A comprehensive pipeline for translational top-down proteomics from a single blood draw
Open accessAbstract
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.
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A range of clinical samples including serum, urine, or biopsy tissue have been studied using top-down proteomics to identify protein profiles characteristic of a disease and generate more reliable biomarkers.
“A range of clinical samples including serum, urine, or biopsy tissue, have been studied using top-down proteomics to identify protein profiles characteristic of a certain disease generating a more reliable biomarker [155–157].”
From Top-down proteomics: challenges, innovations, and applications in basic and clinical research (2020) · cites this paper as [157]
Toby et al. developed a top-down proteomics-based protocol for analyzing human peripheral blood mononuclear cells.
“Recently, Toby et al. developed a top-down proteomics-based protocol for the analysis of human peripheral blood mononuclear cells [157].”
From Top-down proteomics: challenges, innovations, and applications in basic and clinical research (2020) · cites this paper as [157]
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Deep Profiling of Plasma Proteoforms with Engineered Nanoparticles for Top-Down Proteomics
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Self-citationNext-Generation Serology by Mass Spectrometry: Readout of the SARS-CoV-2 Antibody Repertoire
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Targeted Quantification of Proteoforms in Complex Samples by Proteoform Reaction Monitoring
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Multi‐Omics Studies in Historically Excluded Populations: The Road to Equity
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State-of-the-Art and Future Directions in Structural Proteomics
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Top‐Down Proteomics: Why and When?
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Methylation of KRAS by SETD7 promotes KRAS degradation in non-small cell lung cancer
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Secretome Proteomic Approaches for Biomarker Discovery: An Update on Colorectal Cancer
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Mass spectrometry-based proteomics for advancing solid organ transplantation research
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Self-citationA Conformation-Specific Approach to Native Top-down Mass Spectrometry
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Divergent Antibody Repertoires Found for Omicron versus Wuhan SARS-CoV-2 Strains Using Ig-MS
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Self-citationProteoform characterization based on top-down mass spectrometry
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Self-citationTopDownApp: An open and modular platform for analysis and visualisation of top‐down proteomics data
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Endotypes of Paediatric Cough—Do They Exist and Finding New Techniques to Improve Clinical Outcomes
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Advances in Sample Preparation for Protein Top-Down Mass Spectrometry
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Keeping It Native: Non‐Denaturing Approaches to Top‐Down Protein Mass Spectrometry
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Quantitative Top‐Down Proteomics: A Review
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- Authors
- Toby, T. K., Fornelli, L., Srzentić, K., DeHart, C. J., Levitsky, J., Friedewald, J., & Kelleher, N. L.
- Affiliations
- Northwestern University (US)
- Corresponding author
- Kelleher, Neil L.
- ORCID
- Venue
- Nature Protocols
- Published
- 2018-12-05
- Volume
- 14
- Issue
- 1
- Pages
- 119-152
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- PMC
- PMC6439476
- OpenAlex
- W2569599813
- Cited by
- 86
- References
- 69
- Citations by year
- 2026: 6 · 2025: 9 · 2024: 10 · 2023: 13 · 2022: 16 · 2021: 11 · 2020: 12 · 2019: 7 · 2015: 1
- FWCI
- 3.24
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Protocols (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institute of General Medical Sciences (P41GM108569, T32GM105538)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolomics and Mass Spectrometry Studies
- Keywords
- top-down proteomics, proteoforms, peripheral blood mononuclear cells, cell lysis, sample preparation, liquid chromatography-tandem mass spectrometry, clinical samples
- MeSH terms
- Amino Acid Sequence, Chromatography, Liquid, Electrophoresis, Polyacrylamide Gel, Fourier Analysis, Humans, Leukocytes, Mononuclear, Software, Phlebotomy, Proteome, Proteomics, Complex Mixtures, Tandem Mass Spectrometry
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