We employed a cell surface “”shaving”" technique with either trypsin or
proteinase-K combined with LC-MS/MS. Trypsin-derived data were controlled using a “”false-positive”" strategy where cells were incubated without protease, removed by centrifugation and the resulting supernatants digested. Peptides check details identified in this fraction most likely result from cell lysis and were removed from the trypsin-shaved data set. We identified 42 predicted S. aureus COL surface proteins from 260 surface-exposed peptides. Trypsin and proteinase-K digests were highly complementary with ten proteins identified by both, 16 specific to proteinase-K treatment, 13 specific to trypsin and three identified in the control. The use of a subtracted false-positive strategy improved enrichment of surface-exposed peptides in the trypsin data set to approximately 80% (124/155 peptides). Predominant surface proteins were those associated with methicillin resistance surface protein SACOL0050 (pls) and
penicillin-binding protein 2′ (mecA), as well as bifunctional autolysin and the extracellular matrix-binding protein Ebh. The cell shaving strategy is a rapid method for identifying surface-exposed peptide epitopes that may be useful in the design of novel vaccines against S. aureus.”
“An emphasis of current proteomic research is the validation this website of plasma protein biomarkers. The process of blood collection itself is critical to the accuracy and reproducibility of quantitative biomarker assays. We have developed selected reaction monitoring (SRM) assays to analyse thirteen abundant PTK6 plasma proteins and evaluated the impact of three different blood collection tubes on the levels of these proteins. We also assessed the implications
of the time taken to analyse plasma samples by evaluating the recovery of these proteins. We showed that SRM detects minor differences in the levels of some proteins which can be attributed to collection tube type. The average recovery for 12 of 18 assays was higher for proteins that were collected in tubes containing protease inhibitors compared to conventional collection tubes. For five of the assays, the differential recovery was statistically significant. Delaying MS analysis of a freeze-thawed sample for 1 hour showed greatly reduced recovery of these analytes; however differences attributed to tube type were only evident at the baseline timepoint. Finally, we assessed the natural variation of circulating levels of these proteins in a cohort of seven healthy individuals. This study provides useful information for researchers contemplating blood collection for undertaking protein biomarker studies.