Selective Chemoprecipitation and Subsequent Release of Tagged Species for the Analysis of Nitropeptides by Liquid Chromatography-Tandem Mass Spectrometry
News May 17, 2011
Tyrosine nitration is a low-abundance posttranslational protein modification that requires appropriate enrichment techniques to enable proteomic analyses. We report a simple yet highly specific method to enrich nitropeptides by chemoprecipitation involving only two straightforward chemical modifications of the nitropeptides prior to capturing the obtained derivatives with a strategically designed solid-phase active ester reagent (SPAER). Specifically, capping of the aliphatic amines in the peptides is done first by reductive methylation to preserve the charge state of peptides for electrospray ionization mass spectrometric analysis, followed by reduction of nitrotyrosines to the corresponding aminotyrosines. These peptides are then immobilized on SPAER, while other peptides carrying no free amino groups are separated from the SPAER-immobilized species by thoroughly washing the beads from which the tagged peptide derivatives can easily be released by acid-catalyzed hydrolysis at room temperature. The benefits of selective enrichment from a matrix of unmodified peptides for liquid chromatography-tandem mass spectrometry are demonstrated on three synthetic nitropeptides that are nitrated fragments of biologically relevant proteins. Identification of several in vitro nitrated human plasma proteins, also implicated under various pathological processes, by database searches from the enriched and tagged tryptic nitropeptides is presented as a practical application. We also show that converting the nitro-group to the small 4-formylbenzoylamido tag does not significantly alter fragmentation properties upon collision-induced dissociation compared to those of the native nitropeptides, and at the same time this derivatization actually improves electron capture dissociation due to conversion of the electron-predator nitro-group to this novel tag.
The article is published online in Molecular & Cellular Proteomics and is free to access.
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