title: Selective chemoprecipitation to enrich nitropeptides from complex proteomes for mass-spectrometric analysis.
abstract: Post-translational protein nitration has attracted interest owing to its involvement in cellular signaling, effects on protein function and potential as biomarker of nitroxidative stress. We describe a procedure for enriching nitropeptides for mass spectrometry (MS)-based proteomics that is a simple and reliable alternative to immunoaffinity-based methods. The starting material for this procedure is a proteolytic digest. The peptides are reacted with formaldehyde and sodium cyanoborohydride to dimethylate all the N-terminal and side chain amino groups. Sodium dithionite is added subsequently to reduce the nitro groups to amines; in theory, the only amino groups present will have originally been nitro groups. The peptide sample is then applied to a solid-phase active ester reagent (SPAER), and those peptides with amino groups will be selectively and covalently captured. Release of the peptides on hydrolysis with trifluoroacetic acid (TFA) results in peptides that have a 4-formyl-benzamido group where the nitro group used to be. In qualitative setups, the procedure can be used to identify proteins modified by reactive nitrogen species and to determine the specific sites of their nitration. Quantitative measurements can be performed by stable-isotope labeling of the peptides in the reductive dimethylation step. Preparation of the SPAER takes about 1 d. Enrichment of nitropeptides requires about 2 d, and sample preparations need 1-30 h, depending on the experimental design. LC-MS/MS assays take from 4 h to several days and data processing can be done in 1-7 d.
authors.affiliation: 1] Department of Molecular Biology & Immunology, University of North Texas (UNT) Health Science Center, Fort Worth, Texas, USA. [2] Department of Pharmacology and Neuroscience, UNT Health Science Center, Fort Worth, Texas, USA.
authors.forename: Laszlo
authors.initials: L
authors.lastname: Prokai
authors.affiliation: Department of Molecular Biology & Immunology, University of North Texas (UNT) Health Science Center, Fort Worth, Texas, USA.
authors.forename: Jia
authors.initials: J
authors.lastname: Guo
authors.affiliation: 1] Department of Pharmacology and Neuroscience, UNT Health Science Center, Fort Worth, Texas, USA. [2] Department of Pharmaceutical Sciences, UNT System College of Pharmacy, UNT Health Science Center, Fort Worth, Texas, USA.
authors.forename: Katalin
authors.initials: K
authors.lastname: Prokai-Tatrai
mesh_terms: D011232:Chemical Precipitation; D005557:Formaldehyde / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D011499:Protein Processing, Post-Translational; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014269:Trifluoroacetic Acid / Q000737:chemistry
chemical_list: D010455:Peptides; D020543:Proteome; D005557:Formaldehyde; D014269:Trifluoroacetic Acid