Proteomic characterization of inter-alpha inhibitor proteins from human plasma
Proteomic characterization of inter-alpha inhibitor proteins from human plasma
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DOI：
10.1002/pmic.200500563
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发表时间：
2006-05-01
期刊：
影响因子：
3.4
通讯作者：
Hixson, DC
中科院分区：
文献类型：
--
作者：
Josic, D;Brown, MK;Hixson, DC
α间抑制蛋白（IaIp）是在人血浆中以相对高的浓度发现的结构相关的丝氨酸蛋白酶抑制剂家族。最近的研究已经暗示了IaIp在脓毒症中的作用，并且已经证明了它们作为脓毒症和癌症中的生物标志物的潜力。为了表征纯化工艺最后步骤期间分离的IAPs蛋白和污染蛋白，使用SELDI-TOF MS和HPLC-ESI-MS/MS。经SDS PAGE或2-DE分离后，从凝胶上切下80、125和250 kDa的多肽条带，并用胰蛋白酶消化。通过两种MS方法分析胰蛋白酶肽。纯化过程中的主要污染物为80 kDa条带，主要含有IaIp重链（HC）H3。HC H1和HC H2也出现在该带中。此外，还鉴定了一些维生素K依赖性凝血因子和抑制剂以及其他血浆蛋白。发现代表前α抑制剂的125-kDa条带含有bikunin和HC H3。还通过SELDI-TOF MS检测到其他HC H1、H2和最近描述的HC H4的存在。通过ESI-MS/MS确认了125 kDa条带中存在HC H1、H2和H3，但不存在H4。三个多肽，H1和H2与bikunin一起，被确定在250 kDa的带，代表ITI，通过两种MS技术。同样，仅通过SELDI-TOF MS在该条带中检测到H4的存在，但是相应肽的数量仍然不足以最终鉴定该多肽。蛋白质组学方法的应用的重要性进行了讨论，以适当的评价基于人血浆的治疗药物。
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 SDSPAGE 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.