted ionization (MAI) is demonstrated to be a robust and sensitive analytical method capable of analyzing proteins such as cholera toxin B-subunit and pertussis toxin mutant from conditions containing relatively high amounts of inorganic salts, buffers, and preservatives without the need for prior sample clean-up or concentration. By circumventing some of the sample preparation steps, MAI simplifies and accelerates the analytical workflow for biological samples in complex media. The benefits of multiply charged ions characteristic of electrospray ionization (ESI) and the robustness of matrix-assisted laser desorption/ionization (MALDI) can be obtained from a single method, making it well suited for analysis of proteins and other biomolecules at ultra-high resolution as demonstrated on an Orbitrap Fusion where protein subunits were resolved for which MALDI-time-of-flight failed. MAI results are compared with those obtained with ESI, MALDI, and laserspray ionization methods and fundamental commonalities discussed.Graphical abstractDownload full-size image