Splitless nanoflow hplc system

Selected Reaction Monitoring (SRM) Mass Spectrometry was used to quantify protein abundance as previously described (22 (link)). 8pmol of BSA was added to each 20μg protein sample as an internal standard. Samples were analyzed using a TSQ Vantage triple quadrupole mass spectrometer (Thermo Scientific), operated in the selected reaction monitoring mode with a splitless nanoflow HPLC system (Eksigent). Data were processed using Pinpoint to find and integrate the correct peptide chromatographic peaks. To quantify protein abundance, the relative quantity of each protein was first normalized to the BSA internal standard and then to the geometric mean of three stable cellular reference proteins: α-actin (ACTA), ribosomal protein S27a (RPS27A) and peptidyl-prolyl isomerase A (PPIA).

We utilized Selected Reaction Monitoring (SRM) Mass Spectrometry to quantify protein abundance from 2 pooled explants per animal as previously described^{23 (link)}. We determined cartilage protein concentration by the BCA Protein Assay (Thermo Scientific) following manufacturer instructions for small volumes. 3pmol equine serum albumin (ESA) (Rockland Immunochemicals) was added to each 20μg protein sample as an internal standard. Samples were separated by short-gel electrophoresis, trypsin digested, and analyzed using a TSQ Vantage triple quadrupole mass spectrometer (Thermo Scientific) operated in the selected reaction monitoring mode with a splitless nanoflow HPLC system (Eksigent). Data were processed using Pinpoint to find and integrate the correct peptide chromatographic peaks. To quantify protein abundance, the relative quantity of each protein was first normalized to the ESA internal standard and then to the geometric mean of six stable cellular reference proteins: (ATP5B, GAPDH, PPIA, RPS27A, VDAC, VIM). Loaded and tBHP treated samples were analyzed relative to the average of the site-matched unloaded control samples. The following proteins were not consistently detected in a sufficient number of samples to include in the final analysis: AKR1B1, CALR, GPX1, GPX3, GPX4, GSTA3, GSTM1, GSTP1, MSRA, NNT, PHB, PHB2.