Towards a universal method for middle-down analysis of antibodies via proton transfer charge reduction-Orbitrap mass spectrometry.

Modern mass spectrometry technology allows for extensive sequencing of the ~ 25 kDa subunits of monoclonal antibodies (mAbs) produced by IdeS proteolysis followed by disulfide bond reduction, an approach known as middle-down mass spectrometry (MD MS). However, the spectral congestion of tandem mass spectra of large polypeptides dramatically complicates fragment ion assignment. Here, we report the development and benchmark of an MD MS strategy based on the combination of different ion fragmentation techniques with proton transfer charge reduction (PTCR) to simplify the gas-phase sequencing of mAb subunits. Applied on the liquid chromatography time scale using an Orbitrap Tribrid mass spectrometer, PTCR produces easy-to-interpret mass spectra with limited ion signal overlap. We demonstrate that the accurate estimation of the number of charges submitted to the Orbitrap mass analyzer after PTCR allows for the detection of charge-reduced product ions over a wide mass-over-charge (m/z) window with low parts per million m/z accuracy. Therefore, PTCR-based MD MS analysis increases not only sequence coverage, number of uniquely identified fragments, and number of assigned complementary ion pairs, but also the general confidence in the assignment of subunit fragments. This data acquisition method can be readily applied to any class of mAbs without an apparent need for optimization, and benefits from the high resolving power of the Orbitrap mass analyzer to return sequence coverage of individual subunits exceeding 80% in a single run, and > 90% when just two experiments are combined.

Top-down mass spectrometry analysis of capsid proteins of recombinant adeno-associated virus using multiple ion activations and proton transfer charge reduction.

Adeno-associated viruses (AAVs) are common vectors for emerging gene therapies due to their lack of pathogenicity in humans. Here, we present our investigation of the viral proteins (i.e., VP1, VP2, and VP3) of the capsid of AAVs via top-down mass spectrometry (MS). These proteins, ranging from 59 to 81 kDa, were chromatographically separated using hydrophilic interaction liquid chromatography and characterized in the gas-phase by high-resolution Orbitrap Fourier transform MS. Complementary ion dissociation methods were utilized to improve the overall sequence coverage. By reducing the overlap of product ion signals via proton transfer charge reduction on the Orbitrap Ascend BioPharma Tribrid mass spectrometer, the sequence coverage of each VP was significantly increased, reaching up to ∼40% in the case of VP3. These results showcase the improvements in the sequencing of proteins >30 kDa that can be achieved by manipulating product ions via gas-phase reactions to obtain easy-to-interpret fragmentation mass spectra.

Characterization of the Antibody Response to SARS-CoV-2 Infection in COVID-19 Transplant versus Nontransplant Recipients by Ig-MS.

Solid organ transplant recipients with immunosuppressant regimens to prevent rejection are less able to mount effective immune responses to pathogenic infection. Here, we apply a recently reported mass spectrometry-based serological approach known as Ig-MS to characterize immune responses against infection with SARS-CoV-2 in cohorts of transplant recipients and immunocompetent controls, both at a single early time point following COVID-19 diagnosis as well as over the course of one-month postdiagnosis. We found that the antibody repertoires generated by transplant recipients against SARS-CoV-2 do not differ significantly compared to immunocompetent individuals with regard to repertoire titer, clonality, or glycan composition. Importantly, our study is the first to characterize the evolution of antibody glycan profiles in transplant recipients with COVID-19 disease, presenting evidence that the evolution of glycan composition in these immunocompromised individuals is similar to that in immunocompetent people.

Automated Immunoprecipitation, Sample Preparation, and Individual Ion Mass Spectrometry Platform for Proteoforms.

Charge detection mass spectrometry (CDMS) is a well-established technique that provides direct mass spectral outputs regardless of analyte heterogeneity or molecular weight. Over the past few years, it has been demonstrated that CDMS can be multiplexed on Orbitrap analyzers utilizing an integrated approach termed individual ion mass spectrometry (I2MS). To further increase adaptability, robustness, and throughput of this technique, here, we present a method that utilizes numerous integrated equipment components including a Kingfisher system, SampleStream platform, and Q Exactive mass spectrometer to provide a fully automated workflow for immunoprecipitation, sample preparation, injection, and subsequent I2MS acquisition. This automated workflow has been applied to a cohort of 58 test subjects to determine individualized patient antibody responses to SARS-CoV-2 antigens. Results from a range of serum donors include 37 subject I2MS spectra that contained a positive COVID-19 antibody response and 21 I2MS spectra that contained a negative COVID-19 antibody response. This high-throughput automated I2MS workflow can currently process over 100 samples per week and is general for making immunoprecipitation-MS workflows achieve proteoform resolution.

Top-Down Proteomics Identifies Plasma Proteoform Signatures of Liver Cirrhosis Progression.

Cirrhosis, advanced liver disease, affects 2-5 million Americans. While most patients have compensated cirrhosis and may be fairly asymptomatic, many decompensate and experience life-threatening complications such as gastrointestinal bleeding, confusion (hepatic encephalopathy), and ascites, reducing life expectancy from 12 to less than 2 years. Among patients with compensated cirrhosis, identifying patients at high risk of decompensation is critical to optimize care and reduce morbidity and mortality. Therefore, it is important to preferentially direct them towards specialty care which cannot be provided to all patients with cirrhosis. We used discovery Top-down Proteomics (TDP) to identify differentially expressed proteoforms (DEPs) in the plasma of patients with progressive stages of liver cirrhosis with the ultimate goal to identify candidate biomarkers of disease progression. In this pilot study, we identified 209 DEPs across three stages of cirrhosis (compensated, compensated with portal hypertension, and decompensated), of which 115 derived from proteins enriched in the liver at a transcriptional level and discriminated the three stages of cirrhosis. Enrichment analyses demonstrated DEPs are involved in several metabolic and immunological processes known to be impacted by cirrhosis progression. We have preliminarily defined the plasma proteoform signatures of cirrhosis patients, setting the stage for ongoing discovery and validation of biomarkers for early diagnosis, risk stratification, and disease monitoring.

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.

Target Identification of a Class of Pyrazolone Protein Aggregation Inhibitor Therapeutics for Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no cure, and current treatment options are very limited. Previously, we performed a high-throughput screen to identify small molecules that inhibit protein aggregation caused by a mutation in the gene that encodes superoxide dismutase 1 (SOD1), which is responsible for about 25% of familial ALS. This resulted in three hit series of compounds that were optimized over several years to give three compounds that were highly active in a mutant SOD1 ALS model. Here we identify the target of two of the active compounds (6 and 7) with the use of photoaffinity labeling, chemical biology reporters, affinity purification, proteomic analysis, and fluorescent/cellular thermal shift assays. Evidence is provided to demonstrate that these two pyrazolone compounds directly interact with 14-3-3-E and 14-3-3-Q isoforms, which have chaperone activity and are known to interact with mutant SOD1G93A aggregates and become insoluble in the subcellular JUNQ compartment, leading to apoptosis. Because protein aggregation is the hallmark of all neurodegenerative diseases, knowledge of the target compounds that inhibit protein aggregation allows for the design of more effective molecules for the treatment of ALS and possibly other neurodegenerative diseases.

Automated imaging and identification of proteoforms directly from ovarian cancer tissue.

The molecular identification of tissue proteoforms by top-down mass spectrometry (TDMS) is significantly limited by throughput and dynamic range. We introduce AutoPiMS, a single-ion MS based multiplexed workflow for top-down tandem MS (MS2) directly from tissue microenvironments in a semi-automated manner. AutoPiMS directly off human ovarian cancer sections allowed for MS2 identification of 73 proteoforms up to 54 kDa at a rate of <1 min per proteoform. AutoPiMS is directly interfaced with multifaceted proteoform imaging MS data modalities for the identification of proteoform signatures in tumor and stromal regions in ovarian cancer biopsies. From a total of ~1000 proteoforms detected by region-of-interest label-free quantitation, we discover 303 differential proteoforms in stroma versus tumor from the same patient. 14 of the top proteoform signatures are corroborated by MSI at 20 micron resolution including the differential localization of methylated forms of CRIP1, indicating the importance of proteoform-enabled spatial biology in ovarian cancer.

Immunocomplexed Antigen Capture and Identification by Native Top-Down Mass Spectrometry.

Antibody-antigen interactions are central to the immune response. Variation of protein antigens such as isoforms and post-translational modifications can alter their antibody binding sites. To directly connect the recognition of protein antigens with their molecular composition, we probed antibody-antigen complexes by using native tandem mass spectrometry. Specifically, we characterized the prominent peanut allergen Ara h 2 and a convergent IgE variable region discovered in patients who are allergic to peanuts. In addition to measuring the antigen-induced dimerization of IgE antibodies, we demonstrated how immunocomplexes can be isolated in the gas phase and activated to eject, identify, and characterize proteoforms of their bound antigens. Using tandem experiments, we isolated the ejected antigens and then fragmented them to identify their chemical composition. These results establish native top-down mass spectrometry as a viable platform for precise and thorough characterization of immunocomplexes to relate structure to function and enable the discovery of antigen proteoforms and their binding sites.

Real-Time Spectral Library Matching for Sample Multiplexed Quantitative Proteomics.

Sample multiplexed quantitative proteomics assays have proved to be a highly versatile means to assay molecular phenotypes. Yet, stochastic precursor selection and precursor coisolation can dramatically reduce the efficiency of data acquisition and quantitative accuracy. To address this, intelligent data acquisition (IDA) strategies have recently been developed to improve instrument efficiency and quantitative accuracy for both discovery and targeted methods. Toward this end, we sought to develop and implement a new real-time spectral library searching (RTLS) workflow that could enable intelligent scan triggering and peak selection within milliseconds of scan acquisition. To ensure ease of use and general applicability, we built an application to read in diverse spectral libraries and file types from both empirical and predicted spectral libraries. We demonstrate that RTLS methods enable improved quantitation of multiplexed samples, particularly with consideration for quantitation from chimeric fragment spectra. We used RTLS to profile proteome responses to small molecule perturbations and were able to quantify up to 15% more significantly regulated proteins in half the gradient time compared to traditional methods. Taken together, the development of RTLS expands the IDA toolbox to improve instrument efficiency and quantitative accuracy for sample multiplexed analyses.

Mass spectrometry characterization of antibodies at the intact and subunit levels: from targeted to large-scale analysis.

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 (e.g., 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 a priori knowledge.

Middle-Down Mass Spectrometry Reveals Activity-Modifying Phosphorylation Barcode in a Class C G Protein-Coupled Receptor.

G protein-coupled receptors (GPCRs) are the largest family of membrane receptors in humans. They mediate nearly all aspects of human physiology and thus are of high therapeutic interest. GPCR signaling is regulated in space and time by receptor phosphorylation. It is believed that different phosphorylation states are possible for a single receptor, and each encodes for unique signaling outcomes. Methods to determine the phosphorylation status of GPCRs are critical for understanding receptor physiology and signaling properties of GPCR ligands and therapeutics. However, common proteomic techniques have provided limited quantitative information regarding total receptor phosphorylation stoichiometry, relative abundances of isomeric modification states, and temporal dynamics of these parameters. Here, we report a novel middle-down proteomic strategy and parallel reaction monitoring (PRM) to quantify the phosphorylation states of the C-terminal tail of metabotropic glutamate receptor 2 (mGluR2). By this approach, we found that mGluR2 is subject to both basal and agonist-induced phosphorylation at up to four simultaneous sites with varying probability. Using a PRM tandem mass spectrometry methodology, we localized the positions and quantified the relative abundance of phosphorylations following treatment with an agonist. Our analysis showed that phosphorylation within specific regions of the C-terminal tail of mGluR2 is sensitive to receptor activation, and subsequent site-directed mutagenesis of these sites identified key regions which tune receptor sensitivity. This study demonstrates that middle-down purification followed by label-free quantification is a powerful, quantitative, and accessible tool for characterizing phosphorylation states of GPCRs and other challenging proteins.

Divergent Antibody Repertoires Found for Omicron versus Wuhan SARS-CoV-2 Strains Using Ig-MS.

SARS-CoV-2 Omicron (B.1.1.529) and its subvariants are currently the most common variants of concern worldwide, featuring numerous mutations in the spike protein and elsewhere that collectively make Omicron variants more transmissible and more resistant to antibody-mediated neutralization provided by vaccination, previous infections, and monoclonal antibody therapies than their predecessors. We recently reported the creation and characterization of Ig-MS, a new mass spectrometry-based serology platform that can define the repertoire of antibodies against an antigen of interest at single proteoform resolution. Here, we applied Ig-MS to investigate the evolution of plasma antibody repertoires against the receptor-binding domain (RBD) of SARS-CoV-2 in response to the booster shot and natural viral infection. We also assessed the capacity for antibody repertoires generated in response to vaccination and/or infection with the Omicron variant to bind to both Wuhan- and Omicron-RBDs. Our results show that (1) the booster increases antibody titers against both Wuhan- and Omicron- RBDs and elicits an Omicron-specific response and (2) vaccination and infection act synergistically in generating anti-RBD antibody repertoires able to bind both Wuhan- and Omicron-RBDs with variant-specific antibodies.

Venom characterization of the Brazilian Pampa snake Bothrops pubescens by top-down and bottom-up proteomics.

The envenomation from the Bothrops genus is characterized by systemic and local effects caused by the main toxin families in the venom. In Bothrops pubescens venom we were able to identify 89 protein groups belonging to 13 toxin families with the bottom-up proteomics approach and 40 unique proteoforms belonging to 6 toxin families with the top-down proteomics approach. We also identified multi-proteoform complexes of dimeric L-amino acid oxidase using native top-down mass spectrometry.

Mapping the Proteoform Landscape of Five Human Tissues.

A functional understanding of the human body requires structure-function studies of proteins at scale. The chemical structure of proteins is controlled at the transcriptional, translational, and post-translational levels, creating a variety of products with modulated functions within the cell. The term "proteoform" encapsulates this complexity at the level of chemical composition. Comprehensive mapping of the proteoform landscape in human tissues necessitates analytical techniques with increased sensitivity and depth of coverage. Here, we took a top-down proteomics approach, combining data generated using capillary zone electrophoresis (CZE) and nanoflow reversed-phase liquid chromatography (RPLC) hyphenated to mass spectrometry to identify and characterize proteoforms from the human lungs, heart, spleen, small intestine, and kidneys. CZE and RPLC provided complementary post-translational modification and proteoform selectivity, thereby enhancing the overall proteome coverage when used in combination. Of the 11,466 proteoforms identified in this study, 7373 (64%) were not reported previously. Large differences in the protein and proteoform level were readily quantified, with initial inferences about proteoform biology operative in the analyzed organs. Differential proteoform regulation of defensins, glutathione transferases, and sarcomeric proteins across tissues generate hypotheses about how they function and are regulated in human health and disease.

Rational Design, Synthesis, and Mechanism of (3S,4R)-3-Amino-4-(difluoromethyl)cyclopent-1-ene-1-carboxylic Acid: Employing a Second-Deprotonation Strategy for Selectivity of Human Ornithine Aminotransferase over GABA Aminotransferase.

Human ornithine aminotransferase (hOAT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that contains a similar active site to that of γ-aminobutyric acid aminotransferase (GABA-AT). Recently, pharmacological inhibition of hOAT was recognized as a potential therapeutic approach for hepatocellular carcinoma. In this work, we first studied the inactivation mechanisms of hOAT by two well-known GABA-AT inactivators (CPP-115 and OV329). Inspired by the inactivation mechanistic difference between these two aminotransferases, a series of analogues were designed and synthesized, leading to the discovery of analogue 10b as a highly selective and potent hOAT inhibitor. Intact protein mass spectrometry, protein crystallography, and dialysis experiments indicated that 10b was converted to an irreversible tight-binding adduct (34) in the active site of hOAT, as was the unsaturated analogue (11). The comparison of kinetic studies between 10b and 11 suggested that the active intermediate (17b) was only generated in hOAT and not in GABA-AT. Molecular docking studies and pKa computational calculations highlighted the importance of chirality and the endocyclic double bond for inhibitory activity. The turnover mechanism of 10b was supported by mass spectrometric analysis of dissociable products and fluoride ion release experiments. Notably, the stopped-flow experiments were highly consistent with the proposed mechanism, suggesting a relatively slow hydrolysis rate for hOAT. The novel second-deprotonation mechanism of 10b contributes to its high potency and significantly enhanced selectivity for hOAT inhibition.

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.

Next-Generation Serology by Mass Spectrometry: Readout of the SARS-CoV-2 Antibody Repertoire.

Methods of antibody detection are used to assess exposure or immunity to a pathogen. Here, we present Ig-MS, a novel serological readout that captures the immunoglobulin (Ig) repertoire at molecular resolution, including entire variable regions in Ig light and heavy chains. Ig-MS uses recent advances in protein mass spectrometry (MS) for multiparametric readout of antibodies, with new metrics like Ion Titer (IT) and Degree of Clonality (DoC) capturing the heterogeneity and relative abundance of individual clones without sequencing of B cells. We applied Ig-MS to plasma from subjects with severe and mild COVID-19 and immunized subjects after two vaccine doses, using the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 as the bait for antibody capture. Importantly, we report a new data type for human serology, that could use other antigens of interest to gauge immune responses to vaccination, pathogens, or autoimmune disorders.