Yu Haiqiang, Murata Kenji, Hedrick Jerry L, T Almaraz Ruben, Xiang Fan, Franz Andreas H
Department of Chemistry, University of the Pacific, 3601 Pacific Avenue, Stockton, CA 95211, USA.
Arch Biochem Biophys. 2007 Jul 1;463(1):1-11. doi: 10.1016/j.abb.2007.02.002. Epub 2007 Feb 23.
The disulfide bonds in the galactose-specific lectin SEL 24K from the egg of the Chinook salmon Oncorhynchus tshawytscha were determined by mass spectrometry. Four predictive in silico tools were used to determine the oxidation state of cysteines in the sequence and possible location of the disulfide bonds. A combination of tryptic digestion, HPLC separation, and chemical modifications were used to establish the location of seven disulfide bonds and one pair of free cysteines. After proteolysis, peptides containing one or two disulfide bonds were identified by reduction and mass spectral comparison. MALDI mass spectrometry was supported by chemical modification (iodoacetamide) and in silico digestion. The assignments of disulfide bonds were further confirmed by mass spectral fragmentation studies including in-source dissociation (ISD) and collision-induced dissociation (CID). The experimentally determined disulfide bonds and free Cys residues were only partially consistent with those generated by several automated public-domain algorithms.
通过质谱法测定了奇努克鲑鱼（Oncorhynchus tshawytscha）卵中半乳糖特异性凝集素SEL 24K中的二硫键。使用了四种预测性计算机工具来确定序列中半胱氨酸的氧化状态以及二硫键的可能位置。采用胰蛋白酶消化、高效液相色谱分离和化学修饰相结合的方法确定了七个二硫键和一对游离半胱氨酸的位置。蛋白水解后，通过还原和质谱比较鉴定了含有一个或两个二硫键的肽段。基质辅助激光解吸电离质谱法（MALDI）得到了化学修饰（碘乙酰胺）和计算机模拟消化的支持。通过包括源内裂解（ISD）和碰撞诱导裂解（CID）在内的质谱裂解研究进一步证实了二硫键的归属。实验确定的二硫键和游离半胱氨酸残基仅与几种自动化公共领域算法生成的结果部分一致。
