The Hydroxyproline Proteome of HeLa Cells with Emphasis on the Active Sites of Protein Disulfide Isomerases.

Hydroxyproline-containing proteins (other than collagens) are rare and difficult to identify. Only 14 such proteins have been found in the human proteome by biochemical methods despite the fact that the list includes examples of biological importance such as hypoxia-inducible factor and the 40S ribosomal protein S23 (RPS23), both of which have significant biological function of the post-translational modification. Comparison of multinotch search software Global-PTM-Discovery to conventional proteomic database search software gave a nine-fold improvement in correctly identifying non-collagen peptides containing hydroxyproline. Manual interpretation of MS-MS spectra refined this list to discover 36 unique peptides representing 24 unique hydroxyproline sites in 21 proteins, of which only Pro62 of RPS23 had been reported previously in UniProt. Eight of the sites were found to be conserved as prolines in nine species examined, ranging from humans to yeast. These include sites 51 and 395 in protein disulfide-isomerase (PDIA1) and sites 204 and 553 in protein disulfide-isomerase A4 (PDIA4). The apparent occupancy of these sites ranged from 72-89%, suggesting a structural and possibly functional role of these PTMs. Fifteen of the sites most likely contain 4-hydroxyproline (Pro30 of serpin H1, Pro520 of aspartyl/asparaginyl β-hydroxylase, Pro223 of neutral α-glucosidase AB, Pro977 of hypoxia upregulated protein 1, Pro378 of protein ERGIC-53, Pro252 of protein CASC4, Pro545 of bromodomain-containing protein 2, Pro488 of bromodomain-containing protein 3, Pro130 of nucleolar RNA helicase 2, Pro51 of PDIA1, Pro395 of PDIA1, Pro404 of PDIA3, Pro89 of PDIA4, Pro204 of PDIA4, and Pro553 of PDIA4). The remaining sites could be either 4-hydroxyproline or 3-hydroxyproline. Recommendations are made to improve automated interpretation of proteomic data to improve future proteomic research whose goal is to mine more of the remaining dark matter of the proteome.