Protein identification using sequential ion/ion reactions and tandem mass spectrometry
Protein identification using sequential ion/ion reactions and tandem mass spectrometry
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DOI：
10.1073/pnas.0503189102
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发表时间：
2005-07-05
影响因子：
11.1
通讯作者：
Hunt, DF
中科院分区：
文献类型：
--
作者：
Coon, JJ;Ueberheide, B;Hunt, DF
描述了一种通过在线色谱从 IN 和 C 末端 (1-2 s) 同时快速测序完整蛋白质的方法，并将其应用于组蛋白 H3.1 翻译后修饰的表征和 H2A 基因家族其他成员的鉴定。通过电喷雾电离将蛋白质转化为气相多电荷正离子，然后与荧蒽自由基阴离子反应。电子转移到多电荷蛋白质会促进蛋白质主链 N-C α 键的随机解离。然后，多电荷碎片离子在第二次离子/离子反应中与苯甲酸的羧酸根阴离子去质子化。所得单电荷和双电荷离子的 m1z 值用于读取蛋白质 IN 和 C 末端的 15-40 个氨基酸序列。该信息与完整蛋白质的测量质量一起用于搜索蛋白质或核苷酸数据库以寻找可能的匹配，检测翻译后修饰，并确定可能的剪接变体。
A method for rapid sequencing of intact proteins simultaneously from the IN and C termini (1-2 s) with online chromatography is described and applied to the characterization of histone H3.1 posttranslational modifications and the identification of an additional member of the H2A gene family. Proteins are converted to gas-phase multiply charged positive ions by electrospray ionization and then allowed to react with fluoranthene radical anions. Electron transfer to the multiply charged protein promotes random dissociation of the N-C alpha bonds of the protein backbone. Multiply charged fragment ions are then deprotonated in a second ion/ion reaction with the carboxylate anion of benzoic acid. The m1z values for the resulting singly and doubly charged ions are used to read a sequence of 15-40 aa at both the IN and C termini of the protein. This information, with the measured mass of the intact protein, is used to search protein or nucleotide databases for possible matches, detect posttranslational modifications, and determine possible splice variants.