Department of Chemistry, Washington State University, Pullman, Washington 99164, USA.
Anal Chem. 2010 May 1;82(9):3556-66. doi: 10.1021/ac902615g.
In this paper, we present the results of proof-of-concept experiments using a novel photocleavable and mass spectrometry identifiable cross-linker pcPIR (photocleavable protein interaction reporter). pcPIR can be dissociated under UV irradiation either off- or online before the introduction to the mass spectrometers. Photo dissociation of cross-linkers is different from either the gas phase or the chemical cleavage of cross-linkers. Different types of cross-links can be identified using the pcPIR mass relationships, where the mass of cross-linked precursor equals the sum of the masses of the released products and reporter. Since pcPIR is cleaved prior to the entrance to the mass spectrometer, the released peptides are available to be sequenced with routine collision-induced dissociation (CID) MS/MS experiments and database search algorithms. In this report, the pcPIR strategy of identifying the cross-linked peptides with on- and off-line photocleavage coupled with novel targeted data dependent LC-MS/MS is demonstrated with the use of standard peptides, bovine serum albumin (BSA), and human hemoglobin tetramer protein complex.
在本文中，我们展示了使用一种新型光可裂解和质谱可识别交联剂 pcPIR（光可裂解蛋白质相互作用报告器）进行概念验证实验的结果。pcPIR 可在引入质谱仪之前，无论是离线还是在线，在 UV 照射下解离。光解交联剂不同于气相或化学切割交联剂。使用 pcPIR 质量关系可以识别不同类型的交联剂，其中交联前体的质量等于释放产物和报告器的质量之和。由于 pcPIR 在进入质谱仪之前被切割，因此释放的肽可以通过常规的碰撞诱导解离 (CID) MS/MS 实验和数据库搜索算法进行测序。在本报告中，使用标准肽、牛血清白蛋白 (BSA) 和人血红蛋白四聚体蛋白质复合物，展示了 pcPIR 策略，通过在线和离线光裂解结合新型靶向数据相关 LC-MS/MS 鉴定交联肽。
