Abstract
One strategy for improving the throughput of human plasma proteomic discovery analysis while maintaining good depth of analysis is to multiplex using isobaric tags. At present, the greatest multiplexing that is commercially available uses the TMT10plex kit. As an example of this approach, we describe efficient shotgun discovery proteomics of large numbers of human plasma to identify potential biomarkers. In the analysis strategy, a common pooled reference was used to enable comparisons across multiple experiments. Duplicate samples showed excellent overall reproducibility across different TMT experiments. Data filters that improved the quality of individual peptide and protein quantitation included using a filter for purity of the targeted precursor ion in the isolation window and using only unique peptides.
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
References
Li Z, Adams RM, Chourey K, Hurst GB, Hettich RL, Pan C (2012) Systematic comparison of label-free, metabolic labeling, and isobaric chemical labeling for quantitative proteomics on LTQ Orbitrap Velos. J Proteome Res 11(3):1582–1590
Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1(5):376–386
Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, Hattan S, Khainovski N, Pillai S, Dey S, Daniels S, Purkayastha S, Juhasz P, Martin S, Bartlet-Jones M, He F, Jacobson A, Pappin DJ (2004) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 3(12):1154–1169
Thompson A, Schäfer J, Kuhn K, Kienle S, Schwarz J, Schmidt G, Neumann T, Hamon C (2003) Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal Chem 75(8):1895–1904
Dayon L, Hainard A, Licker V, Turck N, Kuhn K, Hochstrasser DF, Burkhard PR, Sanchez J-C (2008) Relative quantification of proteins in human cerebrospinal fluids by MS/MS using 6-plex isobaric tags. Anal Chem 80(8):2921–2931
McAlister GC, Huttlin EL, Haas W, Ting L, Jedrychowski MP, Rogers JC, Kuhn K, Pike I, Grothe RA, Blethrow JD, Gygi SP (2012) Increasing the multiplexing capacity of TMTs using reporter ion isotopologues with isobaric masses. Anal Chem 84(17):7469–7478
Werner T, Becher I, Sweetman G, Doce C, Savitski MM, Bantscheff M (2012) High-resolution enabled TMT 8-plexing. Anal Chem 84(16):7188–7194
Viner R, Bomgarden R, Blank M, Rogers J (2013) Increasing the multiplexing of protein quantitation from 6- to 10-plex with reporter ion isotopologues. PN_AMAS_W617_RViner_R1
Everley RA, Kunz RC, McAllister FE, Gygi SP (2013) Increasing throughput in targeted proteomics assays: 54-plex quantitation in a single mass spectrometry run. Anal Chem 85(11):5340–5346
Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, Fleming T, Eiermann W, Wolter J, Pegram M, Baselga J, Norton L (2001) Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 344(11):783–792
Telli ML, Hunt SA, Carlson RW, Guardino AE (2007) Trastuzumab-related cardiotoxicity: calling into question the concept of reversibility. J Clin Oncol 25(23):3525–3533
Villen J, Gygi SP (2008) The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry. Nat Protoc 3(10):1630–1638
Cao Z, Tang H-Y, Wang H, Liu Q, Speicher DW (2012) Systematic comparison of fractionation methods for in-depth analysis of plasma proteomes. J Proteome Res 11(6):3090–3100
Cox J, Hein MY, Luber CA, Paron I, Nagaraj N, Mann M (2014) Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol Cell Proteomics 13(9):2513–2526
Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26(12):1367–1372
Michalski A, Cox J, Mann M (2011) More than 100,000 detectable peptide species elute in single shotgun proteomics runs but the majority is inaccessible to data-dependent LC−MS/MS. J Proteome Res 10(4):1785–1793
Ting L, Rad R, Gygi SP, Haas W (2011) MS3 eliminates ratio distortion in isobaric multiplexed quantitative proteomics. Nat Methods 8(11):937–940
Wenger CD, Lee MV, Hebert AS, McAlister GC, Phanstiel DH, Westphall MS, Coon JJ (2011) Gas-phase purification enables accurate, multiplexed proteome quantification with isobaric tagging. Nat Methods 8(11):933–935
Acknowledgments
This work was supported by NIH Grants RO1HD076279, RO1CA131582, and WW Smith Charitable Trust Grants H1205 and H1305 (D.W. Speicher), PA Department of Health Commonwealth Universal Research Enhancement (CURE) Program Grant (B. Ky) as well as CA10815 (NCI core grant to the Wistar Institute).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Liu, P., Beer, L.A., Ky, B., Barnhart, K.T., Speicher, D.W. (2017). Quantitative Comparisons of Large Numbers of Human Plasma Samples Using TMT10plex Labeling. In: Greening, D., Simpson, R. (eds) Serum/Plasma Proteomics. Methods in Molecular Biology, vol 1619. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7057-5_22
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7057-5_22
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7056-8
Online ISBN: 978-1-4939-7057-5
eBook Packages: Springer Protocols