Synthesis/Degradation Ratio Mass Spectrometry for Measuring Relative Dynamic Protein Turnover

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• 作者：Benjamin J. Cargile ; Jonathan L. Bundy ; Amy M. Grunden ; and James L. Stephenson ; Jr
• 刊名：Analytical Chemistry
• 出版年：2004
• 出版时间：January 1, 2004
• 年：2004
• 卷：76
• 期：1
• 页码：86 - 97
• 全文大小：183K
• 年卷期：v.76,no.1(January 1, 2004)
• ISSN：1520-6882

文摘

One of the major unanswered questions in quantitativeproteomics is that of dynamic protein turnover in the cell.Here we present a new approach to quantitative proteomics that measures the relative dynamic turnover of proteins in cellular systems. In this approach, termed synthesis/degradation ratio mass spectrometry, stable isotopelabeling is employed to calculate a relative synthesis/degradation ratio that reflects the relative rate at which¹³C is incorporated into individual proteins in the cell.This synthesis/degradation ratio calculation is based ona Poisson distribution model that is designed to supporthigh-throughput analysis. Protein separation and analysisis accomplished by utilizing one-dimensional SDS-PAGEgel electrophoresis followed by cutting the gel into a seriesof bands for in-gel digestion. The resulting peptide mixtures are analyzed via solid-phase MALDI LC-MS andLC-MS/MS using a tandem time-of-flight mass spectrometer. A portion of the soluble protein fraction from an E.coli K-12 strain was analyzed with synthesis/degradationratios varying from approximately 0.1 to 4.4 for a varietyof different proteins. Unlike other quantitative techniques,synthesis/degradation ratio mass spectrometry requiresonly a single cell culture to obtain useful biologicalinformation about the processes occurring inside a cell.This technique is highly amenable to shotgun proteomics-based approaches and thus should allow relative turnovermeasurements for whole proteomes in the future.