Fully Automated Multidimensional Reversed-Phase Liquid Chromatography with Tandem Anion/Cation Exchange Columns for Simultaneous Global Endogenous Tyrosine Nitration Detection, Integral Membrane Prote

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• 作者：Quan Quan ; Samuel S. W. Szeto ; Henry C. H. Law ; Zaijun Zhang ; Yuqiang Wang ; Ivan K. Chu
• 刊名：Analytical Chemistry
• 出版年：2015
• 出版时间：October 6, 2015
• 年：2015
• 卷：87
• 期：19
• 页码：10015-10024
• 全文大小：427K
• ISSN：1520-6882

文摘

Protein tyrosine nitration (PTN) is a signature hallmark of radical-induced nitrative stress in a wide range of pathophysiological conditions, with naturally occurring abundances at substoichiometric levels. In this present study, a fully automated four-dimensional platform, consisting of high-/low-pH reversed-phase dimensions with two additional complementary, strong anion (SAX) and cation exchange (SCX), chromatographic separation stages inserted in tandem, was implemented for the simultaneous mapping of endogenous nitrated tyrosine-containing peptides within the global proteomic context of a Macaca fascicularis cerebral ischemic stroke model. This integrated RP鈥揝A(C)X鈥揜P platform was initially benchmarked through proteomic analyses of Saccharomyces cerevisiae, revealing extended proteome and protein coverage. A total of 27鈥?44 unique peptides from 3684 nonredundant proteins [1% global false discovery rate (FDR)] were identified from M. fascicularis cerebral cortex tissue. The inclusion of the S(A/C)X columns contributed to the increased detection of acidic, hydrophilic, and hydrophobic peptide populations; these separation features enabled the concomitant identification of 127 endogenous nitrated peptides and 137 transmembrane domain-containing peptides corresponding to integral membrane proteins, without the need for specific targeted enrichment strategies. The enhanced diversity of the peptide inventory obtained from the RP鈥揝A(C)X鈥揜P platform also improved analytical confidence in isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analyses.