Triplex protein quantification based on stable isotope labeling by peptide dimethylation applied to cell and tissue lysates
Abstract
Stable isotope labeling is at present one of the most powerful methods in quantitative proteomics. Stable isotope labeling has been performed at both the protein as well as the peptide level using either metabolic or chemical labeling. Here, we present a straightforward and cost-effective triplex quantification method that is based on stable isotope dimethyl labeling at the peptide level. Herein, all proteolytic peptides are chemically labeled at their alpha- and epsilon-amino groups. We use three different isotopomers of formaldehyde to enable the parallel analysis of three different samples. These labels provide a minimum of 4 Da mass difference between peaks in the generated peptide triplets. The method was evaluated based on the quantitative analysis of a cell lysate, using a typical "shotgun" proteomics experiment. While peptide complexity was increased by introducing three labels, still more than 1300 proteins could be identified using 60 microg of starting material, whereby more than 600 proteins could be quantified using at least four peptides per protein. The triplex labeling was further utilized to distinguish specific from aspecific cAMP binding proteins in a chemical proteomics experiment using immobilized cAMP. Thereby, differences in abundance ratio of more than two orders of magnitude could be quantified.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
Chemical labeling is in principle universally applicable, but because labels are introduced later in sample processing, some robustness advantages are lost.
“Chemical labeling (39–41) is in principle universally applicable, but because the labels are introduced later in the sample processing, some of the advantages in robustness are lost.”
From Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ (2014) · cites this paper as [41]
Cites
Ong, S.-E., Blagoev, B., Kratchmarova, I., Kristensen, D. B., Steen, H., Pandey, A., & Mann, M. (2002) · Molecular & Cellular Proteomics · doi:10.1074/mcp.m200025-mcp200 · Cited by 5,574 · see citing works
Quantitative analysis of complex protein mixtures using isotope-coded affinity tags
Gygi, S. P., Rist, B., Gerber, S. A., Turecek, F., Gelb, M. H., & Aebersold, R. (1999) · Nature Biotechnology · doi:10.1038/13690 · Cited by 4,683 · see citing works
Ross, P. L., Huang, Y. N., Marchese, J. N., Williamson, B., Parker, K., Hattan, S., Khainovski, N., Pillai, S., Dey, S., Daniels, S., et al. (2004) · Molecular & Cellular Proteomics · doi:10.1074/mcp.m400129-mcp200 · Cited by 4,401 · see citing works
Quantitative mass spectrometry in proteomics: a critical review
Bantscheff, M., Schirle, M., Sweetman, G. M. A., Rick, J. M., & Küster, B. (2007) · Analytical and Bioanalytical Chemistry · doi:10.1007/s00216-007-1486-6 · Cited by 1,659 · see citing works
Stable-Isotope Dimethyl Labeling for Quantitative Proteomics
Hsu, J., Huang, S., Chow, N.-H., & Chen, S. (2003) · Analytical Chemistry · doi:10.1021/ac0348625 · Cited by 701 · see citing works
Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics
Blagoev, B., Ong, S., Kratchmarova, I., & Mann, M. (2004) · Nature Biotechnology · doi:10.1038/nbt1005 · Cited by 684 · see citing works
Quantification in Proteomics through Stable Isotope Coding: A Review
Julka, S., & Regnier, F. E. (2004) · Journal of Proteome Research · doi:10.1021/pr0340734 · Cited by 320 · see citing works
Huang, L. J.-S., Durick, K., Weiner, J. A., Chun, J. J. M., & Taylor, S. S. (1997) · Journal of Biological Chemistry · doi:10.1074/jbc.272.12.8057 · Cited by 294 · see citing works
Hydrophilic interaction liquid chromatography (HILIC) in proteomics
Boersema, P. J., Mohammed, S., & Heck, A. J. R. (2008) · Analytical and Bioanalytical Chemistry · doi:10.1007/s00216-008-1865-7 · Cited by 275 · see citing works
Self-citationEight-channel iTRAQ Enables Comparison of the Activity of Six Leukemogenic Tyrosine Kinases
Pierce, A., Unwin, R. D., Evans, C. A., Griffiths, S., Carney, L., Zhang, L.-Q., Jaworska, E., Lee, C.-F., Blinco, D., Okoniewski, M. J., et al. (2007) · Molecular & Cellular Proteomics · doi:10.1074/mcp.m700251-mcp200 · Cited by 256 · see citing works
Controlling Deuterium Isotope Effects in Comparative Proteomics
Zhang, R., Sioma, C. S., Thompson, R. A., Xiong, L., & Regnier, F. E. (2002) · Analytical Chemistry · doi:10.1021/ac025614w · Cited by 238 · see citing works
D-AKAP2, a novel protein kinase A anchoring protein with a putative RGS domain
Huang, L. J.-S., Durick, K., Weiner, J. A., et al. (1997) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.94.21.11184 · Cited by 218 · see citing works
Nuclear PtdIns5P as a Transducer of Stress Signaling: An In Vivo Role for PIP4Kbeta
Jones, D. R., Bultsma, Y., Keune, W., Halstead, J. R., Elouarrat, D., Mohammed, S., Heck, A. J. R., D’Santos, C. S., & Divecha, N. (2006) · Molecular Cell · doi:10.1016/j.molcel.2006.07.014 · Cited by 191 · see citing works
Self-citationEvaluation and Optimization of ZIC-HILIC-RP as an Alternative MudPIT Strategy
Boersema, P. J., Divecha, N., Heck, A. J. R., & Mohammed, S. (2007) · Journal of Proteome Research · doi:10.1021/pr060589m · Cited by 190 · see citing works
Self-citationGuo, K., Ji, C., & Li, L. (2007) · Analytical Chemistry · doi:10.1021/ac0704356 · Cited by 169 · see citing works
Scholten, A., Poh, M. K., van Veen, T. A. B., et al. (2006) · Journal of Proteome Research · doi:10.1021/pr0600529 · Cited by 117 · see citing works
Differential Dimethyl Labeling of N-Termini of Peptides after Guanidination for Proteome Analysis
Ji, C., Guo, N., & Li, L. (2005) · Journal of Proteome Research · doi:10.1021/pr050215d · Cited by 106 · see citing works
Mass spectrometry-based quantitative proteomics
Heck, A. J. R., & Krijgsveld, J. (2004) · Expert Review of Proteomics · doi:10.1586/14789450.1.3.317 · Cited by 85 · see citing works
Self-citationRecent advancements in differential proteomics based on stable isotope coding
Julka, S. (2005) · Briefings in Functional Genomics and Proteomics · doi:10.1093/bfgp/4.2.158 · Cited by 74 · see citing works
Mousson, F., Kolkman, A., Pijnappel, W. W. M. P., Timmers, H. T. M., & Heck, A. J. R. (2008) · Molecular & Cellular Proteomics · doi:10.1074/mcp.m700306-mcp200 · Cited by 67 · see citing works
Self-citationHsu, J., Huang, S., & Chen, S. (2006) · Electrophoresis · doi:10.1002/elps.200600147 · Cited by 64 · see citing works
Scholten, A., van Veen, T. A. B., Vos, M. A., & Heck, A. J. R. (2007) · Journal of Proteome Research · doi:10.1021/pr060601a · Cited by 54 · see citing works
Self-citationJi, C., Li, L., Gebre, M., Pasdar, M., & Li, L. (2005) · Journal of Proteome Research · doi:10.1021/pr050094h · Cited by 40 · see citing works
Flagged first, then by citations
Cited by
Sorted by citations
Cox, J., Hein, M. Y., Luber, C. A., Paron, I., Nagaraj, N., & Mann, M. (2014) · Molecular & Cellular Proteomics · doi:10.1074/mcp.m113.031591 · Cited by 5,180 · see citing works
Increasing the Multiplexing Capacity of TMTs Using Reporter Ion Isotopologues with Isobaric Masses
McAlister, G. C., Huttlin, E. L., Haas, W., Ting, L., Jedrychowski, M. P., Rogers, J. C., Kuhn, K., Pike, I., Grothe, R. A., Blethrow, J. D., et al. (2012) · Analytical Chemistry · doi:10.1021/ac301572t · Cited by 605 · see citing works
Ibebunjo, C., Chick, J. M., Kendall, T., Eash, J. K., Li, C., Zhang, Y., Vickers, C., Wu, Z., Clarke, B. A., Shi, J., et al. (2013) · Molecular and Cellular Biology · doi:10.1128/mcb.01036-12 · Cited by 267 · see citing works
- Authors
- Boersema, P. J., Aye, T. T., van Veen, T. A. B., Heck, A. J. R., & Mohammed, S.
- Affiliations
- Utrecht University (NL)
- Corresponding author
- Mohammed, Shabaz
- Venue
- PROTEOMICS
- Publisher
- Wiley
- Published
- 2008-11
- Volume
- 8
- Issue
- 22
- Pages
- 4624-4632
- ISSN
- 1615-9853 (ISSN-L)
- 1615-9861
- OpenAlex
- W2163021396
- Cited by
- 220
- References
- 23
- Citations by year
- 2026: 2 · 2025: 4 · 2024: 5 · 2023: 4 · 2022: 4 · 2021: 7 · 2020: 13 · 2019: 14 · 2018: 13 · 2017: 13 · 2016: 22 · 2015: 27 · 2014: 20 · 2013: 18 · 2012: 16
- FWCI
- 5.48
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- PROTEOMICS (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Netherlands Proteomics Centre
- 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 › Advanced Biosensing Techniques and Applications
- Keywords
- stable isotope labeling, quantitative proteomics, cell lysate, tissue lysates, shotgun proteomics, chemoproteomics, cAMP-binding protein, protein abundance
- MeSH terms
- Cyclic AMP, Animals, Borohydrides, Chromatography, Ion Exchange, Chromatography, Liquid, Leukemia, Erythroblastic, Acute, Formaldehyde, Isomerism, Isotope Labeling, Methylation, Peptides, Protein Binding, Proteins, Mass Spectrometry, Tumor Cells, Cultured, Rats, Wistar, Spectrometry, Mass, Electrospray Ionization, Proteomics, Rats, Tandem Mass Spectrometry
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
