Development of Novel Free Radical Initiated Peptide Sequencing Reagent: Application to Identification and Characterization of Peptides by Mass Spectrometry
Development of Novel Free Radical Initiated Peptide Sequencing Reagent: Application to Identification and Characterization of Peptides by Mass Spectrometry
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DOI：
10.1007/s13361-018-2114-8
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发表时间：
2019-03-01
影响因子：
3.2
通讯作者：
Gao, Jinshan
中科院分区：
文献类型：
--
作者：
Gaspar, Kaylee;Fabijanczuk, Kimberly;Gao, Jinshan
FRIPs-MS技术通过将高质子亲和力部分引入到电荷定位的自由基起始肽序列(CL-fRIPs)试剂中，将其应用范围扩展到疏水性多肽和不含碱性氨基酸残基(赖氨酸、精氨酸和组氨酸)的多肽。其中，CL-fRIPs试剂有三个部分：(1)吡啶作为定位质子的碱性部位，(2)2，2，6，6-四甲基哌啶-1-氧(TEMPO，一种稳定的自由基)作为自由基前体，在气相中产生新生的自由基，(3)N-羟基琥珀酰亚胺(NHS)活化的羧酸作为多肽N端衍生的偶联部位。CL-fRIPs试剂可以通过产生测序离子、酶裂解样自由基引起的侧链损失以及通过一步碰撞激活同时失去节奏来对多肽进行表征。进一步的酶促裂解样自由基诱导的侧链缺失离子的碰撞活化为多肽的结构测定提供了更多的信息。以牛胰岛素为模型多肽，验证了CL-fRIPs试剂在多肽分析中的应用。得到了牛胰岛素的骨架结构和各链的测序信息。
By incorporating a high proton affinity moiety to the charge localized free radical-initiated peptide sequencing (CL-FRIPS) reagent, FRIPS-MS technique has extended the applicability to hydrophobic peptides and peptides without basic amino acid residues (lysine, arginine, and histidine). Herein, the CL-FRIPS reagent has three moieties: (1) pyridine acting as the basic site to locate the proton, (2) 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO, a stable free radical) acting as the free radical precursor to generate the nascent free radical in the gas phase, and (3) N-hydroxysuccinimide (NHS) activated carboxylic acid acting as the coupling site to derivatize the N-terminus of peptides. The CL-FRIPS reagent allows for the characterization of peptides by generating sequencing ions, enzymatic cleavage-like radical-induced side chain losses, and the loss of TEMPO simultaneously via one-step collisional activation. Further collisional activation of enzymatic cleavage-like radical-induced side chain loss ions provides more information for the structure determination of peptides. The application of CL-FRIPS reagent to characterize peptides is proved by employing bovine insulin as the model peptide. Both scaffold structure of bovine insulin and sequencing information of each chain are achieved.