Fully automated multidimensional reversed-phase liquid chromatography with tandem anion/cation exchange columns for simultaneous global endogenous tyrosine nitration detection, integral membrane protein characterization, and quantitative proteomics mapping in cerebral infarcts.
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-SA(C)X-RP platform was initially benchmarked through proteomic analyses of Saccharomyces cerevisiae, revealing extended proteome and protein coverage. A total of 27 144 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-SA(C)X-RP platform also improved analytical confidence in isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analyses.
Department of Chemistry, The University of Hong Kong , Hong Kong 999077, China.
Quan
Q
Quan
Department of Chemistry, The University of Hong Kong , Hong Kong 999077, China.
Samuel S W
SS
Szeto
Department of Chemistry, The University of Hong Kong , Hong Kong 999077, China.
Henry C H
HC
Law
Institute of New Drug Research and Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine, College of Pharmacy, Jinan University , Guangzhou, Guangdong 510632, China.
Zaijun
Z
Zhang
Institute of New Drug Research and Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine, College of Pharmacy, Jinan University , Guangzhou, Guangdong 510632, China.
Yuqiang
Y
Wang
Department of Chemistry, The University of Hong Kong , Hong Kong 999077, China.
Ivan K
IK
Chu
D000818:Animals; D001921:Brain / Q000378:metabolism / Q000473:pathology*; D002852:Chromatography, Ion Exchange / Q000295:instrumentation / Q000379:methods; D056148:Chromatography, Reverse-Phase / Q000295:instrumentation / Q000379:methods*; D004867:Equipment Design; D008252:Macaca fascicularis; D008297:Male; D008565:Membrane Proteins / Q000032:analysis* / Q000378:metabolism; D009574:Nitro Compounds / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D020521:Stroke / Q000378:metabolism* / Q000473:pathology; D014443:Tyrosine / Q000032:analysis* / Q000378:metabolism
D008565:Membrane Proteins; D009574:Nitro Compounds; D014443:Tyrosine