Quantitative analysis of complex protein mixtures using isotope-coded affinity tags
Abstract
We describe an approach for the accurate quantification and concurrent sequence identification of the individual proteins within complex mixtures. The method is based on a class of new chemical reagents termed isotope-coded affinity tags (ICATs) and tandem mass spectrometry. Using this strategy, we compared protein expression in the yeast Saccharomyces cerevisiae, using either ethanol or galactose as a carbon source. The measured differences in protein expression correlated with known yeast metabolic function under glucose-repressed conditions. The method is redundant if multiple cysteinyl residues are present, and the relative quantification is highly accurate because it is based on stable isotope dilution techniques. The ICAT approach should provide a widely applicable means to compare quantitatively global protein expression in cells and tissues.
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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 [39]
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“The application of stable-isotope-labelled derivatives (Gygi et al., 1999) is having an enormous impact on protein analysis (isotope coded affinity tags, ICAT), a clear indication of the potential utility of chemical derivatization (tagging) in API MS. LC/MS: chromatographic retention data The use…”
BackgroundGriffin, T. J., Gygi, S. P., Ideker, T. E., Rist, B., Eng, J. K., Hood, L., & Aebersold, R. H. (2002) · Molecular & Cellular Proteomics · doi:10.1074/mcp.m200001-mcp200 · Cited by 663 · see citing works
“In an earlier study we used similar metabolic perturbation in yeast to demonstrate the ability of the ICAT reagent method to identify and quantify proteins in complex mixtures ( 25 ).”
BackgroundMethodInfluentialIncreasing 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
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CTCF physically links cohesin to chromatin
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Self-citationQuantifying changes in the thiol redox proteome upon oxidative stress in vivo
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“Described technologies such as isotope-coded aYnity tagging (ICAT) [31, 32] and stable isotope labeling with amino acids in cell culture (SILAC) [33, 34] can be combined with mass spectrometry approaches to produce”
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“The most recent utilized MS in conjunction with two-dimensional gel electrophoresis to identify most of their PSD proteins, with a smaller contribution from ICAT-based LC-MS/MS (82), resulting in identification of 180 proteins.”
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- Authors
- Gygi, S. P., Rist, B., Gerber, S. A., Turecek, F., Gelb, M. H., & Aebersold, R.
- Affiliations
- University of Washington (US)
- Corresponding author
- Aebersold, Ruedi
- Venue
- Nature Biotechnology
- Published
- 1999-10
- Volume
- 17
- Issue
- 10
- Pages
- 994-999
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- OpenAlex
- W2111208057
- Semantic Scholar
- 8929f01bc9a788127bcff7a09432f4f7979c8e6b
- Cited by
- 4,683
- References
- 24
- Citations by year
- 2026: 19 · 2025: 27 · 2024: 31 · 2023: 54 · 2022: 50 · 2021: 63 · 2020: 51 · 2019: 70 · 2018: 77 · 2017: 104 · 2016: 123 · 2015: 171 · 2014: 188 · 2013: 235 · 2012: 296
- FWCI
- 75.18
- Type
- journal article
- Language
- en
- Open access
- closed
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Human Genome Research Institute (T32 HG-00035, T32HG000035)
- National Center for Research Resources (RR-11823, P41RR011823)
- Eunice Kennedy Shriver National Institute of Child Health and Human Development (HD 02274, P30HD002274)
- 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 › Redox biology and oxidative stress
- Keywords
- ICAT, protein quantification, tandem mass spectrometry, protein expression profiling, Saccharomyces cerevisiae, glucose repression, ethanol carbon source, stable isotope dilution, cysteine labeling
- MeSH terms
- Affinity Labels, Amino Acid Sequence, Chromatography, Liquid, Isotope Labeling, Proteins, Mass Spectrometry
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