Proteolytic 18O Labeling for Comparative Proteomics: Model Studies with Two Serotypes of Adenovirus

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• 作者：Xudong Yao ; Amy Freas ; Javier Ramirez ; Plamen A. Demirev ; and Catherine Fenselau
• 刊名：Analytical Chemistry
• 出版年：2001
• 出版时间：July 1, 2001
• 年：2001
• 卷：73
• 期：13
• 页码：2836 - 2842
• 全文大小：93K
• 年卷期：v.73,no.13(July 1, 2001)
• ISSN：1520-6882

文摘

A new method for proteolytic stable isotope labeling isintroduced to provide quantitative and concurrent comparisons between individual proteins from two entireproteome pools or their subfractions. Two ¹⁸O atoms areincorporated universally into the carboxyl termini of alltryptic peptides during the proteolytic cleavage of allproteins in the first pool. Proteins in the second pool arecleaved analogously with the carboxyl termini of theresulting peptides containing two ¹⁶O atoms (i.e., nolabeling). The two peptide mixtures are pooled for fractionation and separation, and the masses and isotoperatios of each peptide pair (differing by 4 Da) are measured by high-resolution mass spectrometry. Short sequences and/or accurate mass measurements combinedwith proteomics software tools allow the peptides to berelated to the precursor proteins from which they arederived. Relative signal intensities of paired peptidesquantify the expression levels of their precursor proteinsfrom proteome pools to be compared, using an equationdescribed in the paper. Observation of individual (unpaired) peptides is mainly interpreted as differentialmodification or sequence variation for the protein fromthe respective proteome pool. The method is evaluatedhere in a comparison of virion proteins for two serotypes(Ad5 and Ad2) of adenovirus, taking advantage of information already available about protein sequences andconcentrations. In general, proteolytic ¹⁸O labeling enables a shotgun approach for proteomic studies withquantitation capability and is proposed as a useful toolfor comparative proteomic studies of very complex proteinmixtures.