Abstract
A key issue in proteomics is to quantify changes in protein levels in complex biological samples under different conditions. Traditional two-dimensional gel (2-DE) electrophoresis-based proteomic approaches are tedious and suffer from several limitations, including difficulties in detecting low abundant and insoluble proteins. Isotope-coded affinity tagging (ICAT®), one of the most employed chemical isotope labeling methods, can address many of the shortcomings of 2-DE. ICAT relies on the sensitivity of mass spectrometry (MS) to quantify relative protein abundance in a mixture of two differentially labeled protein samples. We describe here a detailed protocol for ICAT-based quantification of proteins in two or more biological samples, including sample preparation, ICAT labeling, frac-tionation and purification, and analysis by MS. For the MS analysis, we describe a “targeted” approach, which includes quantification of the samples using MS followed by selective identification of only the differentially expressed ICAT pairs using tandem MS (MS/MS). This approach gives more biologically relevant information than a data-dependent MS/MS analysis. We also describe the steps in data analysis, statistical analysis, and protein database searching.
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References
1. Aebersold R, Mann M (2003) Mass spectrometry-based proteomics. Nature 422:198–207
2. Gygi SP, Rist B, Gerber SA, Turecek F, Gelb, MH, Aebersold R (1999) Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotechnol 17:994–999
3. Haqqani AS, Nesic M, Preston E, Baumann E, Kelly J, Stanimirovic D (2005) Characterization of vascular protein expression patterns in cerebral ischemia/reperfusion using laser capture microdissection and ICAT-nanoLC-MS/MS. FASEB J 19:1809–1821
4. Haqqani AS, Kelly J, Baumann E, Haseloff RF, Blasig IE, Stanimirovic DB (2007) Protein markers of ischemic insult in brain endothelial cells identified using 2D gel electrophoresis and ICAT-based quantitative proteomics. J Proteome Res 6:226–239
5. Han DK, Eng J, Zhou H, Aebersold R (2001) Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry. Nat Biotechnol 19:946–951
6. Haqqani AS, Hutchison JS, Ward R, Stanimirovic DB (2007) Protein biomarkers in serum of pediatric patients with severe traumatic brain injury identified by ICAT-LC-MS/MS. J Neurotrauma 24:54–74
7. Ingebrigtsen T, Romner B (2003) Biochemical serum markers for brain damage: A short review with emphasis on clinical utility in mild head injury. Restor Neurol Neurosci 21:171–176
8. Tusher VG, Tibshirani R, Chu G (2001) Significance analysis of microarrays applied to the ionising radiation response. Proc Natl Acad Sci USA 98:5116–5121
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© 2008 Humana Press, a part of Springer Science+Business Media, LLC
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Haqqani, A.S., Kelly, J.F., Stanimirovic, D.B. (2008). Quantitative Protein Profiling by Mass Spectrometry Using Isotope-Coded Affinity Tags. In: Starkey, M., Elaswarapu, R. (eds) Genomics Protocols. Methods in Molecular Biology™, vol 439. Humana Press. https://doi.org/10.1007/978-1-59745-188-8_16
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DOI: https://doi.org/10.1007/978-1-59745-188-8_16
Publisher Name: Humana Press
Print ISBN: 978-1-58829-871-3
Online ISBN: 978-1-59745-188-8
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