RESUMO
Immune monitoring in cancer immunotherapy involves screening CD8+ T-cell responses against neoantigens, the tumor-specific peptides presented by Major histocompatibility complex Class I (MHCI) on the cell surface. High-throughput immune monitoring requires methods to produce and characterize small quantities of thousands of MHCI-peptide complexes that may be tested for a patient's T-cell response. MHCI synthesis has been achieved using a photocleavable peptide that is exchanged by the neoantigen; however, assays that measure peptide exchange currently disassemble the complex prior to analysisâprecluding direct molecular characterization. Here, we use native mass spectrometry (MS) to profile intact recombinant MHCI complexes and directly measure peptide exchange. Coupled with size-exclusion chromatography or capillary-zone electrophoresis, the assay identified all tested human leukocyte antigen (HLA)/peptide combinations in the nanomole to picomole range with minimal run time, reconciling the synthetic and analytical requirements of MHCI-peptide screening with the downstream T-cell assays. We further show that the assay can be "multiplexed" by measuring exchange of multiple peptides simultaneously and also enables calculation of Vc50, a measure of gas-phase stability. Additionally, MHCI complexes were fragmented by top-down sequencing, demonstrating that the intact complex, peptide sequence, and their binding affinity can be determined in a single analysis. This screening tool for MHCI-neoantigen complexes represents a step toward the application of state-of-the-art MS technology in translational settings. Not only is this assay already informing on the viability of immunotherapy in practice, the platform also holds promise to inspire novel MS readouts for increasingly complex biomolecules used in the diagnosis and treatment of disease.
Assuntos
Antígenos de Histocompatibilidade Classe I , Peptídeos , Humanos , Antígenos de Histocompatibilidade Classe I/metabolismo , Peptídeos/química , Espectrometria de Massas , Antígenos HLA , Antígenos de NeoplasiasRESUMO
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.
Assuntos
Proteômica/métodos , Raios Ultravioleta , Animais , Anidrases Carbônicas , Células Cultivadas , Cromatografia Líquida , Fibroblastos , Proteínas Fúngicas , Humanos , Camundongos , Miócitos Cardíacos , Mioglobina , Fótons , Pseudomonas aeruginosa , Espectrometria de Massas em Tandem , UbiquitinaRESUMO
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.
RESUMO
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.
Assuntos
Complexos Multiproteicos/química , Multimerização Proteica , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Complexos Multiproteicos/genética , Conformação Proteica , Processamento de Proteína Pós-Traducional , Subunidades Proteicas/química , Subunidades Proteicas/genéticaRESUMO
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 13â¯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.
Assuntos
Anticorpos Monoclonais/análise , Análise de Fourier , Conformação Proteica , Processamento de Proteína Pós-Traducional , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Cromatografia Líquida/métodos , Proteoma/análise , Prótons , Pseudomonas aeruginosa/metabolismo , Software , Espectrometria de Massas em Tandem/métodos , Isoformas de ProteínasRESUMO
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.
Assuntos
Espectrometria de Massas/métodos , Proteólise , Proteômica/métodos , Aminoácidos , Indicadores e Reagentes , Peso Molecular , Peptídeos/análise , Peptídeos/química , TiocianatosRESUMO
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.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Leves de Imunoglobulina/química , Espectrometria de Massas em Tandem/métodos , Trastuzumab/química , Animais , Cavalos , Estrutura Molecular , Mioglobina/química , ProteóliseRESUMO
Targeted top-down (TD) and middle-down (MD) mass spectrometry (MS) offer reduced sample manipulation during protein analysis, limiting the risk of introducing artifactual modifications to better capture sequence information on the proteoforms present. This provides some advantages when characterizing biotherapeutic molecules such as monoclonal antibodies, particularly for the class of biosimilars. Here, we describe the results obtained analyzing a monoclonal IgG1, either in its â¼150 kDa intact form or after highly specific digestions yielding â¼25 and â¼50 kDa subunits, using an Orbitrap mass spectrometer on a liquid chromatography (LC) time scale with fragmentation from ion-photon, ion-ion, and ion-neutral interactions. Ultraviolet photodissociation (UVPD) used a new 213 nm solid-state laser. Alternatively, we applied high-capacity electron-transfer dissociation (ETD HD), alone or in combination with higher energy collisional dissociation (EThcD). Notably, we verify the degree of complementarity of these ion activation methods, with the combination of 213 nm UVPD and ETD HD producing a new record sequence coverage of â¼40% for TD MS experiments. The addition of EThcD for the >25 kDa products from MD strategies generated up to 90% of complete sequence information in six LC runs. Importantly, we determined an optimal signal-to-noise threshold for fragment ion deconvolution to suppress false positives yet maximize sequence coverage and implemented a systematic validation of this process using the new software TDValidator. This rigorous data analysis should elevate confidence for assignment of dense MS2 spectra and represents a purposeful step toward the application of TD and MD MS for deep sequencing of monoclonal antibodies.
Assuntos
Antineoplásicos Imunológicos/química , Imunoglobulina G/química , Espectrometria de Massas/métodos , Rituximab/química , Análise de Sequência de Proteína/métodos , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Cromatografia Líquida/métodos , Íons/químicaRESUMO
Lipidomes undergo permanent extensive remodeling, but how the turnover rate differs between lipid classes and molecular species is poorly understood. We employed metabolic 15N labeling and shotgun ultra-high-resolution mass spectrometry (sUHR) to quantify the absolute (molar) abundance and determine the turnover rate of glycerophospholipids and sphingolipids by direct analysis of total lipid extracts. sUHR performed on a commercial Orbitrap Elite instrument at the mass resolution of 1.35 × 106 (m/z 200) baseline resolved peaks of 13C isotopes of unlabeled and monoisotopic peaks of 15N labeled lipids (Δm = 0.0063 Da). Therefore, the rate of metabolic 15N labeling of individual lipid species could be determined without compromising the scope, accuracy, and dynamic range of full-lipidome quantitative shotgun profiling. As a proof of concept, we employed sUHR to determine the lipidome composition and fluxes of 62 nitrogen-containing membrane lipids in human hepatoma HepG2 cells.
Assuntos
Glicerofosfolipídeos/análise , Isótopos de Nitrogênio/metabolismo , Esfingolipídeos/análise , Isótopos de Carbono , Análise de Fourier , Glicerofosfolipídeos/metabolismo , Células Hep G2 , Humanos , Marcação por Isótopo , Cinética , Espectrometria de Massas/métodos , Esfingolipídeos/metabolismoRESUMO
A limited amount and extreme concentration variability of proteomic-related samples require efficient analyte preconcentration and purification prior to the mass spectrometry (MS)-based analysis. Preferably, these steps should be coupled online with chosen fractionation and detection techniques for the minimization of the sample loss. To realize such sample pretreatment, herein, an on-chip solid-phase extraction-gradient elution-tandem mass spectrometry (SPE-GEMS/MS) is introduced. This technique combines in a microfluidic format online sample preconcentration/purification on SPE sorbent with further fractionation and MS/MS analysis. C8-functionalized mesoporous magnetic microspheres are chosen as a sorbent, spatially confined with an applied magnetic field. They ensure a selective enrichment and analysis of large hydrophobic peptides (2.5-7 kDa), matching the desired mass bin of the extended bottom-up proteomic (eBUP, 3-7 kDa) approach. Within less than 35 min and without additional sample purification, SPE-GEMS/MS provided 66.5% of protein sequence coverage from 75 fmol of BSA tryptic digest. Analysis of only 33 fmol of a single monoclonal antibody, digested with secreted aspartic protease 9 (Sap9) to large peptides, yielded 80% of its sequence coverage. A more complex equimolar mixture of six antibodies (55 fmol each), submitted to Sap9 proteolysis, was also successfully processed by SPE-GEMS/MS, resulting in 50-67% of the total antibody sequence coverage. Importantly, for all antibodies, unique peptides containing complementarity determining regions were detected for both heavy and light chains, leading to a correct identification of mixture components despite their high sequence homology. Moreover, SPE-GEMS/MS microchip and chosen magnetic sorbent are cost-effective and can be produced and operated in a disposable manner. Therefore, the present technique could be potentially suitable for a high throughput sequencing of monoclonal antibodies and rapid eBUP-based structural protein analysis, especially when only a limited sample amount is available.
Assuntos
Campos Magnéticos , Nanopartículas de Magnetita/química , Microesferas , Peptídeos/análise , Análise de Sequência de Proteína/métodos , Dióxido de Silício/química , Espectrometria de Massas em Tandem , Tamanho da Partícula , Porosidade , Proteômica , Extração em Fase Sólida , Propriedades de SuperfícieRESUMO
Despite the recent advances in structural analysis of monoclonal antibodies with bottom-up, middle-down, and top-down mass spectrometry (MS), further improvements in analysis accuracy, depth, and speed are needed. The remaining challenges include quantitatively accurate assignment of post-translational modifications, reduction of artifacts introduced during sample preparation, increased sequence coverage per liquid chromatography (LC) MS experiment, and ability to extend the detailed characterization to simple antibody cocktails and more complex antibody mixtures. Here, we evaluate the recently introduced extended bottom-up proteomics (eBUP) approach based on proteolysis with secreted aspartic protease 9, Sap9, for analysis of monoclonal antibodies. Key findings of the Sap9-based proteomics analysis of a single antibody include: (i) extensive antibody sequence coverage with up to 100% for the light chain and up to 99-100% for the heavy chain in a single LC-MS run; (ii) connectivity of complementarity-determining regions (CDRs) via Sap9-produced large proteolytic peptides (3.4 kDa on average) containing up to two CDRs per peptide; (iii) reduced artifact introduction (e. g., deamidation) during proteolysis with Sap9 compared to conventional bottom-up proteomics workflows. The analysis of a mixture of six antibodies via Sap9-based eBUP produced comparable results. Due to the reasons specified above, Sap9-produced proteolytic peptides improve the identification confidence of antibodies from the mixtures compared to conventional bottom-up proteomics dealing with shorter proteolytic peptides.
Assuntos
Anticorpos Monoclonais/química , Ácido Aspártico Endopeptidases/química , Proteínas Fúngicas/química , Imunoglobulina G/química , Peptídeos/isolamento & purificação , Proteômica/métodos , Ácido Aspártico Endopeptidases/isolamento & purificação , Candida albicans/química , Candida albicans/enzimologia , Cromatografia Líquida , Regiões Determinantes de Complementaridade , Proteínas Fúngicas/isolamento & purificação , Humanos , Espectrometria de Massas , ProteóliseRESUMO
Established in recent years as an important approach to unraveling the heterogeneity of intact monoclonal antibodies, native mass spectrometry has been rarely utilized for sequencing these complex biomolecules via tandem mass spectrometry. Typically, top-down mass spectrometry has been performed starting from highly charged precursor ions obtained via electrospray ionization under denaturing conditions (i.e., in the presence of organic solvents and acidic pH). Here we systematically benchmark four distinct ion dissociation methodsânamely, higher-energy collisional dissociation, electron transfer dissociation, electron transfer dissociation/higher-energy collisional dissociation, and 213 nm ultraviolet photodissociationâin their capability to characterize a therapeutic monoclonal antibody, trastuzumab, starting from denatured and native-like precursor ions. Interestingly, native top-down mass spectrometry results in higher sequence coverage than the experiments carried out under denaturing conditions, with the exception of ultraviolet photodissociation. Globally, electron transfer dissociation followed by collision-based activation of product ions generates the largest number of backbone cleavages in disulfide protected regions, including the complementarity determining regions, regardless of electrospray ionization conditions. Overall, these findings suggest that native mass spectrometry can certainly be used for the gas-phase sequencing of whole monoclonal antibodies, although the dissociation of denatured precursor ions still returns a few backbone cleavages not identified in native experiments. Finally, a comparison of the fragmentation maps obtained under denaturing and native conditions strongly points toward disulfide bonds as the primary reason behind the largely overlapping dissociation patterns.
RESUMO
Mass spectrometry (MS)-based bottom-up proteomics (BUP) is currently the method of choice for large-scale identification and characterization of proteins present in complex samples, such as cell lysates, body fluids, or tissues. Technically, BUP relies on MS analysis of complex mixtures of small, <3 kDa, peptides resulting from whole proteome digestion. Because of the extremely high sample complexity, further developments of detection methods and sample preparation techniques are necessary. In recent years, a number of alternative approaches such as middle-down proteomics (MDP, addressing up to 15 kDa peptides) and top-down proteomics (TDP, addressing proteins exceeding 15 kDa) have been gaining particular interest. Here we report on the bioinformatics study of both common and less frequently employed digestion procedures for complex protein mixtures specifically targeting the MDP approach. The aim of this study was to maximize the yield of protein structure information from MS data by optimizing peptide size distribution and sequence specificity. We classified peptides into four categories based on molecular weight: 0.6-3 (classical BUP), 3-7 (extended BUP), 7-15 kDa (MDP), and >15 kDa (TDP). Because of instrumentation-related considerations, we first advocate for the extended BUP approach as the potential near-future improvement of BUP. Therefore, we chose to optimize the number of unique peptides in the 3-7 kDa range while maximizing the number of represented proteins. The present study considers human, yeast, and bacterial proteomes. Results of the study can be further used for designing extended BUP or MDP experimental workflows.
Assuntos
Escherichia coli/química , Peptídeos/análise , Proteoma/análise , Saccharomyces cerevisiae/química , Cromatografia Líquida , Bases de Dados de Proteínas , Humanos , Peso Molecular , Peptídeos/química , Peptídeos/classificação , Proteólise , Proteoma/química , Proteoma/classificação , Proteômica , Espectrometria de Massas em TandemRESUMO
Mass spectrometry (MS) is currently the most sensitive and selective analytical technique for routine peptide and protein structure analysis. Top-down proteomics is based on tandem mass spectrometry (MS/ MS) of intact proteins, where multiply charged precursor ions are fragmented in the gas phase, typically by electron transfer or electron capture dissociation, to yield sequence-specific fragment ions. This approach is primarily used for the study of protein isoforms, including localization of post-translational modifications and identification of splice variants. Bottom-up proteomics is utilized for routine high-throughput protein identification and quantitation from complex biological samples. The proteins are first enzymatically digested into small (usually less than ca. 3 kDa) peptides, these are identified by MS or MS/MS, usually employing collisional activation techniques. To overcome the limitations of these approaches while combining their benefits, middle-down proteomics has recently emerged. Here, the proteins are digested into long (3-15 kDa) peptides via restricted proteolysis followed by the MS/MS analysis of the obtained digest. With advancements of high-resolution MS and allied techniques, routine implementation of the middle-down approach has been made possible. Herein, we present the liquid chromatography (LC)-MS/MS-based experimental design of our middle-down proteomic workflow coupled with post-LC supercharging.
Assuntos
Ácido Aspártico Endopeptidases/análise , Proteínas de Bactérias/metabolismo , Candida albicans/enzimologia , Cromatografia Líquida/métodos , Proteínas Fúngicas/análise , Fragmentos de Peptídeos/análise , Proteômica , Espectrometria de Massas em Tandem/métodosRESUMO
Multispecific antibody constructs are quickly becoming more common constructs in biopharmaceuticals to improve specificity and efficacy. While the advent of this technology has led to improved therapeutics, its development has challenged the analytical tools through which these therapeutics are characterized. Moreover, new critical quality attributes, such as aggregation, have challenged the approaches to characterization even further. Herein, we describe a novel native subunit analysis using IdeS and IgdE analyzed by native size exclusion chromatography coupled with mass spectrometry to interrogate the mechanism of aggregation in a multispecific antibody. Digestion by IdeS and IdgE allows for the retention and detection of noncovalent interactions thereafter. Aggregation was localized to single-chain fragment variables (scFvs) wherein a domain swapping mechanism between VH1/VL2 and VH2/VL1 occurs.
Assuntos
Anticorpos , Espectrometria de Massas/métodos , Cromatografia em GelRESUMO
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.
Assuntos
Células Sanguíneas/química , Proteínas Sanguíneas/química , Células da Medula Óssea/química , Bases de Dados de Proteínas , Isoformas de Proteínas/química , Proteoma/química , Processamento Alternativo , Linfócitos B/química , Proteínas Sanguíneas/genética , Linhagem da Célula , Humanos , Leucócitos Mononucleares/química , Transplante de Fígado , Plasma/química , Isoformas de Proteínas/genética , Processamento de Proteína Pós-Traducional , Proteômica , Linfócitos T/químicaRESUMO
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.
Assuntos
Anticorpos Monoclonais , Espectrometria de Massas/métodos , Proteômica/métodos , Animais , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Regiões Determinantes de Complementaridade/análise , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Humanos , CamundongosRESUMO
Top-down proteomics (TDP) by mass spectrometry (MS) is a technique by which intact proteins are analyzed. It has become increasingly popDesalting and concentrating GELFrEEular in translational research because of the value of characterizing distinct proteoforms of intact proteins. Compared to bottom-up proteomics (BUP) strategies, which measure digested peptide mixtures, TDP provides highly specific molecular information that avoids the bioinformatic challenge of protein inference. However, the technique has been difficult to implement widely because of inherent limitations of existing sample preparation methods and instrumentation. Recent improvements in proteoform pre-fractionation and the availability of high-resolution benchtop mass spectrometers have made it possible to use high-throughput TDP for the analysis of complex clinical samples. Here, we provide a comprehensive protocol for analysis of a common sample type in translational research: human peripheral blood mononuclear cells (PBMCs). The pipeline comprises multiple workflows that can be treated as modular by the reader and used for various applications. First, sample collection and cell preservation are described for two clinical biorepository storage schemes. Cell lysis and proteoform pre-fractionation by gel-eluted liquid fractionation entrapment electrophoresis are then described. Importantly, instrument setup and liquid chromatography-tandem MS are described for TDP analyses, which rely on high-resolution Fourier-transform MS. Finally, data processing and analysis are described using two different, application-dependent software tools: ProSight Lite for targeted analyses of one or a few proteoforms and TDPortal for high-throughput TDP in discovery mode. For a single sample, the minimum completion time of the entire experiment is 72 h.
Assuntos
Leucócitos Mononucleares/química , Proteoma/isolamento & purificação , Proteômica/métodos , Software , Sequência de Aminoácidos , Cromatografia Líquida/normas , Misturas Complexas/química , Eletroforese em Gel de Poliacrilamida/normas , Análise de Fourier , Humanos , Flebotomia/normas , Proteômica/normas , Espectrometria de Massas em Tandem/normasRESUMO
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.