Quantitative analysis of complex protein mixtures using isotope-coded affinity tags

Gygi, Steven P.; Rist, Beate; Gerber, Scott A.; Tureček, František; Gelb, Michael H.; Aebersold, Ruedi

doi:10.1038/13690

Cited by 4,651 publications

(3,549 citation statements)

References 23 publications

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“…2.0 µl of sample was loaded onto the biphasic column. 1,2 Peptides were fractionated from the SCX packing onto C 18 . This plot was from fraction one out of seven.…”

Section: Resultsmentioning

confidence: 99%

Reconstructed protein arrays from 3D HPLC/tandem mass spectrometry and 2D gels: complementary approaches to Porphyromonas gingivalis protein expression

Wang

Zhang

Chen

et al. 2002

Analyst

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We compare typical qualitative protein identification data from two-dimensional (2D) polyacrylamide gel electrophoresis and reconstructed protein arrays, in the context of measuring protein expression by the Gram-negative periodontal pathogen Porphyromonas gingivalis. The arrays were assembled computationally from genome annotations and tandem mass spectrometry data from an off-line HPLC fractionation combined with 2D capillary HPLC analysis of whole proteome enzymatic digests. The 2D separation was carried out with a standard binary gradient HPLC system, modified only slightly with readily available components. Compared to 2D gels, the number of annotated open reading frames identified using the 3D HPLC approach was typically larger by at least a factor of 30. However, the newer technology is currently limited in its ability to reflect the many protein variants derived from posttranscriptional and posttranslational processing.

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“…2.0 µl of sample was loaded onto the biphasic column. 1,2 Peptides were fractionated from the SCX packing onto C 18 . This plot was from fraction one out of seven.…”

Section: Resultsmentioning

confidence: 99%

Reconstructed protein arrays from 3D HPLC/tandem mass spectrometry and 2D gels: complementary approaches to Porphyromonas gingivalis protein expression

Wang

Zhang

Chen

et al. 2002

Analyst

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show abstract

“…Using light or heavy isotope-labeled peptide 'tagging' reagents that differ only in molecular mass, proteins derived from normal/diseased or untreated/treated samples can be quantified and identified using LC-MS/MS. 39 The tagging reagents contain a cysteine-reactive group at one end and a biotin tag at the other. After mixing the differently labeled proteins together, they are digested with trypsin, and the cysteine-containing peptides are purified over an avidin column.…”

Section: Lc/ms Methods and Icatmentioning

confidence: 99%

Pharmacoproteomics in drug development

Witzmann

Grant

2003

Pharmacogenomics J

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The field of proteomics is taking on increased significance as the relevance of investigating and understanding protein expression in disease and drug development is appreciated. Recent advances in proteomics have been driven by the availability of numerous annotated whole-genome sequences and a broad range of technological and bioinformatic developments that underscore the complexity of the proteome. This review briefly addresses some of the various technologies that comprise Expression Proteomics and Functional Proteomics, citing examples where these emerging approaches have been applied to pharmacology, toxicology, and the development of drugs.

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“…Then the biotinylated proteins or peptides can be purified by streptavidin-agarose for further analysis. Isotope-coded affinity tag (ICAT) reagents, reactive to free thiols, have been used extensively in quantitative proteomics (Gygi et al, 1999). Here, we replaced the biotin-HPDP in the original biotin-switch method with ICAT reagents following with mass spectrometry detection, allowing quantification of Snitrosated proteins in samples at the proteomic level.…”

Section: Introductionmentioning

confidence: 99%

Quantitative proteomic analysis of S-nitrosated proteins in diabetic mouse liver with ICAT switch method

et al. 2010

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In this study we developed a quantitative proteomic method named ICAT switch by introducing isotope-coded affinity tag (ICAT) reagents into the biotin-switch method, and used it to investigate S-nitrosation in the liver of normal control C57BL/6J mice and type 2 diabetic KK-Ay mice. We got fifty-eight S-nitrosated peptides with quantitative information in our research, among which thirty-seven had changed S-nitrosation levels in diabetic mouse liver. The S-nitrosated peptides belonged to fortyeight proteins (twenty-eight were new S-nitrosated proteins), some of which were new targets of S-nitrosation and known to be related with diabetes. S-nitrosation patterns were different between diabetic and normal mice. Gene ontology enrichment results suggested that S-nitrosated proteins are more abundant in amino acid metabolic processes. The network constructed for Snitrosated proteins by text-mining technology provided clues about the relationship between S-nitrosation and type 2 diabetes. Our work provides a new approach for quantifying S-nitrosated proteins and suggests that the integrative functions of S-nitrosation may take part in pathophysiological processes of type 2 diabetes.

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Quantitative analysis of complex protein mixtures using isotope-coded affinity tags

Cited by 4,651 publications

References 23 publications

Reconstructed protein arrays from 3D HPLC/tandem mass spectrometry and 2D gels: complementary approaches to Porphyromonas gingivalis protein expression

Reconstructed protein arrays from 3D HPLC/tandem mass spectrometry and 2D gels: complementary approaches to Porphyromonas gingivalis protein expression

Pharmacoproteomics in drug development

Quantitative proteomic analysis of S-nitrosated proteins in diabetic mouse liver with ICAT switch method

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