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.

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.

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.

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.

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.

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.

Best practices and benchmarks for intact protein analysis for top-down mass spectrometry.

One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Top-down mass spectrometry enables the analysis of intact proteins and proteoforms. Here members of the Consortium for Top-Down Proteomics provide a decision tree that guides researchers to robust protocols for mass analysis of intact proteins (antibodies, membrane proteins and others) from mixtures of varying complexity. We also present cross-platform analytical benchmarks using a protein standard sample, to allow users to gauge their proficiency.

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.

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.

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.

Precise characterization of KRAS4b proteoforms in human colorectal cells and tumors reveals mutation/modification cross-talk.

Mutations of the KRAS gene are found in human cancers with high frequency and result in the constitutive activation of its protein products. This leads to aberrant regulation of downstream pathways, promoting cell survival, proliferation, and tumorigenesis that drive cancer progression and negatively affect treatment outcomes. Here, we describe a workflow that can detect and quantify mutation-specific consequences of KRAS biochemistry, namely linked changes in posttranslational modifications (PTMs). We combined immunoaffinity enrichment with detection by top-down mass spectrometry to discover and quantify proteoforms with or without the Gly13Asp mutation (G13D) specifically in the KRAS4b isoform. The workflow was applied first to isogenic KRAS colorectal cancer (CRC) cell lines and then to patient CRC tumors with matching KRAS genotypes. In two cellular models, a direct link between the knockout of the mutant G13D allele and the complete nitrosylation of cysteine 118 of the remaining WT KRAS4b was observed. Analysis of tumor samples quantified the percentage of mutant KRAS4b actually present in cancer tissue and identified major differences in the levels of C-terminal carboxymethylation, a modification critical for membrane association. These data from CRC cells and human tumors suggest mechanisms of posttranslational regulation that are highly context-dependent and which lead to preferential production of specific KRAS4b proteoforms.

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.

Defining the NSD2 interactome: PARP1 PARylation reduces NSD2 histone methyltransferase activity and impedes chromatin binding.

NSD2 is a histone methyltransferase that specifically dimethylates histone H3 lysine 36 (H3K36me2), a modification associated with gene activation. Dramatic overexpression of NSD2 in t(4;14) multiple myeloma (MM) and an activating mutation of NSD2 discovered in acute lymphoblastic leukemia are significantly associated with altered gene activation, transcription, and DNA damage repair. The partner proteins through which NSD2 may influence critical cellular processes remain poorly defined. In this study, we utilized proximity-based labeling (BioID) combined with label-free quantitative MS to identify high confidence NSD2 interacting partners in MM cells. The top 24 proteins identified were involved in maintaining chromatin structure, transcriptional regulation, RNA pre-spliceosome assembly, and DNA damage. Among these, an important DNA damage regulator, poly(ADP-ribose) polymerase 1 (PARP1), was discovered. PARP1 and NSD2 have been found to be recruited to DNA double strand breaks upon damage and H3K36me2 marks are enriched at damage sites. We demonstrate that PARP1 regulates NSD2 via PARylation upon oxidative stress. In vitro assays suggest the PARylation significantly reduces NSD2 histone methyltransferase activity. Furthermore, PARylation of NSD2 inhibits its ability to bind to nucleosomes and further get recruited at NSD2-regulated genes, suggesting PARP1 regulates NSD2 localization and H3K36me2 balance. This work provides clear evidence of cross-talk between PARylation and histone methylation and offers new directions to characterize NSD2 function in DNA damage response, transcriptional regulation, and other pathways.

Top-down characterization of endogenous protein complexes with native proteomics.

Protein complexes exhibit great diversity in protein membership, post-translational modifications and noncovalent cofactors, enabling them to function as the actuators of many important biological processes. The exposition of these molecular features using current methods lacks either throughput or molecular specificity, ultimately limiting the use of protein complexes as direct analytical targets in a wide range of applications. Here, we apply native proteomics, enabled by a multistage tandem MS approach, to characterize 125 intact endogenous complexes and 217 distinct proteoforms derived from mouse heart and human cancer cell lines in discovery mode. The native conditions preserved soluble protein-protein interactions, high-stoichiometry noncovalent cofactors, covalent modifications to cysteines, and, remarkably, superoxide ligands bound to the metal cofactor of superoxide dismutase 2. These data enable precise compositional analysis of protein complexes as they exist in the cell and demonstrate a new approach that uses MS as a bridge to structural biology.

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.

Structural Analysis of Monoclonal Antibodies by Ultrahigh Resolution MALDI In-Source Decay FT-ICR Mass Spectrometry.

The emergence of complex protein therapeutics in general and monoclonal antibodies (mAbs) in particular have stimulated analytical chemists to develop new methods and strategies for their structural characterization. Mass spectrometry plays a key role in providing information on the primary amino acid sequence, post-translational modifications, and other structure characteristics that must be monitored during the manufacturing process and subsequent quality control assessment. In this study, we present a novel method that allows structural characterization of mAbs based on MALDI in-source decay (ISD) fragmentation, coupled with Fourier transform ion cyclotron resonance (FT-ICR) MS. The method benefits from higher resolution of absorption mode FT mass spectra, compared to magnitude mode, which enables simultaneous identification of ISD fragments from both the heavy and light chains with a higher confidence in a wide mass range up to m/ z 13 500. This method was applied to two standard mAbs, namely NIST mAb and trastuzumab, in preparation for method application in an interlaboratory study on mAbs structural analysis coordinated by the Consortium for Top-Down Proteomics. Extensive sequence coverage was obtained from the middle-down analysis (IdeS- and GingisKHAN-digested mAbs) that complemented the top-down analysis of intact mAbs. In addition, MALDI FT-ICR MS of IdeS-digested mAbs allowed isotopic-level profiling of proteoforms with regard to heavy chain N-glycosylation.

An informatic framework for decoding protein complexes by top-down mass spectrometry.

Efforts to map the human protein interactome have resulted in information about thousands of multi-protein assemblies housed in public repositories, but the molecular characterization and stoichiometry of their protein subunits remains largely unknown. Here, we report a computational search strategy that supports hierarchical top-down analysis for precise identification and scoring of multi-proteoform complexes by native mass spectrometry.

Chemical-Mediated Digestion: An Alternative Realm for Middle-down Proteomics?

Protein digestion in mass spectrometry (MS)-based bottom-up proteomics targets mainly lysine and arginine residues, yielding primarily 0.6-3 kDa peptides for the proteomes of organisms of all major kingdoms. Recent advances in MS technology enable analysis of complex mixtures of increasingly longer (>3 kDa) peptides in a high-throughput manner supporting the development of a middle-down proteomics (MDP) approach. Generating longer peptides is a paramount step in launching an MDP pipeline, but the quest for the selection of a cleaving agent that would provide the desired 3-15 kDa peptides remains open. Recent bioinformatics studies have shown that cleavage at the rarely occurring amino acid residues such as methionine (Met), tryptophan (Trp), or cysteine (Cys) would be suitable for MDP approach. Interestingly, chemical-mediated proteolytic cleavages uniquely allow targeting these rare amino acids, for which no specific proteolytic enzymes are known. Herein, as potential candidates for MDP-grade proteolysis, we have investigated the performance of chemical agents previously reported to target primarily Met, Trp, and Cys residues: CNBr, BNPS-Skatole (3-bromo-3-methyl-2-(2-nitrophenyl)sulfanylindole), and NTCB (2-nitro-5-thiobenzoic acid), respectively. Figures of merit such as digestion reproducibility, peptide size distribution, and occurrence of side reactions are discussed. The NTCB-based MDP workflow has demonstrated particularly attractive performance, and NTCB is put forward here as a potential cleaving agent for further MDP development.

A Targeted, Differential Top-Down Proteomic Methodology for Comparison of ApoA-I Proteoforms in Individuals with High and Low HDL Efflux Capacity.

Top-down proteomics (TDP) allows precise determination/characterization of the different proteoforms derived from the expression of a single gene. In this study, we targeted apolipoprotein A-I (ApoA-I), a mediator of high-density-lipoprotein cholesterol efflux (HDL-E), which is inversely associated with coronary heart disease risk. Absolute ApoA-I concentration and allelic variation only partially explain interindividual HDL-E variation. Therefore, we hypothesize that differences in HDL-E are associated with the abundances of different ApoA-I proteoforms. Here, we present a targeted TDP methodology to characterize ApoA-I proteoforms in serum samples and compare their abundances between individuals. We characterized 18 ApoA-I proteoforms using selected-ion monitoring coupled to electron-transfer dissociation mass spectrometry. We then compared the abundances of these proteoforms between two groups of four participants, representing the individuals with highest and lowest HDL-E values within the Chicago Healthy Aging Study ( n = 420). Six proteoforms showed significantly ( p < 0.0005) higher intensity in high HDL-E individuals: canonical ApoA-I [fold difference (fd) = 1.17], carboxymethylated ApoA-I (fd = 1.24) and, with highest difference, four fatty acylated forms: palmitoylated (fd = 2.16), oleoylated (fd = 2.08), arachidonoylated (fd = 2.31) and one bearing two modifications: palmitoylation and truncation (fd = 2.13). These results demonstrate translational potential for targeted TDP in revealing, with high sensitivity, associations between interindividual proteoform variation and physiological differences underlying disease risk.

Multiplexed Middle-Down Mass Spectrometry as a Method for Revealing Light and Heavy Chain Connectivity in a Monoclonal Antibody.

Pairing light and heavy chains in monoclonal antibodies (mAbs) using top-down (TD) or middle-down (MD) mass spectrometry (MS) may complement the sequence information on single chains provided by high-throughput genomic sequencing and bottom-up proteomics, favoring the rational selection of drug candidates. The 50 kDa F(ab) subunits of mAbs are the smallest structural units that contain the required information on chain pairing. These subunits can be enzymatically produced from whole mAbs and interrogated in their intact form by TD/MD MS approaches. However, the high structural complexity of F(ab) subunits requires increased sensitivity of the modern TD/MD MS for a comprehensive structural analysis. To address this and similar challenges, we developed and applied a multiplexed TD/MD MS workflow based on spectral averaging of tandem mass spectra (MS/MS) across multiple liquid chromatography (LC)-MS/MS runs acquired in reduced or full profile mode using an Orbitrap Fourier transform mass spectrometer (FTMS). We first benchmark the workflow using myoglobin as a reference protein, and then validate it for the analysis of the 50 kDa F(ab) subunit of a therapeutic mAb, trastuzumab. Obtained results confirm the envisioned benefits in terms of increased signal-to-noise ratio of product ions from utilizing multiple LC-MS/MS runs for TD/MD protein analysis using mass spectral averaging. The workflow performance is compared with the earlier introduced multiplexed TD/MD MS workflow based on transient averaging in Orbitrap FTMS. For the latter, we also report on enabling absorption mode FT processing and demonstrate its comparable performance to the enhanced FT (eFT) spectral representation.