Stable isotope methods for high-precision proteomics. Drug Discov. Today 10, 353-363

Target Discovery Inc., 4015 Fabian Way, Palo Alto, CA 94303, USA.
Drug Discovery Today (Impact Factor: 6.69). 04/2005; 10(5):353-63. DOI: 10.1016/S1359-6446(05)03381-7
Source: PubMed


Stable isotope tagging methods provide a useful means of determining the relative expression level of individual proteins between samples in a mass spectrometer with high precision (coefficients of variation less than 10%). Because two or more samples tagged with different numbers of stable isotopes can be mixed before any processing steps, sample-to-sample recovery differences are eliminated. Mass spectrometry also allows post-translational modifications, splice variations and mutations (often unnoticed in immunoassays) to be detected and identified, increasing the clinical relevance of the assay and avoiding the issues of non-specific binding and cross-reactivity observed in immunoassays. Several stable isotope tagging methods are available for use in proteomics research. We discuss the advantages and disadvantages of each technique with respect to biomarker discovery, target validation, efficacy and toxicology screening and clinical diagnostic applications.

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Available from: Luke V Schneider, Jun 13, 2014
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    • "As mentioned previously, the TD1 and TD2 buffers are fully compatible with the common protein labeling chemistries (data not provided). We have applied the described immunoaffinity/MS method using isotope-differentiated binding energy shift tags in our laboratory (data not shown) [65]. This intact protein capture approach allows the detection of novel protein isoforms (either sequence variants or posttranslational modifications) that may be lost in other biomarker validation methods such as multireaction monitoring (MRM) [66] or the use of stable isotope standards with antipeptide antibody enrichment (SISCAPA) [67]. "
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    • "Following experimental manipulation, cell culture extracts or conditioned media are combined, processed and the relative abundance of ''heavy'' to ''light'' peptide may be assessed to identify differentially expressed proteins. Other recent variants of MCL include iTRAQ [48] and isotope-differentiated binding energy shift tags (IDBEST) [49]. With iTRAQ up to four different protein samples may be coded by reporter-specific reactive groups that covalently bind to lysine side chains and N-termini. "
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    • "Stable Isotope Labeling by Amino Acid in Cell Culture) (Ong et al. 2002), GIST (Global Internal Standard Technology) (Chakraborty and Regnier 2002), I-CAT (Isotope-Coded Affinity Tags) (Gygi et al. 1999), and iTRAQ (Isobaric Tagging for Relative and Absolute Protein Quantitation) (Ross et al. 2004; Schneider and Hall 2005 "
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