Josic Djuro, Brown Mari Kino, Huang Feilei, Lim Yow-Pin, Rucevic Marijana, Clifton James G, Hixson Douglas C
Proteomics Core, COBRE Center for Cancer Research Development, Rhode Island Hospital, Providence, RI 02904, USA.
Proteomics. 2006 May;6(9):2874-85. doi: 10.1002/pmic.200500563.
Inter-alpha inhibitor proteins (IaIp) are a family of structurally related serine protease inhibitors found in relatively high concentrations in human plasma. Recent studies have implicated a role for IaIp in sepsis, and have demonstrated their potential as biomarkers in sepsis and cancer. For characterization of isolated IaI proteins and contaminating proteins during the last steps of the purification process, SELDI-TOF MS and HPLC-ESI-MS/MS were used. After separation by SDS-PAGE or 2-DE, polypeptide bands of 80, 125 and 250 kDa were excised from gels and digested by trypsin. The tryptic peptides were analyzed by both MS methods. The main contamination during the purification process, a band of 80 kDa, contains mainly IaIp heavy chain (HC) H3. HC H1 and H2 were also found in this band. In addition, some vitamin K-dependent clotting factors and inhibitors and other plasma proteins were identified. The 125-kDa band, representing the pre-alpha inhibitor, was found to contain both bikunin and HC H3. The presence of other HC H1, H2 and the recently described HC H4 was also detected by SELDI-TOF MS. The presence of HC H1, H2, and H3 in the 125-kDa band was confirmed by ESI-MS/MS, but not the presence of the H4. Three polypeptides, H1 and H2 together with bikunin, were identified in the 250-kDa band, representing the ITI, by both MS techniques. Once again, the presence of H4 was detected in this band only by SELDI-TOF MS, but the number of corresponding peptides was still not sufficient for final identification of this polypeptide. The importance of the application of proteomic methods for the proper evaluation of therapeutic drugs based on human plasma is discussed.
α-抑制剂间蛋白（IaIp）是一类结构相关的丝氨酸蛋白酶抑制剂家族，在人血浆中浓度相对较高。最近的研究表明IaIp在脓毒症中发挥作用，并证明了它们作为脓毒症和癌症生物标志物的潜力。在纯化过程的最后步骤中，为了鉴定分离出的IaI蛋白和污染蛋白，使用了表面增强激光解吸电离飞行时间质谱（SELDI-TOF MS）和高效液相色谱-电喷雾串联质谱（HPLC-ESI-MS/MS）。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）或双向电泳（2-DE）分离后，从凝胶中切下80、125和250 kDa的多肽条带，并用胰蛋白酶消化。通过两种质谱方法分析胰蛋白酶肽段。纯化过程中的主要污染物，一条80 kDa的条带，主要包含IaIp重链（HC）H3。在该条带中也发现了HC H1和H2。此外，还鉴定出一些维生素K依赖性凝血因子和抑制剂以及其他血浆蛋白。代表前α抑制剂的125 kDa条带被发现同时含有比基尼和HC H3。表面增强激光解吸电离飞行时间质谱还检测到其他HC H1、H2和最近描述的HC H4的存在。电喷雾串联质谱证实了125 kDa条带中存在HC H1、H2和H3，但未证实H4的存在。通过两种质谱技术在代表间α胰蛋白酶抑制剂（ITI）的250 kDa条带中鉴定出三种多肽，即H1、H2和比基尼。同样，仅通过表面增强激光解吸电离飞行时间质谱在该条带中检测到H4的存在，但相应肽段的数量仍不足以最终鉴定该多肽。讨论了蛋白质组学方法在基于人血浆正确评估治疗药物方面的重要性。
