Characterization and quantitation of protein adducts using mass spectrometry

摘要

Protein modifications by reactive intermediates may have a causal role in cellular toxicity but information about which proteins are modified is limited. In order to address identification of protein targets a novel method for detection of adducted peptides based on adduct-specific fragmentations in tandem mass spectrometry (MS/MS) was developed. This method consists of characterizing the MS/MS fragmentation of adducted-model peptides to identify adduct specific features and screening MS/MS spectra for characteristic features of adduction. Benzoquinone (BZQ) and glutathione-conjugated benzoquinone (GS-BZQ) were selected as model electrophiles to develop this method and adducts were prepared with model peptides to identify characteristic features of adduct fragmentation in MS/MS experiments. BZQ-adducted peptides fragmented to give adduct-derived fragments of ion pairs of 141/142 and 211 and a neutral loss of 142. GS-BZQ-adducted peptides fragmented to give adduct-derived fragments of neutral losses of 74, 129, 273, and 447, charged losses of 274 and 448 and ion pairs of 515 and 129. We suggest that these adduct-specific fragments can be used to detect adducted peptides. Subsequently, the data reduction algorithm SALSA was developed to screen MS/MS spectra for spectral characteristics including neutral losses, charged losses and ion pairs in order to facilitate adduct detection. SALSA scores multiple types of spectral features simultaneously and reports a combined score for each spectrum, and search criteria can be arranged in a hierarchal manner for more selective searching. The SALSA algorithm was used to screen spectra of a BSA digest treated with GS-BZQ for fragment characteristics of GS-BZQ-adduction, and spectra from six GS-BZQ modified peptides were ranked among the top twenty highest scoring spectra. Detection of unanticipated peptide modifications is illustrated using the motif-searching algorithm of SALSA which is described and searches for patterns of product ions without regard to precursor m/ z or position along the m/z axis. Finally, because the effects of adduction may depend on its abundance in the cell, a new stable isotope label for differential quantitation of peptide adducts is described. Relative quantitation using the label is linear across a 10,000 fold range of concentration ratios, standard deviation is less than 20%, and quantitation of multiple peptides in a BSA digest is reported. Styrene oxide adducts of hemoglobin are differentially quantified using the label and a concentration/adduct curve for the formation of eight peptide adducts is plotted.