High-Confidence de Novo Peptide Sequencing Using Positive Charge Derivatization and Tandem MS Spectra Merging
High-Confidence de Novo Peptide Sequencing Using Positive Charge Derivatization and Tandem MS Spectra Merging
复制标题
使用正电荷衍生化和串联 MS 谱图合并进行高置信度从头肽测序。
DOI：
10.1021/ac4001699
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发表时间：
2013-05-07
影响因子：
7.4
通讯作者：
Ji, Jianguo
中科院分区：
文献类型：
--
作者：
An, Mingrui;Zou, Xiao;Ji, Jianguo
从头肽测序在发现新的蛋白质序列和修饰方面具有很大的前景，但经常受到质谱解释成功率低的阻碍，这主要是由于碎片离子类型的多样性和每个离子系列的信息不足。在这里，我们描述了一种新的方法，结合了高效的尖端电荷衍生和串联MS光谱合并，这大大提高了解释的性能。TMPP-Ac-OSu（琥珀酰亚胺基氧羰基甲基三（2，4，6-三甲氧基苯基）溴化鳞）用于在尖端上的N-末端处衍生肽以降低质谱复杂性。然后，采用一种新的光谱合并方法，将碰撞诱导解离（CID）和电子转移解离（ETD）的优点联合收割机结合起来。我们将这种方法应用于大鼠C6胶质瘤细胞和鲤鱼，并在蛋白质数据库中搜索所得肽序列。然后，我们获得了数千个高置信度的肽序列，这是传统从头测序方法无法达到的水平。接下来，我们通过同源性搜索完全覆盖骨架但在数据库搜索期间不匹配的序列，鉴定了数十种新的肽序列。此外，我们随机选择了在大鼠C6细胞中发现的34个序列，并对其进行验证。最后，我们得出结论，这种新的方法，结合尖端正电荷衍生和串联质谱合并将大大促进新蛋白质的发现和非模式生物的蛋白质组分析。
De novo peptide sequencing holds great promise in discovering new protein sequences and modifications but has often been hindered by low success rate of mass spectra interpretation, mainly due to the diversity of fragment ion types and insufficient information for each ion series. Here, we describe a novel methodology that combines highly efficient on-tip charge derivatization and tandem MS spectra merging, which greatly boosts the performance of interpretation. TMPP-Ac-OSu (succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl)phosphonium bromide) was used to derivatize peptides at N-termini on tips to reduce mass spectra complexity. Then, a novel approach of spectra merging was adopted to combine the benefits of collision-induced dissociation (CID) and electron transfer dissociation (ETD) fragmentation. We applied this methodology to rat C6 glioma cells and the Cyprinus carpio and searched the resulting peptide sequences against the protein database. Then, we achieved thousands of high-confidence peptide sequences, a level that conventional de novo sequencing methods could not reach. Next, we identified dozens of novel peptide sequences by homology searching of sequences that were fully backbone covered but unmatched during the database search. Furthermore, we randomly chose 34 sequences discovered in rat C6 cells and verified them. Finally, we conclude that this novel methodology that combines on-tip positive charge derivatization and tandem MS spectra merging will greatly facilitate the discovery of novel proteins and the proteome analysis of nonmodel organisms.