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Targeted Quantification of Proteoforms in Human Samples by Proteoform Reaction Monitoring

ABSTRACT: 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 Proteoform Finder specifically designed for easy analysis of PfRM-MS data.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Luca Fornelli

PROVIDER: MSV000091774 | MassIVE | Fri Apr 21 14:37:00 BST 2023

REPOSITORIES: MassIVE

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Publications

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

Huang Che-Fan CF Kline Jake T JT Negrão Fernanda F Robey Matthew T MT Toby Timothy K TK Durbin Kenneth R KR Fellers Ryan T RT Friedewald John J JJ Levitsky Josh J Abecassis Michael M I MMI Melani Rafael D RD Kelleher Neil L NL Fornelli Luca L

Analytical chemistry 20240214 8

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 circumve ...[more]

PMID: 38354049

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

Project description: Not available

| S-EPMC11008684 | biostudies-literature

5xFAD transgenic mouse Abeta proteoforms

Project description:In this study, we have evaluated amyloidosis-relevant Aβ proteoform flux using immunoenrichment and quantitative Top-Down Mass Spectrometry in a well-studied 5xFAD mouse model of age-dependent Aβ amyloidosis.

2023-09-27 | PXD042921 | Pride

Evaluation of cleavable crosslinking for characterization of proteoform structural differences by top-down mass spectrometry

Project description:Cleavable crosslinking has traditionally been employed in bottom-up mass spectrometry to elucidate protein structure and protein-protein interactions through identification of peptides bearing characteristic mass adducts. Here, we demonstrate the application of cleavable crosslinking in top-down mass spectrometry to enhance fragmentation efficiency and enable precise localization of crosslink sites. We first validated this approach using cytochrome c, a well-characterized model protein. Subsequently, we extended top-down cleavable crosslinking to transthyretin, a natively homotetrameric protein exhibiting extensive proteoform heterogeneity, to investigate whether proteoform variations induce structural changes detectable by this method. Our results confirm that cleavable crosslinks can be detected and characterized by top-down mass spectrometry, with crosslinker cleavage under collisional activation significantly enhancing fragmentation. Application to transthyretin (intramolecular crosslinks) yielded complex crosslinking patterns that precluded complete identification of crosslinks. However, the crosslinking data provided valuable information on solvent-accessible residues, functioning effectively as a covalent labeling strategy. This work establishes cleavable crosslinking as a viable chemical crosslinking approach for top-down mass spectrometry applications.

2026-01-17 | PXD073175 | JPOST Repository

Revealing Corynebacterium glutamicum proteoforms through top-down proteomics

Project description:Corynebacterium glutamicum is a bacterium widely employed in the industrial production of amino acids as well as a broad range of other biotechnological products. The present study describes the characterization of C. glutamicum proteoforms, and their post-translational modifications (PTMs) employing top-down proteomics. Despite previous evidence of PTMs having roles in the regulation of C. glutamicum metabolism, this is the first top-down proteome analysis of this organism. We identified 1125 proteoforms from 273 proteins, with 60 % of proteins presenting at least one mass shift, suggesting the presence of PTMs, including several acetylated, oxidized and formylated proteoforms. Furthermore, proteins relevant to amino acid production, protein secretion, and oxidative stress were identified with mass shifts suggesting the presence of uncharacterized PTMs and proteoforms that may affect biotechnologically relevant processes in this industrial workhorse. For instance, the membrane proteins mepB and SecG were identified as a cleaved and a formylated proteoform, respectively. While in the central metabolism, OdhI was identified as two proteoforms with potential biological relevance: a cleaved proteoform and a proteoform with PTMs corresponding to a 70 Da mass shift.

2022-12-10 | PXD038038 | Pride

Proteoform Imaging by Combining MALDI MSI and High-sensitivity Top-down Proteomics

Project description:Spatially resolved characterization of proteoforms has great potential to significantly advance our understanding of physiological and disease mechanisms. However, it faces challenges regarding throughput and coverage. Herein, we developed a robust method for high-throughput proteoform imaging (HTPi) by combining matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) and region-specific top-down proteomic analysis.

2024-12-03 | PXD058552 |

Nanoproteomics enables proteoform-resolved analysis of low-abundance proteins in human serum

Project description:Top-down mass spectrometry (MS)-based proteomics provides a comprehensive analysis of proteoforms to achieve a proteome-wide understanding of protein functions. However, the MS detection of low-abundance proteins from the blood remains an unsolved challenge due to the complexity and extraordinary dynamic range of the blood proteome. Here we develop an integrated ‘nanoproteomics’ method coupling peptide-functionalized superparamagnetic nanoparticles (NPs) with top-down MS for the enrichment and comprehensive analysis of low-abundance cardiac troponin I (cTnI), a gold-standard biomarker for cardiovascular diseases, directly from human serum. These NPs allow for the sensitive enrichment of cTnI (< 1 ng/mL) with high specificity and reproducibility, while simultaneously depleting highly abundant blood proteins such as human serum albumin (>10 orders of magnitude more abundant than cTnI). We demonstrate for the first time that top-down nanoproteomics can provide high-resolution proteoform-resolved molecular fingerprints of diverse cTnI proteoforms to establish proteoform-pathophysiology relationships. This scalable and reproducible antibody-free strategy can generally enable the proteoform-resolved analysis of low-abundance proteins directly from serum to reveal previously unachievable molecular details.

2020-09-11 | PXD019712 | Pride

Positional proteomics reveals differences in N-terminal proteoform stability

Project description:To understand the impact of alternative translation initiation on a proteome, we performed the first study on protein turnover using positional proteomics and ribosome profiling to distinguish between N-terminal proteoforms of individual genes. Overall, we monitored the stability of 1,941 human N-terminal proteoforms, including 147 N-terminal proteoform pairs that originate from alternative translation initiation, alternative splicing or incomplete processing of the initiator methionine. Ribosome profiling of lactimidomycin and cycloheximide treated human Jurkat T-lymphocytes

2015-10-23 | E-GEOD-74279 | biostudies-arrayexpress

Positional proteomics reveals differences in N-terminal proteoform stability

2015-10-23 | GSE74279 | GEO

Predicting electrophoretic mobility of proteoforms for large-scale top-down proteomics

Project description:Large-scale top-down proteomics characterizes proteoforms in cells globally with high confidence and high throughput using reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS) or capillary zone electrophoresis (CZE)-MS/MS. The false discovery rate (FDR) from the target-decoy database search is typically deployed to filter identified proteoforms to ensure high-confidence identifications (IDs). It has been demonstrated that the FDRs in top-down proteomics can be drastically underestimated. An alternative approach to the FDR can be useful for further evaluating the confidence of proteoform IDs after database search. We argue that predicting retention/migration time of proteoforms from the RPLC/CZE separation accurately and comparing their predicted and experimental separation time could be a useful and practical approach. Based on our knowledge, there is still no report in the literature about predicting separation time of proteoforms using large top-down proteomics datasets. In this pilot study, for the first time, we evaluated various semi-empirical models for predicting proteoforms’ electrophoretic mobility (µef) using large-scale top-down proteomics datasets from CZE-MS/MS. We achieved a linear correlation between experimental and predicted µef of E. coli proteoforms (R2=0.98) with a simple semi-empirical model, which utilizes the number of charges and molecular mass of each proteoform as the parameters. Our modeling data suggest that the complete unfolding of proteoforms during CZE separation benefits the prediction of their µef. Our results also indicate that N-terminal acetylation and phosphorylation both decrease proteoforms’ charge by roughly one charge unit.

2020-02-17 | PXD017265 | Pride

Qualitative and quantitative top-down proteomics of human colorectal cancer cell lines identified 23000 proteoforms and revealed drastic proteoform-level differences between metastatic and non-metastatic cancer cells

Project description:Understanding cancer metastasis at the proteoform level is crucial for discovering new protein biomarkers for cancer diagnosis and drug development. Proteins are the primary effectors of function in biology and proteoforms from the same gene can have drastically different biological functions. Here, we present the first qualitative and quantitative top-down proteomics (TDP) study of a pair of isogenic human metastatic and non-metastatic colorectal cancer (CRC) cell lines (SW480 and SW620). This study pursues a global view of human CRC proteome before and after metastasis in a proteoform-specific manner. We identified 23,319 proteoforms of 2,297 genes from the CRC cell lines using capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS), representing nearly one order of magnitude improvement in the number of proteoform identifications from human cell lines compared to literature data. We identified 111 proteoforms containing single amino acid variants (SAAVs) using a proteogenomic approach and revealed drastic differences between the metastatic and non-metastatic cell lines regarding SAAVs profiles. Quantitative TDP analysis unveiled statistically significant differences in proteoform abundance between the SW480 and SW620 cell lines on a proteome scale for the first time. Ingenuity Pathway Analysis (IPA) disclosed that many differentially expressed genes at the proteoform level had diversified functions and were closely related to cancer. Our study represents a milestone in TDP towards the definition of human proteome in a proteoform-specific manner, which will transform basic and translational biomedical research.

2023-01-05 | PXD029703 | Pride

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