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We here present a new method to measure the degree of protein-bound methionine sulfoxide formation at a proteome-wide scale. In human Jurkat cells that were stressed with hydrogen peroxide, over 2000 oxidationsensitive methionines in more than 1600 different proteins were mapped and their extent of oxidation was quantified. Meta-analysis of the sequences surrounding the oxidized methionine residues revealed a high preference for neighboring polar residues. Using synthetic methionine sulfoxide containing peptides designed according to the observed sequence preferences in the oxidized Jurkat proteome, we discovered that the substrate specificity of the cellular methionine sulfoxide reductases is a major determinant for the steady-state of methionine oxidation. This was supported by a structural modeling of the MsrA catalytic center. Finally, we applied our method onto a serum proteome from a mouse sepsis model and identi- Reactive oxygen species (ROS)1 are involved in a broad range of processes including signal transduction and gene expression (1), receptor activation (2), antimicrobial and cytotoxic actions of immune cells (3), and aging and age-related degenerative diseases (4). Cellular oxidative stress is associated with increased levels of reactive oxygen species and the molecular damages they cause (5). Of interest here is that some reactive oxygen species specifically modify targeted biomolecules, whereas others cause nonspecific damage. Peroxides for instance are generally more selective compared with hydroxyl radicals (6). Major ROS targets are proteins, with oxidation occurring both at the peptide backbone and at amino acid side-chains (6). The major oxidation product of protein-bound methionine is methionine sulfoxide, further oxidation of which can lead to methionine sulfone, albeit to a much lesser extent (7). The (patho)physiological importance of this modification is reflected by the methionine sulfoxide reductases (Msr) that are present in nearly all organisms (8, 9): decreased activity of these enzymes was associated with aging and Alzheimer disease (10), and abnormal dopamine signaling was recently found in the methionine sulfoxide reductase A knockout mouse (11). Oxidation of methionine can lead to loss of enzyme activity as shown for a brain voltagedependent potassium channel (12). Other studies suggest that methionine oxidation prevents methylation (13) or has an effect on phosphorylation on serines and threonines proximate to the oxidized site (14). In this respect, protein kinases are also targeted by methionine oxidation affecting their activity (e.g. (15)). Further, oxidation of surface methionines increases the protein surface hydrophobicity (16) and may perturb native protein folding, and such oxidized proteins further often become targets for degradation by the proteasome (17).Although methionines are utmost susceptible to oxidation by several types of ROS (18), no adequate proteomic methodologies exist to characterize the exact sites of oxidation and quantify the degree of oxidation. ...

Using a targeted peptide-centric proteomics approach, we performed in vitro protease substrate profiling of the apoptotic serine protease granzyme B resulting in the delineation of more than 800 cleavage sites in 322 human and 282 mouse substrates, encompassing the known substrates Bid, caspase-7, lupus La protein, and fibrillarin. Triple SILAC (stable isotope labeling by amino acids in cell culture) further permitted intra-experimental evaluation of species-specific variations in substrate selection by the mouse or human granzyme B ortholog. For the first time granzyme B substrate specificities were directly mapped on a proteomic scale and revealed unknown cleavage specificities, uncharacterized extended specificity profiles, and macromolecular determinants in substrate selection that were confirmed by molecular modeling. We further tackled a substrate hunt in an in vivo setup of natural killer cell-mediated cell death confirming in vitro Because macromolecular properties present in protease substrates guide cleavage recognition, specificity, and efficiency beyond canonical cleavage sites, the necessity to determine protease substrates directly in a natural proteome and even in a species-specific context strikingly became important to fully elucidate proteolytic actions. Together with recent advances in the development of protease-targeted activity-based probes, systematic high throughput methods with broad applicability for the identification of (individual) in vitro and in vivo protease substrate repertoires have recently emerged (1).Granzyme B (GrB), 1 a serine protease that recognizes aspartic acid in the substrate P1 position, is contained within the secretory granules of cytotoxic T lymphocytes and natural killer (NK) cells (2) and gains entry into transformed or virally infected target cells by the pore-forming protein perforin (3). Once delivered in targets cells, GrB can promote apoptosis either by activation of the caspase cascade (4) or by directly cleaving substrate proteins (5-10). Next to a few reported extracellular (11) and viral substrates (12) only about 60 possible cellular mammalian GrB substrates have been identified to date mainly by non-systematic approaches. Only for a few of these, physiological relevance was shown and occasionally in a species-specific context (13-15) as it was only recently found that human and mouse granzyme B signal via overlapping as well as distinct apoptotic pathways.The substrate specificity of mouse, human, and rat GrB was profiled previously by positional scanning combinatorial libraries of short tetrapeptides from P4 to P1 and, using phage display, for mouse and human GrB somewhat extended to P2Ј (13,(15)(16)(17). By further showing that Bid is a very poor substrate for mGrB, in sharp contrast to its very efficient cleavage by hGrB, contradictory results obtained by using recombinant GrB from different species were elucidated (13-15). Next to GrB, GrA was also reported to display altered substrate specificity and functionality fueled by structural differences...