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.

Figure 1. Posttranslational protein nitration

Posttranslational nitration modifies (a) tyrosine¹, forming 3NT, and (b) tryptophan residues (sites of possible modifications are numbered⁷). RNS, reactive nitrogen species

Figure 2

Reaction scheme for the preparation of SPAER. Steps: (a) Succinylation of aminopropyl-modified controlled pore glass (CPG-C₃H₇-NH₂) with succinic anhydride (SA); (b) Fmoc-hydrazide formation via N-hydroxysuccinimide active ester in situ generated from N-hydroxysuccinimide (HOSu) and N,N-diisopropylcarbodiimide (DIC) followed by coupling with 9-fluorenylmethyl carbazate (Fmoc-NH-NH₂); (c) Removal of Fmoc protecting group with piperidine generates CPG-hydrazide; (d) Condensation of this immobilized hydrazide reagent with 4-formylbenzoic acid N-hydroxysuccinimide active ester (4FB-OSu) produces SPAER. The red-colored group is the active ester (AE) function, while the reaction of magenta-colored groups results in the immobilization of the AE on the surface of the CPG as hydrazide.

Figure 3

Schematic illustration of the SPAER-based enrichment procedure. This involves (a) pre- enrichment derivatizations, (b) chemoprecipitation by covalent capture of the dimethylated and reduced nitropeptides and removal of non-nitro species, and (c) producing a solution of tagged peptides by acid-catalyzed hydrolysis.

Figure 4

Scheme illustrating Steps 30 to 41 of the protocol. (Red arrow: addition of SPAER; blue arrow: transfer of solution or suspension; thin blue arrow: remove or replace; straight block arrow: move to next procedure; curved block arrow: vortex or rotate; curved and double-headed block arrow: shake.)

Figure 5

Example of anticipated results: LC–ESI-MS base-peak chromatograms of tryptic digest from in vitro nitrated human plasma proteins (a) before and (b) after SPAER-based enrichment. The latter shows significantly reduced sample complexity thereby permitting the identification of, e.g., Tyr-227 of hemopexin as a target for nitration. Without enrichment, DY^*FMPC^@PGR (where Y* represents 3NT and C^@ denotes carbamidomethylated Cys) was most likely suppressed and/or not selected for data-dependent CID-MS/MS acquisition. This was due to overwhelming matrix of non-nitro peptides burdening the instrument’s productive working cycles thereby eluding identification of this low-abundance nitropeptide. On the other hand, the enriched peptide ^#DY^ΔFMPC^@PGR (where Y^Δ denotes the tagged 3AT obtained from 3NT after enrichment and # indicates dimethylation) was the major constituent of the chromatographic peak eluting at 51.8 min, when post-run extracted-ion chromatogram (XIC) was retrieved to determine its retention time (RT). This resulted in the acquisition of CID-MS/MS spectrum from the doubly-protonated precursor m/z 659.2767, which was matched to the dimethylated and tagged peptide upon searching a human protein database using both Mascot and SEQUEST algorithms. Annotation and inspection of the MS/MS spectrum validated the identification of ^#DY^ΔFMPC^@PGR.

Figure 6

Example for relative quantification by light and heavy dimethyl labeling via the SPAER-based nitropeptide enrichment: Nitroubiquitin was added to human plasma and, after tryptic digestion, the peptides were labeled in two samples differentially by reductive dimethylation using HCHO/NaBH₃CN and D¹³CDO/NaBD₃CN, respectively, as reagents. After enrichment, the relative concentrations of the tagged light (blue shading and line) and heavy dimethyl-labeled ^#TLSDY^ΔNIQK^# (red shading and line) reflected nitroubiquitin concentrations accurately both in the (a) XICs and in the (b) narrow-range ESI-FTMS spectrum obtained through averaging over their elution period. No chromatographic isotope effect was observed.