基于质谱技术的二次化学衍生标记新方法的构建与研究

Construction and Research of New Labeling Method Using Continuous Two Chemical Derivatization Based on Mass Spectrometry

采用具有双官能团活性马来酸酐作为新的标记试剂,创新性的采用轻重马来酸酐和轻重巯基乙醇对多肽进行二次化学衍生化,利用LC-MS/MS对一次和二次标记产物进行色谱和质谱分析。通过对比标记产物色谱行为发现,马来酰化衍生明显增加了肽段的疏水性,延长了多肽在色谱柱上的保留时间,保留时间从15. 16 min延长到23. 78 min。质谱结果表明,经巯基乙醇二次标记后,衍生化产物为一分子苯丙氨酸（F）、一分子马来酸酐（MA）和一分子β-巯基乙醇（β-ME）的分子量之和,说明标记位点发生在N端氨基上并成功实现了双分子标记。利用该方法,成功实现了对Hela细胞全蛋白酶解多肽进行多重标记。通过对肽段性质研究表明,95%肽段是C端为精氨酸（R）的肽段,5%肽段是C端为赖氨酸（K）的肽段。

Maleic anhydride which has bifunctional reactive was used as a new kind of labeling reagent.Isotopic maleic anhydride and isotopic mercapto ethanol were innovatively used for two chemical derivatization,and the first and second labelled products were analyzed by chromatograph and mass spectrometry（ LC-MS/MS）. The result of the chromatographic behavior indicated that the hydrophobicity of the peptide was significantly increased by the maleic derivatization,thus the retention time of the peptide on the chromatographic column was prolonged,as a result,the retention time was extended from 15.16 min to 23.78 min. The mass spectrometry showed that the derivative product was a sum of molecular weights of phenylalanine（ F）,a molecule of maleic anhydride（ MA） and a molecule of beta mercapto ethanol（ β-ME）,which indicated that the labeling occurred on the N terminal amino group and successfully realized the double molecular labeling.Total protein modification of Hela cell were successfully realized by using this method.The results of peptide analysis showed that 95% of the peptides detected were C terminal R peptide and5% C terminal K peptide.