Many powerful methods in mass spectrometry rely on activation of ions by high-energy collisions with gas particles. For example, multiple Collision Induced Dissociation (CID) has been used for many years to determine structural information for ions ranging from small organics to large, native-like protein complexes. More recently, Collision Induced Unfolding (CIU) has proved to be a very powerful method for understanding high-order protein structure and detecting differences between similar proteins. Quantifying the thermochemistry underlying dissociation/unfolding in these experiments can be quite challenging without reliable models of ion heating and cooling. Established physical models of CID are valuable in predicting ion heating but do not explicitly include mechanisms for cooling, which may play a large part in CID/CIU in modern instruments. and Molecular Dynamics methods are extremely computationally expensive for modeling CID/CIU of large analytes such as biomolecular ions. In this tutorial perspective, limiting behaviors of ion kinetic energy damping, heating, and cooling set by "extreme" cases are explored, and an Improved Impulsive Collision Theory and associated software ("Ion Simulations of the Physics of Activation", IonSPA) are introduced that can model all of these for partially inelastic collisions. Finally, examples of modeled collisional activation of native-like protein ions under realistic experimental conditions are discussed, with an outlook toward the use of IonSPA in accessing the thermochemical information hidden in CID breakdown curves and CIU fingerprints.

The ability to observe intact proteins by native mass spectrometry allows measurements of size, oligomeric state, numbers and types of ligands and post translational modifications bound, among many other characteristics. These studies have the potential to, and in some cases are, advancing our understanding of the role of structure in protein biology and biochemistry. However, there are some long-unresolved questions about to what extent solution-like structures persist without solvent in the vacuum of the mass spectrometer. Strong evidence from multiple sources over the years has demonstrated that well-folded proteins maintain native-like states if care is taken during sample preparation, ionization, and transmission through the gas phase. For partially unfolded states, dynamic and disordered proteins, and other important landmarks along the protein folding/unfolding pathway, caution has been urged in the interpretation of the results of native ion mobility/mass spectrometric data. New gas-phase tools allow us to provide insight into these questions with labeling reactions delivered through ion/ion chemistry. This Young Scientist Perspective demonstrates the robustness of these tools in describing native-like structure as well as possible deviations from native-like structure during native ion mobility/mass spectrometry. This Perspective illustrates some of the changes in structure produced by the removal of solvent and details some of the challenges and potential of the field.

Single-frequency ion parking, a useful technique in electrospray mass spectrometry (ESI-MS), involves gas-phase charge-reduction ion/ion reactions in an electrodynamic ion trap in conjunction with the application of a supplementary oscillatory voltage to selectively inhibit the reaction rate of an ion of interest. The ion parking process provides a means for limiting the extent of charge reduction in a controlled fashion and allows for ions distributed over a range of charge states to be concentrated into fewer charge states (a single charge state under optimal conditions). As charge reduction inherently leads to an increase in the mass-to-charge () ratio of the ions, it is important that the means for storing and analyzing ions be able to accommodate ions of high ratios. The so-called 'digital ion trap' (DIT), which uses a digital waveform as the trapping RF, has been demonstrated to be well-suited for the analysis of high ions by taking advantage of its ability to manipulate the waveform frequency. In this study, the feasibility of ion parking in a 3D quadrupole ion trap operated as a DIT using a slow-amplitude single-frequency sine-wave for selective inhibition of an ion/ion reaction is demonstrated. A recently described model that describes ion parking has been adjusted for the DIT case and is used to interpret experimental data for proteins ranging in mass from 8600 Da to 467,000 Da.

Capillary vibrating sharp-edge spray ionization (cVSSI) combined with hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has been utilized to characterize different solution-phase DNA conformers including DNA G-quadruplex topologies as well as triplex DNA and duplex DNA. In general, G-quadruplex DNA shows a wide range of protection of hydrogens extending from ~12% to ~21% deuterium incorporation. Additionally, the DNA sequences selected to represent parallel, antiparallel, and hybrid G-quadruplex topologies exhibit slight differences in deuterium uptake levels which appear to loosely relate to overall conformer stability. Notably, the exchange level for one of the hybrid sequence sub topologies of G-quadruplex DNA (24 TTG) is significantly different (compared with the others studied here) despite the DNA sequences being highly comparable. For the quadruplex-forming sequences, correlation analysis suggests protection of base hydrogens involved in tetrad hydrogen bonding. For duplex DNA ~19% deuterium incorporation is observed while only ~16% is observed for triplex DNA. This increased protection of hydrogens may be due to the added backbone scaffolding and Hoogsteen base pairing of the latter species. These experiments lay the groundwork for future studies aimed at determining the structural source of this protection as well as the applicability of the approach for ascertaining different oligonucleotide folds, co-existing conformations, and/or overall conformer flexibility.

Through optimization of terminal frequencies and effective sampling rates, we have developed nonlinear sawtooth-shaped frequency sweeps for efficient Fourier transform ion mobility mass spectrometry (FT-IM-MS) experiments. This is in contrast to conventional FT-IM-MS experiments where ion gates are modulated according to a linear frequency sweep. Linear frequency sweeps are effective but can be hindered by the amount of useful signal obtained using a single sweep over a large frequency range imposed by ion gating inefficiencies, particularly small ion packets, and gate depletion. These negative factors are direct consequences of the inherently low gate pulse widths of high-frequency ion gating events, placing an upper bound on FT-IM-MS performance. Here, we report alternative ion modulation strategies. Sawtooth frequency sweeps may be constructed for the purpose of either extending high-SNR transients or conducting efficient signal-averaging experiments for low-SNR transients. The data obtained using this approach show high-SNR signals for a set of low-mass tetraalkylammonium salts (<1000 m/z) where resolving powers in excess of 500 are achieved. Data for low-SNR obtained for multimeric protein complexes streptavidin (53 kDa) and GroEL (800 kDa) also reveal large increases in the signal-to-noise ratio for reconstructed arrival time distributions.

The demand for analytical tools for the analysis of low-concentration volume-limited samples has driven researchers to explore new analytical approaches. Mass spectrometry excels at trace analysis due to its high sensitivity and specificity, whereas ambient methods simplify, or completely eliminate sample preparation. Herein, we report a triboelectric nanogenerator-coated blade spray ambient mass spectrometry (TENG-CBS MS) method for the extraction, elution, and ionization of volume-limited, low-concentration small molecule drug samples with minimum sample preparation. Using a TENG device as the CBS power supply, we show it is possible to extract and analyze drug samples in a pulsed fashion at sub-nanogram to picogram levels with good stability and reproducibility. A wide range of analytes polarities were tested. Results indicated this method could also be useful for the analysis of low-level analytes in precious, volume limited samples in a simple single step.

Increasing the dimensionality of ion mobility (IM) presents an enticing opportunity to increase the information content and selectivity of many analyses. However, for implementations of IM that use constant electrostatic gradients to separate ions in a buffer gas, technical challenges have limited the adoption of the technique and number of dimensions within individual experiments. Here, we introduce a strategy to "reset" the potentials of ions between IM dimensions. To achieve this, mobility-selected ions are trapped between dimensions of IM, using a combination of RF and electrostatic fields, while the subsequent dimension of IM is devoid of any drift field. By applying an incremental voltage ramp, the potential of the trapping region is elevated, simultaneously establishing the drift field in the subsequent dimension of IM. The trapped ions are then released and separated. We measured similar arrival-time distributions of protein ions using this strategy and a method without potential resetting, suggesting that potential resetting can be performed without additional losses or activation of ions. The findings of those experiments were corroborated by ion trajectory simulations, which exhibited a very small changes in ion position and no significant changes in effective temperatures during potential resetting. Finally, we demonstrate that IM information can be preserved during potential resetting by selecting subpopulations of 9+ cytochrome ions, resetting their potential, subjecting them to a second-dimension IM separation, and observing the retention of conformers within each subpopulation. We anticipate that this strategy will be useful for advancing flexible, multidimensional experiments on electrostatic IM instruments.

Peptide/protein quantitation using mass spectrometry (MS) is advantageous due to its high sensitivity. Traditional absolute peptide quantitation methods rely on making calibration curves using peptide standards or isotope-labelled peptide standards, which are expensive and take time to synthesize. A method which can eliminate the need for using standards would be beneficial. Recently, we developed coulometric mass spectrometry (CMS) which can be used to quantify peptides that are oxidizable (e.g., those containing tyrosine or tryptophan), without using peptide standard. The method is based on electrochemical oxidation of peptides followed by MS to measure the oxidation yield. However, it cannot be directly used to quantify peptides without oxidizable residues. To extend this method for quantifying peptides/proteins in general, in this study, we adopted a derivatization strategy, in which a target peptide is first tagged with an electroactive reagent such as monocarboxymethylene blue NHS ester (MCMB-NHS ester), followed with quantitation by CMS. To illustrate the power of this method, we have analyzed peptides MG and RPPGFSPFR. The quantification error was less than 5%. Using RPPGFSPFR as an example, the quantitation sensitivity of the technique was found to be 0.25 pmol. Furthermore, we also used the strategy to quantify proteins cytochrome C and β-casein with an error of 2-26%.

Electrospray ionization (ESI) is one of the most popular methods to generate ions for mass spectrometry (MS). When compared with other ionization techniques, it can generate ions from liquid-phase samples without additives, retaining covalent and non-covalent interactions of the molecules of interest. When hyphenated to liquid chromatography, it greatly expands the versatility of MS analysis of complex mixtures. However, despite the extensive growth in the application of ESI, the technique still suffers from some drawbacks when powered by direct current (DC) power supplies. Triboelectric nanogenerators promise to be a new power source for the generation of ions by ESI, improving on the analytical capabilities of traditional DC ESI. In this review we highlight the fundamentals of ESI driven by DC power supplies, its contrasting qualities to triboelectric nanogenerator power supplies, and its applications to three distinct fields of research: forensics, metabolomics, and protein structure analysis.

Increasing studies associating glycerophospholipids with various pathological conditions highlight the need for their thorough characterization. However, the intricate composition of the lipidome due to the presence of lipid isomers poses significant challenges to structural lipidomics. This study uses the anodic corrosion of two metals in a single theta nESI emitter as a tool to simultaneously characterize lipids at multiple isomer levels. Anodic corrosion of cobalt and copper in the positive ion mode generates the metal-adducted lipid complexes, [M+Co] and [M+Cu], respectively. Optimization of parameters such as the distances of the electrodes from the nESI tip allowed the achievement of the formation of one metal-adducted lipid product at a time. Collision-induced dissociation (CID) of [M+Co] results in preferential loss of the fatty acyl (FA) chain at the 2 position, thus generating singly charged -specific fragment ions. Whereas, multistage fragmentation of [M+Cu] via CID generated a C=C bond position-specific characteristic ion pattern induced by the π-Cu interaction. The feasibility of the method was tested on PC lipid extract from egg yolk to identify lipids on multiple isomer levels. Thus, the application of dual metal anodic corrosion allows lipid isomer identification with reduced sample preparation time, no signal suppression by counter anions, low sample consumption, and no need for an extra apparatus.

Antibodies are one of the most formidable molecular weapons available to our immune system. Their high specificity against a target (antigen) and capability of triggering different immune responses (, complement system activation and antibody-dependent cell-mediated cytotoxicity) make them ideal drugs to fight many different human diseases. Currently, both monoclonal antibodies and more complex molecules based on the antibody scaffold are used as biologics. Naturally, such highly heterogeneous molecules require dedicated analytical methodologies for their accurate characterization. Mass spectrometry (MS) can define the presence and relative abundance of multiple features of antibodies, including critical quality attributes. The combination of small and large variations within a single molecule can only be determined by analyzing intact antibodies or their large (25 to 100 kDa) subunits. Hence, top-down (TD) and middle-down (MD) MS approaches have gained popularity over the last decade. In this Young Scientist Feature we discuss the evolution of TD and MD MS analysis of antibodies, including the new frontiers that go beyond biopharma applications. We will show how this field is now moving from the "quality control" analysis of a known, single antibody to the high-throughput investigation of complex antibody repertoires isolated from clinical samples, where the ultimate goal is represented by the complete gas-phase sequencing of antibody molecules without the need of any knowledge.

Although protein footprinting results are commonly obtained by ESI-based LC-MS/MS, a more rapid-turnaround alternative approach is desirable to expand the scope of protein footprinting and facilitate routine analysis such as monitoring protein high order structure in quality control or checking epitope maps. Considering that MALDI is a faster procedure that can be easily adapted for high-throughput analysis, we explore here the feasibility of developing a MALDI-based analysis "portfolio" of bottom-up peptide mass mapping for footprinting. The approach was applied to several model proteins that were submitted to two footprinting strategies, FPOP and GEE labeling, and their performance was evaluated. We found adequate coverage that can be improved with automatic off-line separation and spotting, demonstrating the capability to footprint accurately protein conformational change, showing that MALDI may be useful for selected applications in protein footprinting.

Comprehensive structural characterization of phosphatidylcholines (PCs) is essential to understanding their biological functions and roles in metabolism. Electron induced dissociation (EID) of protonated PCs directly generated from biological tissues has previously been shown to provide in-depth structural information on the lipid headgroup, regiosiomerism of fatty acyl tails and double bond positions. Although phosphatidylcholine ions formed via alkali metal cationization ( [M + Na] and [M + K]) are commonly generated during matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry experiments, the gas-phase ion chemistry behavior of EID on sodium- and potassium-cationized phosphatidylcholine ion types has not been studied for ions generated directly from tissue. Herein, we demonstrate EID on [M + Na] and [M + K] ion types in a MALDI imaging mass spectrometry workflow for lipid structural characterization. Briefly, near-complete structural information can be obtained upon EID of sodium- and potassium-cationized PCs, including diagnostic fragmentation of the lipid headgroup as well as identification of fatty acyl chain positions and double bond position. EID of cationized lipids generates s-specific glycerol backbone cleavages as well as a favorable combined loss of -2 fatty acid with choline over -1, allowing for facile differentiation and relative quantification of PC regioisomers. Moreover, relative quantification of positional isomers from biological tissue reveals that the relative percentages of sodium- and potassium-cationized -positional isomers varies significantly in different regions of rat brain tissue.

Human milk oligosaccharides (HMOs) are a class of glycans that are highly abundant in human milk and contribute to the healthy growth of an infant's immune system. While new advancements in analytical methodologies have been made in glycomics, the high degree of isomeric heterogeneity and lack of authentic standards have made the high-resolution separation and accurate characterization of linkage positioning of all HMO species very challenging. Herein, we present an evaluation of the use of host-guest chemistry in conjunction with cyclic ion mobility spectrometry-mass spectrometry (cIMS-MS)-based separations for the identification of linkage positioning in three pairs of di-, tetra-, and hexasaccharide HMO isomers that only differ in the positioning of one glycosidic linkage (β1,3 versus β1,4). Suitable hosts, such as α/β cyclodextrins, cucurbit[]urils ( = 5, 7), crown ethers, cyclic peptides, and an ionophore, were used to assess host-guest inclusion complex formation as well as linkage-specific cIMS-MS trends. Our results indicated a linkage-specific trend for the [M + 2α + 2H] cyclodextrin-based host-guest inclusion complexes where the β1,3 linkage-containing isomers were always higher mobility than the β1,4 linkage-containing ones as well one for the [M + α + β + 2H] complexes where the β1,4 linkage-containing isomers were always higher mobility than the β1,3 linkage-containing ones. We also observed diagnostic mobility fingerprints for the cucurbituril-based complexes. We anticipate that linkage-specific and mobility fingerprint trends can potentially aid in identifying linkage positioning for other HMO isomers as well as in complex human milk samples.

Cellular heterogeneity is commonly investigated using single-cell genomics and transcriptomics to investigate biological questions such as disease mechanism, therapeutic screening, and genomic and transcriptomic diversity between cellular populations and subpopulations at the cellular level. Single-cell mass spectrometry (MS)-based proteomics enables the high-throughput examination of protein expression at the single-cell level with wide applicability, and with spatial and temporal resolution, applicable to the study of cellular development, disease, effect of treatment, etc. The study of single-cell proteomics has lagged behind genomics and transcriptomics largely because proteins from single-cell samples cannot be amplified as DNA and RNA can using well established techniques such as PCR. Therefore, analytical methods must be robust, reproducible, and sensitive enough to detect the very small amount of protein within a single cell. To this end, nearly every step of the proteomics process has been extensively altered and improved to facilitate the proteomics analysis of single cells including cell counting and sorting, lysis, protein digestion, sample cleanup, separation, MS data acquisition, and data analysis. Here, we have reviewed recent advances in single-cell protein separation using nano reversed phase liquid chromatography (nRPLC) and capillary electrophoresis (CE) to inform application driven selection of separation techniques in the laboratory setting.

Characterization of phospholipid isomers is challenging due to their identical masses and similarities in structures. Here, we report that copper (I) ion complexed with phospholipids can be used to characterize both carbon-carbon double-bond (C=C bond) positional and geometric isomers. We investigate the distinct fragmentation patterns induced by the π-Cu interaction and developed strategies to rapidly characterize the isomerism of phospholipids. The multi-stage fragmentation of Cu-adducted lipids by collision-induced dissociation can generate diagnostic ions to locate C=C bonds in unsaturated lipids. Furthermore, the collision cross sections of Cu-adducted parent lipids and product ions can be used as molecular descriptors in distinguishing C=C bond geometric isomers. A bovine heart lipid extract containing Z-configuration lipids spiked with an E-configuration lipid was analyzed to demonstrate rapidness and effectiveness of the method in distinguishing lipid geometric isomers from a real sample.

A commercial quadrupole/time-of-flight tandem mass spectrometer has been modified and evaluated for its performance in conducting ion/ion reaction studies involving high mass (>100 kDa) ions. Modifications include enabling the application of dipolar AC waveforms to opposing rods in three quadrupole arrays in the ion path. This modification allows for resonance excitation of ions to effect ion activation, selective ion isolation, and ion parking. The other set of opposing rods in each array is enabled for the application of dipolar DC voltages for the purpose of broad-band (non-selective) ion heating. The plates between each quadrupole array are enabled for the application of either DC or AC (or both) voltages. The use of AC voltages allows for the simultaneous storage of ions of opposite polarity, thereby enabling mutual storage ion/ion reactions. Ions derived from nano-electrospray ionization of GroEL and β-galactosidase under native conditions were used to evaluate limits of instrument performance, in terms of range, ion isolation, and ion storage. After adjustment of the pulser frequency, ions as high in as 400,000 were detected. Significant losses in efficiency were noted above 250,000 that is likely due to roll-over in the ion detector efficiency and possibly also due to limitations in ion transfer efficiency from the collision quadrupole to the pulser region of the mass analyzer. No measurable decrease in the apparent mass resolving power was noted upon charge state reduction of the model ions. Resonance ejection techniques that employ the dipolar AC capabilities of the quadrupoles allow for ion isolation at values much greater than the RF/DC limitation of Q1 of = 2100. For example, at the highest low-mass cutoff achievable in the collision quadrupole ( = 500), it is possible to isolate ions of as high as 62,000. This is limited by the lowest dipolar AC frequency (5 kHz) that can be applied. A simple model is included to provide for an estimate of the ion cloud radius based on ion , ion , and ion trap operating conditions. The model predicts that singly charged ions of 1 MDa and thermal energy can be contained in the ion trap at the maximum low-mass cutoff, although such an ion would not be detected efficiently. Doubly charged GroEL ions were observed experimentally. Collectively, the performance characteristics at high , the functionality provided by the standard instrument capabilities, the modifications described above, and highly flexible instrument control software provide for a highly versatile platform for the study of high mass ion/ion reactions.

Native mass spectrometry analysis of membrane proteins has yielded many useful insights in recent years with respect to membrane protein-lipid interactions, including identifying specific interactions and even measuring binding affinities based on observed abundances of lipid-bound ions after collision-induced dissociation (CID). However, the behavior of non-covalent complexes subjected to extensive CID can in principle be affected by numerous factors related to gas-phase chemistry, including gas-phase basicity (GB) and acidity, shared-proton bonds, and other factors. A recent report from our group showed that common lipids span a wide range of GB values. Notably, phosphatidylcholine (PC) and sphingomyelin lipids are more basic than arginine, suggesting they may strip charge upon dissociation in positive ion mode, while phosphoserine lipids are slightly less basic than arginine and may form especially strong shared-proton bonds. Here, we use CID to probe the strength of non-specific gas-phase interactions between lipid head groups and several proteins, used to deliberately avoid possible physiological protein-lipid interactions. The strengths of the protein-head group interactions follow the trend predicted based solely on lipid and amino acid GBs: phosphoserine (PS) head group forms the strongest bonds with these proteins and out-competes the other head groups studied, while glycerophosphocholine (GPC) head groups form the weakest interactions and dissociate carrying away a positive charge. These results indicate that gas-phase thermochemistry can play an important role in determining which head groups remain bound to protein ions with native-like structures and charge states in positive ion mode upon extensive collisional activation.

Ovarian cancer is one of the leading causes of cancer related deaths affecting United States women. Early-stage detection of ovarian cancer has been linked to increased survival, however, current screening methods, such as biomarker testing, have proven to be ineffective in doing so. Therefore, further developments are necessary to be able to achieve positive patient prognosis. Ongoing efforts are being made in biomarker discovery towards clinical applications in screening for early-stage ovarian cancer. In this perspective, we discuss and provide examples for several workflows employing mass spectrometry-based proteomics towards protein biomarker discovery and characterization in the context of ovarian cancer; workflows include protein identification and characterization as well as intact protein profiling. We also discuss the opportunities to merge these workflows for a multiplexed approach for biomarkers. Lastly, we provide our insight as to future developments that may serve to enhance biomarker discovery workflows while also considering translational potential.

The detection of glycans and glycoconjugates has gained increasing attention in biological fields. Traditional mass spectrometry (MS)-based methods for glycoconjugate analysis are challenged with poor intensity when dealing with complex biological samples. We developed a desalting paper spray mass spectrometry (DPS-MS) strategy to overcome the issue of signal suppression of carbohydrates in salted buffer. Glycans and glycoconjugates (i.e., glycopeptides, nucleotide sugars, .) in non-volatile buffer (e.g., Tris buffer) can be loaded on the paper substrate from which buffers can be removed by washing with ACN/HO (90/10 v/v) solution. Glycans or glycoconjugates can then be eluted and spray ionized by adding ACN/HO/formic acid (FA) (10/90/1 v/v/v) solvent and applying a high voltage (HV) to the paper substrate. This work also showed that DPS-MS is applicable for direct detection of intact glycopeptides and nucleotide sugars as well as determination of glycosylation profiling of antibody, such as NIST monoclonal antibody IgG (NISTmAb). NISTmAb was deglycosylated with PNGase F to release -linked oligosaccharides. Twenty-six -linked oligosaccharides were detected by DPS-MS within a 5-minute timeframe without the need for further enrichment or derivatization. This work demonstrates that DPS-MS allows fast and sensitive detection of glycans/oligosaccharides and glycosylated species in complex matrices and has great potential in bioanalysis.

The combined use of electrospray ionization run in so-called "native mode" with top-down mass spectrometry (nTDMS) is enhancing both structural biology and discovery proteomics by providing three levels of information in a single experiment: the intact mass of a protein or complex, the masses of its subunits and non-covalent cofactors, and fragment ion masses from direct dissociation of subunits that capture the primary sequence and combinations of diverse post-translational modifications (PTMs). While intact mass data are readily deconvoluted using well-known software options, the analysis of fragmentation data that result from a tandem MS experiment - essential for proteoform characterization - is not yet standardized. In this tutorial, we offer a decision-tree for the analysis of nTDMS experiments on protein complexes and diverse bioassemblies. We include an overview of strategies to navigate this type of analysis, provide example data sets, and highlight software for the hypothesis-driven interrogation of fragment ions for localization of PTMs, metals, and cofactors on native proteoforms. Throughout we have emphasized the key features (deconvolution, search mode, validation, other) that the reader can consider when deciding upon their specific experimental and data processing design using both open-access and commercial software.