Targeted Quantification of Proteoforms in Complex Samples by Proteoform Reaction Monitoring.

Existing mass spectrometric assays used for sensitive and specific measurements of target proteins across multiple samples, such as selected/multiple reaction monitoring (SRM/MRM) or parallel reaction monitoring (PRM), are peptide-based methods for bottom-up proteomics. Here, we describe an approach based on the principle of PRM for the measurement of intact proteoforms by targeted top-down proteomics, termed proteoform reaction monitoring (PfRM). We explore the ability of our method to circumvent traditional limitations of top-down proteomics, such as sensitivity and reproducibility. We also introduce a new software program, Proteoform Finder (part of ProSight Native), specifically designed for the easy analysis of PfRM data. PfRM was initially benchmarked by quantifying three standard proteins. The linearity of the assay was shown over almost 3 orders of magnitude in the femtomole range, with limits of detection and quantification in the low femtomolar range. We later applied our multiplexed PfRM assay to complex samples to quantify biomarker candidates in peripheral blood mononuclear cells (PBMCs) from liver-transplanted patients, suggesting their possible translational applications. These results demonstrate that PfRM has the potential to contribute to the accurate quantification of protein biomarkers for diagnostic purposes and to improve our understanding of disease etiology at the proteoform level.

Orbitrap Mass Spectrometry and High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Enable the in-Depth Analysis of Human Serum Proteoforms.

Blood serum and plasma are arguably the most commonly analyzed clinical samples, with dozens of proteins serving as validated biomarkers for various human diseases. Top-down proteomics may provide additional insights into disease etiopathogenesis since this approach focuses on protein forms, or proteoforms, originally circulating in blood, potentially providing access to information about relevant post-translational modifications, truncations, single amino acid substitutions, and many other sources of protein variation. However, the vast majority of proteomic studies on serum and plasma are carried out using peptide-centric, bottom-up approaches that cannot recapitulate the original proteoform content of samples. Clinical laboratories have been slow to adopt top-down analysis, also due to higher sample handling requirements. In this study, we describe a straightforward protocol for intact proteoform sample preparation based on the depletion of albumin and immunoglobulins, followed by simplified protein fractionation via polyacrylamide gel electrophoresis. After molecular weight-based fractionation, we supplemented the traditional liquid chromatography-tandem mass spectrometry (LC-MS2) data acquisition with high-field asymmetric waveform ion mobility spectrometry (FAIMS) to further simplify serum proteoform mixtures. This LC-FAIMS-MS2 method led to the identification of over 1000 serum proteoforms < 30 kDa, outperforming traditional LC-MS2 data acquisition and more than doubling the number of proteoforms identified in previous studies.

Proteomics evaluation of five economical commercial abundant protein depletion kits for enrichment of diseases-specific biomarkers from blood serum.

Blood serum is arguably the most analyzed biofluid for disease prediction and diagnosis. Herein, we benchmarked five different serum abundant protein depletion (SAPD) kits with regard to the identification of disease-specific biomarkers in human serum using bottom-up proteomics. As expected, the IgG removal efficiency among the SAPD kits is highly variable, ranging from 70% to 93%. A pairwise comparison of database search results showed a 10%-19% variation in protein identification among the kits. Immunocapturing-based SAPD kits against IgG and albumin outperformed the others in the removal of these two abundant proteins. Conversely, non-antibody-based methods (i.e., kits using ion exchange resins) and kits leveraging a multi-antibody approach were proven to be less efficient in depleting IgG/albumin from samples but led to the highest number of identified peptides. Notably, our results indicate that different cancer biomarkers could be enriched up to 10% depending on the utilized SAPD kit compared with the undepleted sample. Additionally, functional analysis of the bottom-up proteomic results revealed that different SAPD kits enrich distinct disease- and pathway-specific protein sets. Overall, our study emphasizes that a careful selection of the appropriate commercial SAPD kit is crucial for the analysis of disease biomarkers in serum by shotgun proteomics.

Improved Label-Free Quantification of Intact Proteoforms Using Field Asymmetric Ion Mobility Spectrometry.

The high-throughput quantification of intact proteoforms using a label-free approach is typically performed on proteins in the 0-30 kDa mass range extracted from whole cell or tissue lysates. Unfortunately, even when high-resolution separation of proteoforms is achieved by either high-performance liquid chromatography or capillary electrophoresis, the number of proteoforms that can be identified and quantified is inevitably limited by the inherent sample complexity. Here, we benchmark label-free quantification of proteoforms of Escherichia coli by applying gas-phase fractionation (GPF) via field asymmetric ion mobility spectrometry (FAIMS). Recent advances in Orbitrap instrumentation have enabled the acquisition of high-quality intact and fragmentation mass spectra without the need for averaging time-domain transients prior to Fourier transform. The resulting speed improvements allowed for the application of multiple FAIMS compensation voltages in the same liquid chromatography-tandem mass spectrometry experiment without increasing the overall data acquisition cycle. As a result, the application of FAIMS to label-free quantification based on intact mass spectra substantially increases the number of both identified and quantified proteoforms without penalizing quantification accuracy in comparison to traditional label-free experiments that do not adopt GPF.

Comprehensive characterization of the Hsp70 interactome reveals novel client proteins and interactions mediated by posttranslational modifications.

Hsp70 interactions are critical for cellular viability and the response to stress. Previous attempts to characterize Hsp70 interactions have been limited by their transient nature and the inability of current technologies to distinguish direct versus bridged interactions. We report the novel use of cross-linking mass spectrometry (XL-MS) to comprehensively characterize the Saccharomyces cerevisiae (budding yeast) Hsp70 protein interactome. Using this approach, we have gained fundamental new insights into Hsp70 function, including definitive evidence of Hsp70 self-association as well as multipoint interaction with its client proteins. In addition to identifying a novel set of direct Hsp70 interactors that can be used to probe chaperone function in cells, we have also identified a suite of posttranslational modification (PTM)-associated Hsp70 interactions. The majority of these PTMs have not been previously reported and appear to be critical in the regulation of client protein function. These data indicate that one of the mechanisms by which PTMs contribute to protein function is by facilitating interaction with chaperones. Taken together, we propose that XL-MS analysis of chaperone complexes may be used as a unique way to identify biologically important PTMs on client proteins.

Structural and Functional Characterization of a Novel Scorpion Toxin that Inhibits NaV1.8 via Interactions With the DI Voltage Sensor and DII Pore Module.

Voltage-gated sodium channel NaV1.8 regulates transmission of pain signals to the brain. While NaV1.8 has the potential to serve as a drug target, the molecular mechanisms that shape NaV1.8 gating are not completely understood, particularly mechanisms that couple activation to inactivation. Interactions between toxin producing animals and their predators provide a novel approach for investigating NaV structure-function relationships. Arizona bark scorpions produce Na+ channel toxins that initiate pain signaling. However, in predatory grasshopper mice, toxins inhibit NaV1.8 currents and block pain signals. A screen of synthetic peptide toxins predicted from bark scorpion venom showed that peptide NaTx36 inhibited Na+ current recorded from a recombinant grasshopper mouse NaV1.8 channel (OtNaV1.8). Toxin NaTx36 hyperpolarized OtNaV1.8 activation, steady-state fast inactivation, and slow inactivation. Mutagenesis revealed that the first gating charge in the domain I (DI) S4 voltage sensor and an acidic amino acid (E) in the DII SS2 - S6 pore loop are critical for the inhibitory effects of NaTx36. Computational modeling showed that a DI S1 - S2 asparagine (N) stabilizes the NaTx36 - OtNaV1.8 complex while residues in the DI S3 - S4 linker and S4 voltage sensor form electrostatic interactions that allow a toxin glutamine (Q) to contact the first S4 gating charge. Surprisingly, the models predicted that NaTx36 contacts amino acids in the DII S5 - SS1 pore loop instead of the SS2 - S6 loop; the DII SS2 - S6 loop motif (QVSE) alters the conformation of the DII S5 - SS1 pore loop, enhancing allosteric interactions between toxin and the DII S5 - SS1 pore loop. Few toxins have been identified that modify NaV1.8 gating. Moreover, few toxins have been described that modify sodium channel gating via the DI S4 voltage sensor. Thus, NaTx36 and OtNaV1.8 provide tools for investigating the structure-activity relationship between channel activation and inactivation gating, and the connection to alternative pain phenotypes.

Characterization of large intact protein ions by mass spectrometry: What directions should we follow?

Theoretically, the gas-phase interrogation of whole proteoforms via mass spectrometry, known as top-down proteomics, bypasses the protein inference problem that afflicts peptide-centric proteomic approaches. Despite this obvious advantage, the application of top-down proteomics remains rare, mainly due to limited throughput and difficulty of analyzing proteins >30 kDa. Here we will discuss some of the problems encountered during the characterization of large proteoforms, and guided by a combination of theoretical background and experimental evidence we will describe some innovative data acquisition strategies and novel mass spectrometry technologies that can at least partially overcome such limitations.

The Blood Proteoform Atlas: A reference map of proteoforms in human hematopoietic cells.

Human biology is tightly linked to proteins, yet most measurements do not precisely determine alternatively spliced sequences or posttranslational modifications. Here, we present the primary structures of ~30,000 unique proteoforms, nearly 10 times more than in previous studies, expressed from 1690 human genes across 21 cell types and plasma from human blood and bone marrow. The results, compiled in the Blood Proteoform Atlas (BPA), indicate that proteoforms better describe protein-level biology and are more specific indicators of differentiation than their corresponding proteins, which are more broadly expressed across cell types. We demonstrate the potential for clinical application, by interrogating the BPA in the context of liver transplantation and identifying cell and proteoform signatures that distinguish normal graft function from acute rejection and other causes of graft dysfunction.

Structural Analysis of Monoclonal Antibodies with Top-down and Middle-down Electron Transfer Dissociation Mass Spectrometry: The First Decade.

Monoclonal antibodies (mAbs) are protein biotherapeutics with a proven efficacy toward fighting life-threatening diseases. Their exceptional healing potential drives the annual increase in the number of novel mAbs and other antibody-like molecules entering clinical trials and the number of approved mAb-based drugs. Mass spectrometry (MS) offers high selectivity and specificity for the potentially unambiguous identification and comprehensive structural characterization of proteins, including at the proteoform level. It is thus not surprising that MS-based approaches are playing a central role in the biopharma laboratories, complementing and advancing traditional biotherapeutics characterization workflows. A combination of MS approaches is required to comprehensively characterize mAbs' structures: the commonly employed bottom-up MS approaches are efficiently complemented with mass measurements at the intact and subunit (middle-up) levels, together with product ion analysis following gas-phase fragmentation of precursor ions performed at the intact (top-down) and subunit (middle-down) levels. Here we overview our group's contribution to increasing the efficiency of these approaches and the development of the novel strategies over the past decade. Our particular focus has been on the top-down and middle-down MS methods that utilize electron transfer dissociation (ETD) for gas-phase protein ion fragmentation. Several approaches pioneered by our group, particularly an ETD-based middle-down approach, constitute a part of commercial software solutions for the mAb's characterization workflows.

Evolutionary Dynamics of Indels in SARS-CoV-2 Spike Glycoprotein.

SARS-CoV-2, responsible for the current COVID-19 pandemic that claimed over 5.0 million lives, belongs to a class of enveloped viruses that undergo quick evolutionary adjustments under selection pressure. Numerous variants have emerged in SARS-CoV-2, posing a serious challenge to the global vaccination effort and COVID-19 management. The evolutionary dynamics of this virus are only beginning to be explored. In this work, we have analysed 1.79 million spike glycoprotein sequences of SARS-CoV-2 and found that the virus is fine-tuning the spike with numerous amino acid insertions and deletions (indels). Indels seem to have a selective advantage as the proportions of sequences with indels steadily increased over time, currently at over 89%, with similar trends across countries/variants. There were as many as 420 unique indel positions and 447 unique combinations of indels. Despite their high frequency, indels resulted in only minimal alteration of N-glycosylation sites, including both gain and loss. As indels and point mutations are positively correlated and sequences with indels have significantly more point mutations, they have implications in the evolutionary dynamics of the SARS-CoV-2 spike glycoprotein.

Spectrum of Apolipoprotein AI and Apolipoprotein AII Proteoforms and Their Associations With Indices of Cardiometabolic Health: The CARDIA Study.

Background ApoAI (apolipoproteins AI) and apoAII (apolipoprotein AII) are structural and functional proteins of high-density lipoproteins (HDL) which undergo post-translational modifications at specific residues, creating distinct proteoforms. While specific post-translational modifications have been reported to alter apolipoprotein function, the full spectrum of apoAI and apoAII proteoforms and their associations with cardiometabolic phenotype remains unknown. Herein, we comprehensively characterize apoAI and apoAII proteoforms detectable in serum and their post-translational modifications and quantify their associations with cardiometabolic health indices. Methods and Results Using top-down proteomics (mass-spectrometric analysis of intact proteins), we analyzed paired serum samples from 150 CARDIA (Coronary Artery Risk Development in Young Adults) study participants from year 20 and 25 exams. Measuring 15 apoAI and 9 apoAII proteoforms, 6 of which carried novel post-translational modifications, we quantified associations between percent proteoform abundance and key cardiometabolic indices. Canonical (unmodified) apoAI had inverse associations with HDL cholesterol and HDL-cholesterol efflux, and positive associations with obesity indices (body mass index, waist circumference), and triglycerides, whereas glycated apoAI showed positive associations with serum glucose and diabetes mellitus. Fatty-acid‒modified ApoAI proteoforms had positive associations with HDL cholesterol and efflux, and inverse associations with obesity indices and triglycerides. Truncated and dimerized proteoforms of apoAII were associated with HDL cholesterol (positively) and obesity indices (inversely). Several proteoforms had no significant associations with phenotype. Conclusions Associations between apoAI and AII and cardiometabolic indices are proteoform-specific. These results provide "proof-of-concept" that precise chemical characterization of human apolipoproteins will yield improved insights into the complex pathways through which proteins signify and mediate health and disease.

Identification and Characterization of Novel Proteins from Arizona Bark Scorpion Venom That Inhibit Nav1.8, a Voltage-Gated Sodium Channel Regulator of Pain Signaling.

The voltage-gated sodium channel Nav1.8 is linked to neuropathic and inflammatory pain, highlighting the potential to serve as a drug target. However, the biophysical mechanisms that regulate Nav1.8 activation and inactivation gating are not completely understood. Progress has been hindered by a lack of biochemical tools for examining Nav1.8 gating mechanisms. Arizona bark scorpion (Centruroides sculpturatus) venom proteins inhibit Nav1.8 and block pain in grasshopper mice (Onychomys torridus). These proteins provide tools for examining Nav1.8 structure-activity relationships. To identify proteins that inhibit Nav1.8 activity, venom samples were fractioned using liquid chromatography (reversed-phase and ion exchange). A recombinant Nav1.8 clone expressed in ND7/23 cells was used to identify subfractions that inhibited Nav1.8 Na+ current. Mass-spectrometry-based bottom-up proteomic analyses identified unique peptides from inhibitory subfractions. A search of the peptides against the AZ bark scorpion venom gland transcriptome revealed four novel proteins between 40 and 60% conserved with venom proteins from scorpions in four genera (Centruroides, Parabuthus, Androctonus, and Tityus). Ranging from 63 to 82 amino acids, each primary structure includes eight cysteines and a "CXCE" motif, where X = an aromatic residue (tryptophan, tyrosine, or phenylalanine). Electrophysiology data demonstrated that the inhibitory effects of bioactive subfractions can be removed by hyperpolarizing the channels, suggesting that proteins may function as gating modifiers as opposed to pore blockers.

Oncogenic KRAS creates an aspartate metabolism signature in colorectal cancer cells.

Oncogenic mutations in the KRAS gene are found in 30-50% of colorectal cancers (CRC), and recent findings have demonstrated independent and nonredundant roles for wild-type and mutant KRAS alleles in governing signaling and metabolism. Here, we quantify proteomic changes manifested by KRAS mutation and KRAS allele loss in isogenic cell lines. We show that the expression of KRASG13D upregulates aspartate metabolizing proteins including PCK1, PCK2, ASNS, and ASS1. Furthermore, differential expression analyses of transcript-level data from CRC tumors identified the upregulation of urea cycle enzymes in CRC. We find that expression of ASS1 supports colorectal cancer cell proliferation and promotes tumor formation in vitro. We show that loss of ASS1 can be rescued with high levels of several metabolites.

Mass spectrometry-based proteomic platforms for better understanding of SARS-CoV-2 induced pathogenesis and potential diagnostic approaches.

While protein-protein interaction is the first step of the SARS-CoV-2 infection, recent comparative proteomic profiling enabled the identification of over 11,000 protein dynamics, thus providing a comprehensive reflection of the molecular mechanisms underlying the cellular system in response to viral infection. Here we summarize and rationalize the results obtained by various mass spectrometry (MS)-based proteomic approaches applied to the functional characterization of proteins and pathways associated with SARS-CoV-2-mediated infections in humans. Comparative analysis of cell-lines versus tissue samples indicates that our knowledge in proteome profile alternation in response to SARS-CoV-2 infection is still incomplete and the tissue-specific response to SARS-CoV-2 infection can probably not be recapitulated efficiently by in vitro experiments. However, regardless of the viral infection period, sample types, and experimental strategies, a thorough cross-comparison of the recently published proteome, phosphoproteome, and interactome datasets led to the identification of a common set of proteins and kinases associated with PI3K-Akt, EGFR, MAPK, Rap1, and AMPK signaling pathways. Ephrin receptor A2 (EPHA2) was identified by 11 studies including all proteomic platforms, suggesting it as a potential future target for SARS-CoV-2 infection mechanisms and the development of new therapeutic strategies. We further discuss the potentials of future proteomics strategies for identifying prognostic SARS-CoV-2 responsive age-, gender-dependent, tissue-specific protein targets.

Sequential Ion-Ion Reactions for Enhanced Gas-Phase Sequencing of Large Intact Proteins in a Tribrid Orbitrap Mass Spectrometer.

Obtaining extensive sequencing of an intact protein is essential in order to simultaneously determine both the nature and exact localization of chemical and genetic modifications which distinguish different proteoforms arising from the same gene. To effectively achieve such characterization, it is necessary to take advantage of the analytical potential offered by the top-down mass spectrometry approach to protein sequence analysis. However, as a protein increases in size, its gas-phase dissociation produces overlapping, low signal-to-noise fragments. The application of advanced ion dissociation techniques such as electron transfer dissociation (ETD) and ultraviolet photodissociation (UVPD) can improve the sequencing results compared to slow-heating techniques such as collisional dissociation; nonetheless, even ETD- and UVPD-based approaches have thus far fallen short in their capacity to reliably enable extensive sequencing of proteoforms ≥30 kDa. To overcome this issue, we have applied proton transfer charge reduction (PTCR) to limit signal overlap in tandem mass spectra (MS2) produced by ETD (alone or with supplemental ion activation, EThcD). Compared to conventional MS2 experiments, following ETD/EThcD MS2 with PTCR MS3 prior to m/z analysis of deprotonated product ions in the Orbitrap mass analyzer proved beneficial for the identification of additional large protein fragments (≥10 kDa), thus improving the overall sequencing and in particular the coverage of the central portion of all four analyzed proteins spanning from 29 to 56 kDa. Specifically, PTCR-based data acquisition led to 39% sequence coverage for the 56 kDa glutamate dehydrogenase, which was further increased to 44% by combining fragments obtained via HCD followed by PTCR MS3.

Interlaboratory Study for Characterizing Monoclonal Antibodies by Top-Down and Middle-Down Mass Spectrometry.

The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product's primary structure accurately and reproducibly. The major objective of the present study is to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up (BU) approaches with regard to confirming protein integrity, sequencing variable domains, avoiding artifacts, and revealing modifications and their locations. We also aim to gather information on the common TD/MD MS methods and practices in the field. A panel of three mAbs was selected and centrally provided to 20 laboratories worldwide for the analysis: Sigma mAb standard (SiLuLite), NIST mAb standard, and the therapeutic mAb Herceptin (trastuzumab). Various MS instrument platforms and ion dissociation techniques were employed. The present study confirms that TD/MD MS tools are available in laboratories worldwide and provide complementary information to the BU approach that can be crucial for comprehensive mAb characterization. The current limitations, as well as possible solutions to overcome them, are also outlined. A primary limitation revealed by the results of the present study is that the expert knowledge in both experiment and data analysis is indispensable to practice TD/MD MS.

Elucidating Proteoform Dynamics Underlying the Senescence Associated Secretory Phenotype.

Primary diploid cells exit the cell cycle in response to exogenous stress or oncogene activation through a process known as cellular senescence. This cell-autonomous tumor-suppressive mechanism is also a major mechanism operative in organismal aging. To date, temporal aspects of senescence remain understudied. Therefore, we use quantitative proteomics to investigate changes following forced HRASG12V expression and induction of senescence across 1 week in normal diploid fibroblasts. We demonstrate that global intracellular proteomic changes correlate with the emergence of the senescence-associated secretory phenotype and the switch to robust cell cycle exit. The senescence secretome reinforces cell cycle exit, yet is largely detrimental to tissue homeostasis. Previous studies of secretomes rely on ELISA, bottom-up proteomics or RNA-seq. To date, no study to date has examined the proteoform complexity of secretomes to elucidate isoform-specific, post-translational modifications or regulated cleavage of signal peptides. Therefore, we use a quantitative top-down proteomics approach to define the molecular complexity of secreted proteins <30 kDa. We identify multiple forms of immune regulators with known activities and affinities such as distinct forms of interleukin-8, as well as GROα and HMGA1, and temporally resolve secreted proteoform dynamics. Together, our work demonstrates the complexity of the secretome past individual protein accessions and provides motivation for further proteoform-resolved measurements of the secretome.

Thorough Performance Evaluation of 213 nm Ultraviolet Photodissociation for Top-down Proteomics.

Top-down proteomics studies intact proteoform mixtures and offers important advantages over more common bottom-up proteomics technologies, as it avoids the protein inference problem. However, achieving complete molecular characterization of investigated proteoforms using existing technologies remains a fundamental challenge for top-down proteomics. Here, we benchmark the performance of ultraviolet photodissociation (UVPD) using 213 nm photons generated by a solid-state laser applied to the study of intact proteoforms from three organisms. Notably, the described UVPD setup applies multiple laser pulses to induce ion dissociation, and this feature can be used to optimize the fragmentation outcome based on the molecular weight of the analyzed biomolecule. When applied to complex proteoform mixtures in high-throughput top-down proteomics, 213 nm UVPD demonstrated a high degree of complementarity with the most employed fragmentation method in proteomics studies, higher-energy collisional dissociation (HCD). UVPD at 213 nm offered higher average proteoform sequence coverage and degree of proteoform characterization (including localization of post-translational modifications) than HCD. However, previous studies have shown limitations in applying database search strategies developed for HCD fragmentation to UVPD spectra which contains up to nine fragment ion types. We therefore performed an analysis of the different UVPD product ion type frequencies. From these data, we developed an ad hoc fragment matching strategy and determined the influence of each possible ion type on search outcomes. By paring down the number of ion types considered in high-throughput UVPD searches from all types down to the four most abundant, we were ultimately able to achieve deeper proteome characterization with UVPD. Lastly, our detailed product ion analysis also revealed UVPD cleavage propensities and determined the presence of a product ion produced specifically by 213 nm photons. All together, these observations could be used to better elucidate UVPD dissociation mechanisms and improve the utility of the technique for proteomic applications.

Proton Transfer Charge Reduction Enables High-Throughput Top-Down Analysis of Large Proteoforms.

Despite the recent technological advances in Fourier transform mass spectrometry (FTMS) instrumentation, top-down proteomics (TDP) is currently mostly applied to the characterization of proteoforms <30 kDa due to the poor performance of high-resolution FTMS for the analysis of larger proteoforms and the high complexity of intact proteomes in the 30-60 kDa mass range. Here, we propose a novel data acquisition method based on ion-ion proton transfer, herein termed proton transfer charge reduction (PTCR), to investigate large proteoforms of Pseudomonas aeruginosa in a high-throughput fashion. We designed a targeted data acquisition strategy, named tPTCR, which applies two consecutive gas phase fractionation steps for obtaining intact precursor masses: first, a narrow (1.5 m/z-wide) quadrupole filter m/z transmission window is used to select a subset of charge states from all ionized proteoform cations; second, this aliquot of protein cations is subjected to PTCR in order to reduce their average charge state: upon m/z analysis in an Orbitrap, proteoform mass spectra with minimal m/z peak overlap and easy-to-interpret charge state distributions are obtained, simplifying the proteoform mass calculation. Subsequently, the same quadrupole-selected narrow m/z region of analytes is subjected to collisional dissociation to obtain proteoform sequence information, which used in combination with intact mass information leads to proteoform identification through an off-line database search. The newly proposed method was benchmarked against the previously developed "medium/high" data-dependent acquisition strategy and doubled the number of UniProt entries and proteoforms >30 kDa identified on the liquid chromatography time scale.

Direct measurement of light and heavy antibody chains using ion mobility and middle-down mass spectrometry.

The analysis of monoclonal antibodies (mAbs) by a middle-down mass spectrometry (MS) approach is a growing field that attracts the attention of many researchers and biopharmaceutical companies. Usually, liquid fractionation techniques are used to separate mAbs polypeptides chains before MS analysis. Gas-phase fractionation techniques such as high-field asymmetric waveform ion mobility spectrometry (FAIMS) can replace liquid-based separations and reduce both analysis time and cost. Here, we present a rapid FAIMS tandem MS method capable of characterizing the polypeptide sequence of mAbs light and heavy chains in an unprecedented, easy, and fast fashion. This new method uses commercially available instruments and takes ~24 min, which is 40-60% faster than regular liquid chromatography-MS/MS analysis, to acquire fragmentation data using different dissociation methods.