De Novo Sequencing of Tryptic Phosphopeptides Using Matrix-Assisted Laser Desorption/Ionization Based Tandem Mass Spectrometry with Hydrogen Atom Attachment
De Novo Sequencing of Tryptic Phosphopeptides Using Matrix-Assisted Laser Desorption/Ionization Based Tandem Mass Spectrometry with Hydrogen Atom Attachment
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DOI：
10.1021/acs.analchem.7b04635
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发表时间：
2018-02-20
影响因子：
7.4
通讯作者：
Tanaka，Koichi
中科院分区：
文献类型：
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作者：
Asakawa，Daiki;Takahashi，Hidenori;Tanaka，Koichi
磷酸化是最丰富的蛋白质修饰，基于自由基碎片化技术的串联质谱（MS/MS）已被证明是一种很有前途的磷酸化蛋白质组学应用方法，因为它能够确定蛋白质上的磷酸化位点。自由基诱导断裂技术是指在离子阱质谱仪中将氢吸附或提取到肽上，这一过程被称为氢吸附/提取解离（HAD），这是最近才发展起来的。在本研究中，我们使用HAD-MS/MS结合基质辅助激光解吸/电离（MALDI）分析了模型磷酸肽和磷蛋白消化，以证明基于HAD-MS/MS的分析方法的有效性。将色氨酸肽分为精氨酸端肽和赖氨酸端肽，MALDI hd -MS/MS可以选择性地观察到c端侧片段离子，从而促进了精氨酸端色氨酸肽的测序。相比而言，MALDI hd -MS/MS对赖氨酸端色氨酸肽同时产生N端和c端片段，质谱较复杂。多肽的胍基化使赖氨酸转化为精氨酸，有利于MALDI hd -MS/MS质谱的解释。该方法可用于胰蛋白酶肽的从头测序。
Phosphorylation is the most abundant protein modification, and tandem mass spectrometry (MS/MS) with radical-based fragmentation techniques has proven to be a promising method for phosphoproteomic applications, owing to its ability to determine phosphorylation sites on proteins. The radical-induced fragmentation technique involves the attachment or abstraction of hydrogen to peptides in an ion trap mass spectrometer, in a process called hydrogen attachment/abstraction dissociation (HAD), which has only been recently developed. In the present investigation, we have analyzed model phosphopeptides and phosphoprotein digests using HAD-MS/MS, combined with matrix-assisted laser desorption/ionization (MALDI), in order to demonstrate the usefulness of the HAD-MS/MS-based analytical method. The tryptic peptides were categorized as arginine- and lysine-terminated peptides, and MALDI HAD-MS/MS is found to facilitate the sequencing of arginine-terminated tryptic peptides, because of the selective observation of C-terminal side fragment ions. In contrast, MALDI HAD-MS/MS of lysine-terminated tryptic peptides produced both N- and C-terminal side fragments, such that the mass spectra were complex. The guanidination of peptide converted lysine into homoarginine, which facilitated the interpretation of MALDI HAD-MS/MS mass spectra. The present method was useful for de novo sequencing of tryptic phosphopeptides.