TI - PHOSphopeptide Mapping of Rex-1 Using LC-MS/MS . AB - Multiple strategies were employed to identify the PHOSphorylation sites within Rex-1 . The affinity purified S-tagged Rex-1 band was excised and treated as follows . First , the protein was subjected to trypsin enzymatic digestion . The tryptic peptides that were too large to detect were either digested further with elastase or independently digested with elastase . This combined analytical approach allowed us to obtain a detailed physical map covering 100% of the Rex-1 sequence (Fig 3A) . Our analysis identified four serine PHOSphorylation sites at Ser-36 , Ser-70 , Ser-97 and Ser-106 . We also identified three PHOSphorylated threonine residues at Thr-22 , Thr-37 and Thr-174 . Figure 3B shows a representative MS/MS spectrum of the tryptic phosPHOpeptide , which identified PHOSphorylation at Thr-174 . We did not identify tyrosine site -specific PHOSphorylation , which is consistent with an earlier report [39] .