【作者】 赵望泓； 张韬文； 宋容； 张瑞； 魏世成； 林久祥；

【Author】 Wanghong Zhao~1,Taowen Zhang~2,Rong Song~2,Rui Zhang~2,Shicheng Wei~(3*),Jiuxiang Lin~(3*)(1 College of Stomatology,Southern Medical University,Guangzhou 510515, China,2 School of Stomatology,Lanzhou University,Lanzhou 730000,China,3School of Stomatology,Peking University,Beijing 100081,China)

【机构】 南方医科大学口腔医学院； 兰州大学口腔医学院； 北京大学口腔医学院；

【摘要】 目的:血清蛋白在特殊生理或病理状态下往往经过剪切、翻译后修饰加工为成熟蛋白发挥生物学功能,但目前的质谱鉴定只能粗略鉴定蛋白,并不能确定蛋白的亚型及剪切片段。使用succinimidyloxyca rbo nyl methyltris(2,4,6-trimethoxyphenyl)phosphoniumbromide(TMPP-Ac-OSu)专一地对蛋白质N端进行化学标记,结合质谱分析,在给出蛋白鉴定结果的同时,也确定了蛋白质N端,达到精确鉴定蛋白的目的。方法:以C57BL/6J系孕鼠血清为例,首先利用糖苷酶酶切血清中大分子蛋白的糖链,以便于在双向电泳中分离到目标蛋白,双向电泳分离蛋白,在胶内酶解之前,使用TMPP-Ac-OSu试剂处理样品,经过简单的洗脱步骤,高效地标记蛋白N端,通过LTQ-orbitrap液质联用仪鉴定蛋白。结果:通过常规质谱鉴定的Haptoglobin、pregnancy zone protein蛋白,用我们的方法进一步精确为haptoglobinβ-subunit、pregnancy zone protein 35 kDa亚型,并发现了四种蛋白为其理论序列的剪切片段:其中三种是剪切掉信号肽的成熟蛋白,另外一种蛋白complementcompo nent 3发现了新的剪切形式。结论:通过在质谱过程中添加几步操作,在质谱鉴定的同时确定蛋白的亚型,有助于确定蛋白亚型及寻找新的剪切形式。更多还原

【Abstract】 Objective:The mass spectrometry technology has been widely used in proteins identification.However,the identification results are not very accurate,often hardly discriminate different protein subunits or determine protein isoforms being which region of the theoretical sequence,further leading to the wrong prediction of protein function, location and so on.Thus,we aimed to accurately identify proteins by determining their N-termini.Methods:In this study,2-DE separated proteins were processed using succinimidylo xycarbonylmethyl tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-Ac-OSu)before in-gel digestion.Proteins N-termini were specifically derivatized, after which they were analyzed on an LTQ-Orbitrap mass spectrometer.Results:By routine mass spectrometry analysis,two proteins from the serum of C57BL/6J pregnant mice were identified as haptoglobin and pregnancy zone protein;with our new method which were accurately determined as haptoglobinβ-subunit and pregnancy zone protein 35 kDa subunit.In addition,four proteins were found part of the theoretical sequence, three of which were determined as mature proteins not the original form with signal peptides in database,another protein named complement component 3 was found having a new N-terminus different from existing isoforms,indicating a potential novel isoform. Conclusion:In summary,we had distinguished protein subunits and protein isoforms by protein N-terminal derivatization coupled with mass spectrometry identification.更多还原