Comparison of Electrokinetics-Based Multidimensional Separations Coupled with Electrospray Ionization-Tandem Mass Spectrometry for Characterization of Human Salivary Proteins

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• 作者：Xueping Fang ; Li Yang ; Weijie Wang ; Tao Song ; Cheng S. Lee ; Don L. DeVoe ; Brian M. Balgley
• 刊名：Analytical Chemistry
• 出版年：2007
• 出版时间：August 1, 2007
• 年：2007
• 卷：79
• 期：15
• 页码：5785 - 5792
• 全文大小：206K
• 年卷期：v.79,no.15(August 1, 2007)
• ISSN：1520-6882

文摘

The foundation for saliva-based diagnostics is the development of a complete catalog of secreted proteins detectable in saliva. Besides protein complexity, the greatestbioanalytical challenge facing comprehensive analysis ofsaliva samples is related to the large variation of proteinrelative abundances including the presence of high-abundance proteins such as amylases, mucins, proline-rich proteins (PRPs), and secretory IgA complex. Amonga number of electrokinetic separation techniques, transient capillary isotachophoresis/capillary zone electrophoresis (CITP/CZE) specifically targets trace amountsof proteins and thus reduces the range of relative proteinabundances for providing unparallel advantages towardthe identification of low-abundance proteins. By employing a CITP/CZE-based multidimensional separation platform coupled with electrospray ionization-tandem massspectrometry (ESI-tandem MS), a total of 6112 fullytryptic peptides are sequenced at a 1% false discovery rate(FDR), leading to the identification of 1479 distincthuman SwissProt protein entries. By comparing withcapillary isoelectric focusing (CIEF) as another electrokinetics-based stacking approach, CITP/CZE not onlyoffers a broad field of application but also is less prone toprotein/peptide precipitation during the analysis. Theultrahigh resolving power of CITP/CZE is evidenced bythe large number of distinct peptide identifications measured from each CITP fraction together with the lowpeptide fraction overlapping among identified peptides.Furthermore, when evaluating the protein sequence coverage by the number of distinct peptides mapping to eachprotein identification, the CITP-based proteome technology similarly achieves the superior performance with 674proteins (46%) having three or more distinct peptides,288 (19%) having two distinct peptides, and 517 (35%)having a single distinct peptide.