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 4R-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 4R-hydroxyproline or 3S-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.

中文翻译：

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HeLa细胞的羟脯氨酸蛋白质组以蛋白质二硫键异构酶的活性位点为重点。

含有羟脯氨酸的蛋白质（胶原蛋白除外）非常罕见，很难鉴定。尽管该清单包括具有生物学重要性的例子，例如缺氧诱导因子和40S核糖体蛋白S23（RPS23），但通过生物化学方法在人蛋白质组中仅发现了14种这样的蛋白质，这两种蛋白质均具有重要的生物学功能。翻译后修饰。多缺口搜索软件Global-PTM-Discovery与常规蛋白质组数据库搜索软件的比较在正确识别包含羟脯氨酸的非胶原蛋白肽方面提高了九倍。MS-MS光谱的手动解释完善了此列表，以发现代表21种蛋白质中24个独特的羟脯氨酸位点的36种独特的肽，其中UniProt以前仅报道了RPS23的Pro62。在从人类到酵母的9个物种中，发现其中8个位点作为脯氨酸被保存下来。这些包括蛋白质二硫键异构酶（PDIA1）中的位点51和395，以及蛋白质二硫键异构酶A4（PDIA4）中的位点204和553。这些站点的表观占有率为72-89％，表明这些PTM具有结构性和功能性作用。15个位点最可能包含4R-羟脯氨酸（丝氨酸蛋白酶抑制剂H1的Pro30，天冬氨酰/天冬酰胺基β-羟化酶的Pro520，中性α-葡萄糖苷酶AB的Pro223，缺氧上调的Pro977，蛋白ERGIC-53的Pro378，蛋白252 CASC4，含溴结构域蛋白3的Pro545，含溴结构域蛋白3的Pro488，核仁RNA解旋酶的Pro130，PDIA1的Pro51，PDIA1的Pro395，PDIA3的Pro404，PDIA4的Pro89，PDIA4的Pro204和PDIA4的Pro553） 。其余位点可以是4R-羟脯氨酸或3S-羟脯氨酸。提出了改善蛋白质组学数据的自动解释的建议，以改进未来的蛋白质组学研究，其目的是挖掘蛋白质组学中剩余的更多暗物质。