Chemical cross-linking with thiol-cleavable reagents combined with differential mass spectrometric peptide mapping -: A novel approach to assess intermolecular protein contacts
Chemical cross-linking with thiol-cleavable reagents combined with differential mass spectrometric peptide mapping -: A novel approach to assess intermolecular protein contacts
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DOI：
10.1110/ps.9.8.1503
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发表时间：
2000-08-01
期刊：
影响因子：
8
通讯作者：
Roepstorff, P
中科院分区：
文献类型：
--
作者：
Bennett, KL;Kussmann, M;Roepstorff, P
采用化学交联和差示MALDI-MS分析相结合的方法，研究了同源二聚体DNA结合蛋白PARR单体之间的分子间接触区以及糖蛋白CD28和CD80之间的相互作用。用硫醇可裂解交联剂3，3‘-二硫代双(琥珀酸亚胺)(DTSSP)体外修饰PAR二聚体，用胰酶消化PAR二聚体，并用MALDI-MS进行肽谱分析。在硫醇试剂存在和不存在的情况下，比较消化的交联型Parr二聚体得到的肽图强烈支持二聚体复合体中单体的“头尾”排列。用DTSSP体外交联CD28-Ig G和CD80-F-ab，用非还原SDS-PAGE鉴定，胰酶原位消化，MALDI-MS(+/-硫醇试剂)肽图谱分析。这些数据揭示了糖蛋白结构的受体区域之间存在分子间的交联键，以及CD28-Ig G的融合结构域和CD80-F-ab的受体结构域之间存在一些意想不到但却具有特异性的相互作用，化学交联结合差异MALDI-MS肽图(+/-硫醇试剂)的策略使得能够定位所研究的络合物的界面区域(S)，并清楚地展示了这种方法在获得相互作用的非共价络合物结构信息方面的有效性。
The intermolecular contact regions between monomers of the homodimeric DNA binding protein ParR and the interaction between the glycoproteins CD28 and CD80 were investigated using a strategy that combined chemical crosslinking with differential MALDI-MS analyses. ParR dimers were modified in vitro with the thiol-cleavable cross-linker 3,3'-dithio-bis(succinimidylproprionate) (DTSSP), proteolytically digested with trypsin and analyzed by MALDI-MS peptide mapping. Comparison of the peptide maps obtained from digested cross-linked ParR dimers in the presence and absence of a thiol reagent strongly supported a "head-to-tail" arrangement of the monomers in the dimeric complex. Glycoprotein fusion constructs CD28-IgG and CD80-F-ab were cross-linked in vitro by DTSSP, characterized by nonreducing SDS-PAGE, digested in situ with trypsin and analyzed by MALDI-MS peptide mapping (+/- thiol reagent). The data revealed the presence of an intermolecular cross-link between the receptor regions of the glycoprotein constructs, as well as a number of unexpected but nonetheless specific interactions between the fusion domains of CD28-IgG and the receptor domain of CD80-F-ab, The strategy of chemical cross-linking combined with differential MALDI-MS peptide mapping (+/- thiol reagent) enabled localization of the interface region(s) of the complexes studied and clearly demonstrates the utility of such an approach to obtain structural information on interacting noncovalent complexes.