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...

False positive peptide identifications are a major concern in the field of peptidecentric, mass spectrometry-driven gel-free proteomics. They occur in regions where the score distributions of true positives and true negatives overlap. Removal of these false positive identifications necessarily involves a trade-off between sensitivity and specificity. Existing postprocessing tools typically rely on a fixed or semifixed set of assumptions in their attempts to optimize both the sensitivity and the specificity of peptide and protein identification using MS/MS spectra. Because of the expanding diversity in available proteomics technologies, however, these postprocessing tools often struggle to adapt to emerging technology-specific peculiarity. Here we present a novel tool named Peptizer that solves this adaptability issue by making use of pluggable assumptions. This research-oriented postprocessing tool also includes a graphical user interface to perform efficient manual validation of suspect identifications for optimal sensitivity recovery. Peptizer is open source software under the Apache2 license and is written in Java.

Objective: Constitutively activating mutations of the thyrotropin receptor (TSHR) have been found in the majority of autonomously functioning thyroid nodules (AFTNs) in European patients. The reported frequency of these mutations varies among reports but amounts to 50 -80%. To date, only one such mutation responsible for AFTNs has been identified in the Japanese population and the pathogenic role of such mutations in Japanese AFTNs has been questioned. In the present study, we evaluated the frequency of activating mutations in the TSHR and Gas in 10 Japanese AFTNs. Design: Genomic DNA was extracted from fresh frozen tissue. The TSHR and the almost entire sequence of the gene coding for the a subunit of Gs have been amplified and sequenced. Results: In sequence analysis, four mutations in the TSHR (T632A, I486M, M453T and L512R) were found. To complete our analysis, we searched mutations in the gene coding for the a subunit of Gs, in the samples negative for TSHR mutations. In one case a mutation (R201H) affecting GTPase activity was found. Conclusions: If we focus on the solitary nodules, we obtain the same mutation proportion as in European patients (70%). The absence of TSHR and Gas mutations in a significant proportion of autonomous adenomas in multinodular goiters suggests that other causes may also play a role in the genesis of these lesions.