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.
Anal Chem
; 87(19): 10015-24, 2015 Oct 06.
Article
em En
| MEDLINE
| ID: mdl-26335518
ABSTRACT
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 27â¯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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Tirosina
/
Encéfalo
/
Acidente Vascular Cerebral
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Cromatografia de Fase Reversa
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Proteínas de Membrana
/
Nitrocompostos
Tipo de estudo:
Diagnostic_studies
Limite:
Animals
Idioma:
En
Ano de publicação:
2015
Tipo de documento:
Article