Yang T, Horejsh D R, Mahan K J, Zaluzec E J, Watson T J, Gage D A
Department of Chemistry, University of Wisconsin-Eau Claire 54702, USA.
Anal Biochem. 1996 Nov 1;242(1):55-63. doi: 10.1006/abio.1996.0427.
The combined use of trypsin digestion and peptide mass mapping by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is reported here as an effective and rapid means for identifying the cross-linking sites in human oxy hemoglobin A (HbA) cross-linked with either bis(3,5-dibromosalicyl)-succinate or -glutarate. MALDI-MS analysis of a nondigested sample of oxy HbA modified with bis(3,5-dibromosalicyl)-glutarate showed that cross-linking only occurred between the beta 1- and beta 2-protomers and not between alpha 1- and alpha 2- or alpha- and beta-protomers, along with a modification reaction on an un-cross-linked beta-chain. Results of the MALDI tryptic peptide mass maps of cross-linked hemoglobins showed several cross-linked peptides having masses consistent with: beta Val67-Lys95-XL-beta Val67-Lys95, beta Val67-Lys95-XL-beta Val67-Arg104, beta Val67-Arg104-XL-beta Val67-Arg104, where XL represents the succinyl or glutaryl bridging span moiety. Each of these peptides contains Lys82, the targeted residue for these reagents, substantiating the cross-linking sites at beta 1Lys82-beta 2Lys82. This approach in general will enable rapid identification of the cross-linking sites in engineered proteins or intracellularly recombinant cross-linked proteins when the mass of the cross-linker and the protein primary structure are known.
本文报道了胰蛋白酶消化与基质辅助激光解吸/电离质谱法（MALDI-MS）肽质量图谱相结合的方法，作为一种有效且快速鉴定与人氧合血红蛋白A（HbA）交联的双（3,5-二溴水杨酸基）琥珀酸酯或戊二酸酯交联位点的手段。对用双（3,5-二溴水杨酸基）戊二酸酯修饰的未消化氧合HbA样品进行MALDI-MS分析表明，交联仅发生在β1和β2亚基之间，而非α1和α2或α和β亚基之间，同时未交联的β链上也有修饰反应。交联血红蛋白的MALDI胰蛋白酶肽质量图谱结果显示，有几种交联肽的质量与以下情况相符：βVal67-Lys95-XL-βVal67-Lys95、βVal67-Lys95-XL-βVal67-Arg104、βVal67-Arg104-XL-βVal67-Arg104，其中XL代表琥珀酰或戊二酰桥连跨度部分。这些肽中的每一个都含有Lys82，即这些试剂的靶向残基，证实了β1Lys82-β2Lys82处的交联位点。当交联剂质量和蛋白质一级结构已知时，这种方法总体上能够快速鉴定工程蛋白或细胞内重组交联蛋白中的交联位点。
