Abstract
High-throughput and in-depth proteomic analysis of plasma and serum samples remains challenging due to the presence of multiple high-abundance proteins. Here, we provide a detailed protocol for proteomic analysis of serum and plasma specimens using a high-abundance protein depletion kit and TMTpro 16-plex reagents. This method requires only 5 μL serum or plasma, identifying and quantifying about 1000 proteins. A batch of 16 samples can be processed in 36 h. On average, each sample consumes about 1.5 h of mass spectrometer instrument time. Overall, our method can identify proteins across six orders of magnitude with high reproducibility (CV < 20%) using a shorter instrument time and less sample volume compared to existing methods. Thus, the method is suitable to be applied to large-scale proteomic studies.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Geyer PE, Holdt LM, Teupser D et al (2017) Revisiting biomarker discovery by plasma proteomics. Mol Syst Biol 13:942
Anderson NL, Anderson NG (2002) The human plasma proteome: history, character, and diagnostic prospects*. Mol Cell Proteomics 1:845–867
Pernemalm M, Lewensohn R, Lehtiö J (2009) Affinity prefractionation for MS-based plasma proteomics. Proteomics 9:1420–1427
Rifai N, Gillette MA, Carr SA (2006) Protein biomarker discovery and validation: the long and uncertain path to clinical utility. Nat Biotechnol 24:971–983
Di Girolamo F, Righetti PG, Soste M et al (2013) Reproducibility of combinatorial peptide ligand libraries for proteome capture evaluated by selected reaction monitoring. J Proteome 89:215–226
Pieper R, Su Q, Gatlin CL et al (2003) Multi-component immunoaffinity subtraction chromatography: an innovative step towards a comprehensive survey of the human plasma proteome. Proteomics 3:422–432
Moulder R, Bhosale SD, Goodlett DR et al (2018) Analysis of the plasma proteome using iTRAQ and TMT-based isobaric labeling. Mass Spectrom Rev 37:583–606
Lee PY, Osman J, Low TY et al (2019) Plasma/serum proteomics: depletion strategies for reducing high-abundance proteins for biomarker discovery. Bioanalysis 11:1799–1812
Pietrowska M, Wlosowicz A, Gawin M et al (2019) MS-based proteomic analysis of serum and plasma: problem of high abundant components and lights and shadows of albumin removal. In: Advances in experimental medicine and biology, pp 57–76
Keshishian H, Burgess MW, Specht H et al (2017) Quantitative, multiplexed workflow for deep analysis of human blood plasma and biomarker discovery by mass spectrometry. Nat Protoc 12:1683–1701
Roche S, Tiers L, Provansal M et al (2009) Depletion of one, six, twelve or twenty major blood proteins before proteomic analysis: the more the better? J Proteome 72:945–951
Smith MPW, Wood SL, Zougman A et al (2011) A systematic analysis of the effects of increasing degrees of serum immunodepletion in terms of depth of coverage and other key aspects in top-down and bottom-up proteomic analyses. Proteomics 11:2222–2235
Bruderer R, Muntel J, Müller S et al (2019) Analysis of 1508 plasma samples by capillary-flow data-independent acquisition profiles proteomics of weight loss and maintenance. Mol Cell Proteomics 18:1242–1254
Deutsch EW, Omenn GS, Sun Z et al (2021) Advances and utility of the human plasma proteome. J Proteome Res 20:5241–5263
Nakayasu ES, Gritsenko M, Piehowski PD et al (2021) Tutorial: best practices and considerations for mass-spectrometry-based protein biomarker discovery and validation. Nat Protoc 16:3737–3760
Wewer Albrechtsen NJ, Geyer PE, Doll S et al (2018) Plasma proteome profiling reveals dynamics of inflammatory and lipid homeostasis markers after Roux-en-Y gastric bypass surgery. Cell Syst 7:601–612.e3
Kulak NA, Geyer PE, Mann M (2017) Loss-less nano-fractionator for high sensitivity, high coverage proteomics. Mol Cell Proteomics 16:694–705
Tognetti M, Sklodowski K, Müller S et al (2022) Biomarker candidates for tumors identified from deep-profiled plasma stem predominantly from the low abundant area. J Proteome Res 21:1718–1735
Li J, Cai Z, Bomgarden RD et al (2021) TMTpro-18plex: the expanded and complete set of TMTpro reagents for sample multiplexing. J Proteome Res 20:2964–2972
Li J, Van Vranken JG, Pontano Vaites L et al (2020) TMTpro reagents: a set of isobaric labeling mass tags enables simultaneous proteome-wide measurements across 16 samples. Nat Methods 17:399–404
Thompson A, Schäfer J, Kuhn K et al (2003) Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal Chem 75:1895–1904
Keshishian H, Burgess MW, Gillette MA et al (2015) Multiplexed, quantitative workflow for sensitive biomarker discovery in plasma yields novel candidates for early myocardial injury. Mol Cell Proteomics 14:2375–2393
Dey KK, Wang H, Niu M et al. (2019) Deep undepleted human serum proteome profiling toward biomarker discovery for Alzheimer’s disease. Clin Proteomics 16(1):16. https://doi.org/10.1186/s12014-019-9237-1
Tang S, Sun R, Xiao Q et al (2020) Proteomics uncovers immunosuppression in COVID-19 patients with long disease course. medRxiv 18:2020.06.14.20131078
Bi X, Liu W, Ding X et al (2022) Proteomic and metabolomic profiling of urine uncovers immune responses in patients with COVID-19. Cell Rep 38:110271
Shen B, Yi X, Sun Y et al (2020) Proteomic and metabolomic characterization of COVID-19 patient sera. Cell 182:59–72.e15
Mertins P, Tang LC, Krug K et al (2018) Reproducible workflow for multiplexed deep-scale proteome and phosphoproteome analysis of tumor tissues by liquid chromatography-mass spectrometry. Nat Protoc 13:1632
Gao H, Zhang F, Liang S et al (2020) Accelerated lysis and proteolytic digestion of biopsy-level fresh-frozen and FFPE tissue samples using pressure cycling technology. J Proteome Res 19:1982–1990
Tu C, Rudnick PA, Martinez MY et al (2010) Depletion of abundant plasma proteins and limitations of plasma proteomics. J Proteome Res 9:4982–4991
Zhu T, Sun R, Zhang F et al (2021) BatchServer: a web server for batch effect evaluation, visualization, and correction. J Proteome Res 20:1079–1086
Chi H, Liu C, Yang H et al (2018) Comprehensive identification of peptides in tandem mass spectra using an efficient open search engine. Nat Biotechnol 36:1059–1061
Acknowledgments
We thank the National Key R&D Program of China (2021YFA1301601, 2021YFA1301602, 2020YFE0202200), the National Natural Science Foundation of China (81972492, 21904107, 81672086), and Zhejiang Provincial Natural Science Foundation for Distinguished Young Scholars (LR19C050001) for their financial support.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Zhou, Y. et al. (2023). High-Throughput and In-Depth Proteomic Profiling of 5 μL Plasma and Serum Using TMTpro 16-Plex. In: Greening, D.W., Simpson, R.J. (eds) Serum/Plasma Proteomics. Methods in Molecular Biology, vol 2628. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2978-9_6
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2978-9_6
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2977-2
Online ISBN: 978-1-0716-2978-9
eBook Packages: Springer Protocols