Mass Spectrometry-Based Analysis of Rat Liver and Hepatocellular Carcinoma Morris Hepatoma 7777 Plasma Membrane Proteome

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• 作者：Lulu Cao ; James G. Clifton ; Werner Reutter ; Djuro Josic
• 刊名：Analytical Chemistry
• 出版年：2013
• 出版时间：September 3, 2013
• 年：2013
• 卷：85
• 期：17
• 页码：8112-8120
• 全文大小：398K
• 年卷期：v.85,no.17(September 3, 2013)
• ISSN：1520-6882

文摘

The gel-based proteomic analysis of plasma membranes from rat liver and chemically induced, malignant hepatocellular carcinoma Morris hepatoma 7777 was systematically optimized to yield the maximum number of proteins containing transmembrane domains (TMDs). Incorporation of plasma membrane proteins into a polyacrylamide 鈥渢ube gel鈥?followed by in-gel digestion of 鈥渢ube gel鈥?pieces significantly improved detection by electrospray ionization鈥搇iquid chromatography鈥搕andem mass spectrometry. Removal of less hydrophobic proteins by washing isolated plasma membranes with 0.1 M sodium carbonate enables detection of a higher number of hydrophobic proteins containing TMDs in both tissues. Subsequent treatment of plasma membranes by a proteolytic enzyme (trypsin) causes the loss of some of the proteins that are detected after washing with sodium carbonate, but it enables the detection of other hydrophobic proteins containing TMDs. Introduction of mass spectrometers with higher sensitivity, higher mass resolution and mass accuracy, and a faster scan rate significantly improved detection of membrane proteins, but the improved sample preparation is still useful and enables detection of additional hydrophobic proteins. Proteolytic predigestion of plasma membranes enables detection of additional hydrophobic proteins and better sequence coverage of TMD-containing proteins in plasma membranes from both tissues.