A method for high-sensitivity peptide sequencing using postsource decay matrix-assisted laser desorption ionization mass spectrometry
A method for high-sensitivity peptide sequencing using postsource decay matrix-assisted laser desorption ionization mass spectrometry
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DOI：
10.1073/pnas.96.13.7131
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发表时间：
1999-06-22
影响因子：
11.1
通讯作者：
Lacey, MP
中科院分区：
文献类型：
--
作者：
Keough, T;Youngquist, RS;Lacey, MP
已经开发出一种使用基质辅助激光解吸电离质谱法进行从头肽测序的方法。该方法将促进需要快速测定肽或蛋白质序列的生物学研究，例如，测定翻译后修饰、鉴定从组合肽库中分离的活性化合物以及选择性鉴定蛋白质作为蛋白质组研究的一部分。该方法包括快速、一步地将磺酸基添加到胰蛋白酶肽的 N 末端，然后采集源后衰变 (PSD) 碎片离子光谱。这些衍生物旨在促进主链酰胺键的有效电荷位点引发断裂，并选择性地增强对包含分子 C 末端（y 离子）的单个片段离子系列的检测。整个方法已应用于 pmol 量的肽，所得的 PSD 片段离子光谱通常表现出 20 个或更多氨基酸残基的不间断序列。然而，Pro C 末端酰胺键处的断裂效率显着降低。光谱足够简单，从头序列标记是常规的。该技术已成功应用于肽混合物、高质量肽（高达 3,600 Da）以及从二维凝胶电泳中分离出的蛋白质的明确鉴定。这些衍生肽的 PSD 谱通常比从天然肽谱中获得的谱图更具有选择性的蛋白质序列数据库搜索。
A method has been developed for de novo peptide sequencing using matrix-assisted laser desorption ionization mass spectrometry. This method will facilitate biological studies that require rapid determination of peptide or protein sequences, e,g,, determination of posttranslational modifications, identification of active compounds isolated from combinatorial peptide libraries, and the selective identification of proteins as part of proteome studies. The method involves fast, one-step addition of a sulfonic acid group to the N terminus of tryptic peptides followed by acquisition of postsource decay (PSD) fragment ion spectra. The derivatives are designed to promote efficient charge site-initiated fragmentation of the backbone amide bonds and to selectively enhance the detection of a single fragment ion series that contains the C terminus of the molecule (y-ions), The overall method has been applied to pmol quantities of peptides, The resulting PSD fragment ion spectra often exhibit uninterrupted sequences of 20 or more amino acid residues. However, fragmentation efficiency decreases considerably at amide bonds on the C-terminal side of Pro. The spectra are simple enough that de novo sequence tagging is routine. The technique has been successfully applied to peptide mixtures, to high-mass peptides (up to 3,600 Da) and to the unambiguous identification of proteins isolated from two-dimensional gel electrophoresis, The PSD spectra of these derivatized peptides often allow far more selective protein sequence database searches than those obtained from the spectra of native peptides.