PROTEIN 3-NY ID METHOD OF AFFINITY LABEL, SOLID PHASE CAPTURE

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 3-Nitrotyrosine is a post-translational modification produced by the reaction of reactive nitrogen species (predominantly peroxynitrite) with tyrosine, present under physiological conditions, and in increased amounts in many diseases. Thus, 3-nitrotyrosine modification in proteins is a potential biomarker for oxidative stress. Many proteins exhibiting this modification (e.g. prostacyclin synthase and manganese superoxide dismutase) display a change in their enzymatic activity. In chemically modified proteins often times the same tyrosines are modified. These results suggest that there may be some specificity in modification. Of the currently available methods, only mass spectrometry is able to locate these sites specifically. However, only a relatively small portion of the population for a specific protein may be modified, which makes detection difficult. Even for abundant, almost fully modified proteins a nitrotyrosine or target protein specific affinity-based purification is necessary. One method of affinity capture involves the reduction of nitrotyrosine with sodium dithionite to aminotyrosine followed by reaction with an amine specific biotin label (Sulfo-NHS-S-S-Biotin) at reduced pH. The pKa of aminotyrosine is 4.7 (compared to the pKa of other protein amines being ca. 7 to 11). When the labeling reaction is performed at a reduced pH (e.g., pH 5), non-tyrosine amines are much less reactive. Once the labeled protein is digested, the labeled peptides can then be captured with an avidin/streptavidin column and eluted by reduction of the disulfide bond in the label. In our hands, this solution phase labeling method proved to have limited sensitivity. Solid-phase capture has been shown to have a higher recovery than solution-phase labeling followed by affinity capture and has the potential to eliminate several purification steps, potentially increasing the sensitivity of the method. Several matrices and linker strategies were attempted, but a modified version of a commercially available thiol capture resin proved to yield the best results. The chemistry of the resins tested was first evaluated by using a synthetic tryptic BSA peptide with an incorporated nitrotyrosine. The tagged peptide was analyzed using MALDI-TOF MS. To test the biological applicability of the resin, tetranitromethane treated bovine serum albumin (BSA) was used as a model protein. The results indicated that the chemistry for the capture of nitrotyrosine containing peptides is feasible, and 10-fold sensitivity increase over solution phase labeling and capture was achieved, but the sensitivity of the method may still not be adequate to detect endogenous levels of nitrotyrosine in a biological system. This method represents a significant step toward the goal of characterizing specific nitrotyrosine modifications in complex protein mixtures, but further optimization is needed. This project formed the core of the Phd thesis of Mr. Heibeck, who has now accepted a postdoctoral position at PNNL.