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

• Published in: Analytical chemistry (Washington) Vol. 87; no. 19; pp. 10015 - 10024
• Main Authors: Quan, Quan, Szeto, Samuel S. W., Law, Henry C. H., Zhang, Zaijun, Wang, Yuqiang, Chu, Ivan K.
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 06.10.2015
• Subjects: Analytical chemistry; Animals; Anion exchanging; Automation; Brain - metabolism; Brain - pathology; Chromatography; Chromatography, Ion Exchange - instrumentation; Chromatography, Ion Exchange - methods; Chromatography, Reverse-Phase - instrumentation; Chromatography, Reverse-Phase - methods; Equipment Design; Exchange; Macaca fascicularis; Male; Membrane Proteins - analysis; Membrane Proteins - metabolism; Membranes; Nitro Compounds - analysis; Nitro Compounds - metabolism; Peptides; Platforms; Proteins; Proteomics; Proteomics - methods; Saccharomyces cerevisiae; Stroke - metabolism; Stroke - pathology; Tyrosine; Tyrosine - analysis; Tyrosine - metabolism
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.5b02619

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.