High-Confidence de Novo Peptide Sequencing Using Positive Charge Derivatization and Tandem MS Spectra Merging

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• 作者：Mingrui An ; Xiao Zou ; Qingsong Wang ; Xuyang Zhao ; Jing Wu ; Li-Ming Xu ; Hong-Yan Shen ; Xueyuan Xiao ; Dacheng He ; Jianguo Ji
• 刊名：Analytical Chemistry
• 出版年：2013
• 出版时间：May 7, 2013
• 年：2013
• 卷：85
• 期：9
• 页码：4530-4537
• 全文大小：420K
• 年卷期：v.85,no.9(May 7, 2013)
• ISSN：1520-6882

文摘

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.