多肽间非共价作用的质谱研究

Study on Non-covalent Interaction of Polypeptides by Mass Spectrometry

为了研究多肽间的非共价作用,将多肽按摩尔比1∶1混合,待平衡后用电喷雾电离质谱分析多肽间的相互作用。结果表明,其中部分五肽相互作用未形成非共价复合物,也有部分五肽相互作用容易形成非共价复合物,通过改变五肽的氨基酸序列和加长肽链后,用电喷雾电离质谱分析多肽间作用的变化,研究巯基、氢键和疏水作用对多肽非共价作用的影响。五肽Ⅱ-5(Ile-Leu-Gly-Tyr-Ile)和IP-5(Ile-Phe-Thr-Thr-Pro)相互作用会得到较强的非共价复合物峰,用甘氨酸Gly替换IP-5中疏水支链氨基酸苯丙氨酸Phe形成五肽IP′-5(Ile-Gly-Thr-Thr-Pro),替换含羟基侧链苏氨酸Thr形成五肽IP″-5(Ile-Phe-Gly-Thr-Pro)后,五肽Ⅱ-5分别与IP′-5和IP″-5相互作用,质谱信号均减弱,表明氢键作用和疏水作用在多肽的相互作用中能增强多肽间的非共价结合。五肽AK-5(Ala-Val-Ile-Phe-Lys)和ED-5(Glu-Ile-Cys-Ala-Asp)相互作用无非共价复合物质谱信号,用不含巯基侧链的异亮氨酸Ile替换ED-5中含巯基侧链的半胖氨酸Cys,氨基酸序列变为ED′-5(Glu-Ile-Ile-Ala-Asp),AK-5与ED′-5混合作用,非共价作用增强。另外,增加肽链中羟基和疏水基团,研究肽链加长对多肽相互作用的影响,加长五肽ED-5为九肽ED-9(Ala-Lys-Glu-Ile-Cys-Ala-Asp-Pro-Lys),五肽AK-5为九肽AK-9(Lys-Glu-Ala-Val-Ile-Phe-Lys-Thr-Ile),AK-5与ED-9、AK-9与ED-5仍无非共价结合,表明含巯基的多肽分子间容易形成二硫键,阻碍多肽分子与其他多肽的非共价结合,增强多肽分子间的氢键和疏水作用后,巯基的阻碍作用仍占主要地位。

In order to study the non-covalent interaction between peptides, the peptides were mixed in the molar ratio of 1∶1, and the interaction between peptides were analyzed by electrospray ionization mass spectrometry after equilibrium. The results showed that some of the pentapeptides did not interact with each other to form non-covalent complexes, and another part of the pentapeptides could easily form non-covalent complexes through interaction. After the amino acid sequence of peptides were changed and the peptide chain were lengthened, the changes of peptides interaction were analyzed by electrospray ionization mass spectrometry, and the effects of sulfhydryl group, hydrogen bond and hydrophobic action on the non-covalent action of peptides were studied. The pentapeptide Ⅱ-5(Ile-Leu-Gly-Tyr-Ile) and IP-5(Ile-Phe-Thr-Thr-Pro) interacted with each other and strong non-covalent complex peaks were obtained. The hydrophobic branching amino acid phenylalanine Phe in IP-5 was replaced by glycine Gly to become pentapeptide IP′-5(Ile-Gly-Thr-Thr-Pro), and the hydroxyside chain threonine Thr was replaced by glycine Gly to become pentapeptide IP″-5(Ile-Phe-Gly-Thr-Pro), the pentapeptide Ⅱ-5 interacted with IP′-5 and IP′-5, respectively, the spectral signals of non-covalent bond composites were weakened. The results showed that hydrogen bonding and hydrophobic interaction could enhance the non-covalent binding between peptides. The pentapeptide AK-5(Ala-Val-Ile-Phe-Lys) and ED-5(Glu-Ile-Cys-Ala-Asp) did not interact with each other. The semipatropine Cys containing sulfhydryl side chain in ED-5 was replaced by Ile without sulfhydryl side chain, and the amino acid sequence was changed to ED′-5(Glu-Ile-Ile-Ala-Asp). When AK-5 was mixed with ED′-5,the non-covalent effect of AK-5 and ED′-5 was stronger than that of AK-5 and ED-5. Increased the hydroxyl group and hydrophobic group in the polypeptide chain, and the influences of the peptide chain′s length on the polypeptide interaction were studied. By lengthening the peptide chain, the pentapeptide ED-5 was changed into nine-peptide ED-9(Ala-Glu-Ala-Ile-Cys-Ala-Asp-Pro-Lys), and AK-5 was changed into AK-9(Lys-Glu-Ala-Val-Ile-Phe-Lys-Thr-Ile). Then AK-5 was mixed with ED-9, AK-9 was mixed with ED-5, there was not non-covalent binding. The results showed that hydrogen bonding and hydrophobicity can enhance the non-covalent binding between peptides, and the thiol group is easy to form disulfide bonds between peptides, which hinders the non-covalent binding between different peptides. Even enhanced the hydrogen bonding and hydrophobicity were enhanced between peptides, the thiol group still plays a dominant role.