Identification of TSG-6, an Anti-Inflammatory Protein Cytokine, by Integrated On-Column Proteolytic Digestion/LC/MSMS

摘要

An on-column protein digestion/LC/MSMS method has been used to identify unique peptide fragments from tryptic digestion of mouse and human TSG-6. This method was capable of distinguishing between hTSG-6 and mTSG-6 by identifying three non-conserved tryptic peptides per proteoform: (1) Overall a digestion time of 4 minutes at 50°C provided the most abundant peptide ions. Digest fragments were monitored by LC/MSMS using a peptide MRM method monitoring three product ions per peptide to compare peptide peak areas under the varying digestion conditions. The MRM method was subsequently used to determine a preliminary LOD for TSG-6 protein of 32 fmoles (1 ng) on column using a 20 μL injection of a 50 ng/mL solution.