Cabras Tiziana, Fanali Chiara, Monteiro Joana A, Amado Francisco, Inzitari Rosanna, Desiderio Claudia, Scarano Emanuele, Giardina Bruno, Castagnola Massimo, Messana Irene
Department of Sciences Applied to Biosystems, Cagliari University, Cagliari, Italy.
J Proteome Res. 2007 Jul;6(7):2472-80. doi: 10.1021/pr0700706. Epub 2007 May 16.
Histatin 1 (His-1) derivatives showing serial mass increases of 80.0 +/- 0.1 Da were detected in human saliva by HPLC-ESI-MS. The same derivatives were also found in granules of submandibular glands and secretions of submandibular/sublingual glands, but not in granules and secretions of parotid glands. Only one phosphate group was present in His-1 and its derivatives, since treatment with alkaline phosphatase provided an 80.0 Da mass decrease. His-1 derivatives were almost completely transformed into His-1 by treatment with 1 M HCl at 100 degrees C, suggesting the presence of O-sulfotyrosine, which is more labile than phospho-Tyr to acidic hydrolysis. CE-MS analysis of pronase extensive digestion of derivatives confirmed the presence of sulfotyrosine. Derivatives were digested by trypsin, proteinase K, and protease V-8 and analyzed by different MS strategies. The results allowed locating sulfation on the last four tyrosines (Tyr 27, 30, 34, and 36). This study is the first report of the gland-specific sulfation of a salivary phosphopeptide in vivo.
通过高效液相色谱-电喷雾电离质谱（HPLC-ESI-MS）在人类唾液中检测到组蛋白1（His-1）衍生物，其分子量呈连续增加，增量为80.0±0.1 Da。在下颌下腺颗粒以及下颌下腺/舌下腺分泌物中也发现了相同的衍生物，但在腮腺颗粒和分泌物中未发现。His-1及其衍生物仅含有一个磷酸基团，因为用碱性磷酸酶处理后分子量减少了80.0 Da。在100℃下用1 M盐酸处理后，His-1衍生物几乎完全转化为His-1，这表明存在O-磺基酪氨酸，其对酸性水解的稳定性低于磷酸化酪氨酸。对衍生物进行链霉蛋白酶广泛消化后的毛细管电泳-质谱（CE-MS）分析证实了磺基酪氨酸的存在。用胰蛋白酶、蛋白酶K和蛋白酶V-8对衍生物进行消化，并通过不同的质谱策略进行分析。结果确定了最后四个酪氨酸（Tyr 27、30、34和36）发生了硫酸化。本研究是体内唾液磷酸肽腺特异性硫酸化的首次报道。
