Proteome Analysis of Microdissected Tumor Tissue Using a Capillary Isoelectric Focusing-Based Multidimensional Separation Platform Coupled with ESI-Tandem MS

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• 作者：Yueju Wang ; Paul A. Rudnick ; Erin L. Evans ; Jie Li ; Zhengping Zhuang ; Don L. DeVoe ; Cheng S. Lee ; and Brian M. Balgley
• 刊名：Analytical Chemistry
• 出版年：2005
• 出版时间：October 15, 2005
• 年：2005
• 卷：77
• 期：20
• 页码：6549 - 6556
• 全文大小：617K
• 年卷期：v.77,no.20(October 15, 2005)
• ISSN：1520-6882

文摘

This study demonstrates the ability to perform sensitiveproteome analysis on the limited protein quantities available through tissue microdissection. Capillary isoelectricfocusing combined with nano-reversed-phase liquid chromatography in an automated and integrated platform notonly provides systematic resolution of complex peptidemixtures based on their differences in isoelectric pointand hydrophobicity but also eliminates peptide loss andanalyte dilution. In comparison with strong cation exchange chromatography, the significant advantages ofelectrokinetic focusing-based separations include highresolving power, high concentration and narrow analytebands, and effective usage of electrospray ionization-tandem MS toward peptide identifications. Through theuse of capillary isoelectric focusing-based multidimensional peptide separations, a total of 6866 fully trypticpeptides were detected, leading to the identification of1820 distinct proteins. Each distinct protein was identified by at least one distinct peptide sequence. These highmass accuracy and high-confidence identifications weregenerated from three proteome runs of a single glioblastoma multiforme tissue sample, each run consuming only10 g of total protein, an amount corresponding to 20 000selectively isolated cells. Instead of performing multipleruns of multidimensional separations, the overall peakcapacity can be greatly enhanced for mining deeper intotissue proteomics by increasing the number of CIEFfractions without an accompanying increase in sampleconsumption.