Assigning Peptide Disulfide Linkage Pattern Among Regio-Isomers via Methoxy Addition to Disulfide and Tandem Mass Spectrometry
Assigning Peptide Disulfide Linkage Pattern Among Regio-Isomers via Methoxy Addition to Disulfide and Tandem Mass Spectrometry
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DOI：
10.1007/s13361-017-1595-1
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发表时间：
2017-06-01
影响因子：
3.2
通讯作者：
Xia, Yu
中科院分区：
文献类型：
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作者：
Durand, Kirt L.;Tan, Lei;Xia, Yu
精确定位二硫键模式是表征由多个二硫键组成的蛋白质和肽的关键。在此，我们报告了一种基于在线二硫修饰和串联质谱（MS/MS）耦合的方法来区分肽二硫区域异构体。这种方法依赖于一种新的二硫键裂解反应，在溶液中，以甲醇为反应物，254 nm紫外线照射。该反应导致二硫键的选择性断裂，并在一个半胱氨酸残基上形成亚砜甲酯（-SOCH3），在另一个半胱氨酸残基上形成巯基（-SH）。在低能碰撞诱导解离（CID）下，半胱氨酸亚砜甲酯基序产生一个特征的甲醇损失（-32 Da），使其能够从其他可能的异构体结构中识别出来，如s -羟甲基（-SCH2OH）和甲基亚砜（-S(O)-CH3）。由于二硫键可以选择性地在甲氧基加成后被切割和修饰，随后的甲氧基加成产物的MS2 CID提供了增强的序列覆盖率，这在牛胰岛素的分析中得到了证明。更重要的是，这一反应不会引起二硫化物的混乱，可能是因为自由基中间体不参与这一过程。一种基于甲氧基加成后MS3 CID的方法已开发用于分配肽二硫区域异构体中的二硫键模式。该方法成功地应用于表征具有两个二硫键和三个二硫键异构体的肽系统：并排，重叠和环内环构型。
Pinpointing disulfide linkage pattern is critical in the characterization of proteins and peptides consisting of multiple disulfide bonds. Herein, we report a method based on coupling online disulfide modification and tandem mass spectrometry (MS/MS) to distinguish peptide disulfide regio-isomers. Such a method relies on a new disulfide bond cleavage reaction in solution, involving methanol as a reactant and 254 nm ultraviolet (UV) irradiation. This reaction leads to selective cleavage of a disulfide bond and formation of sulfenic methyl ester (-SOCH3) at one cysteine residue and a thiol (-SH) at the other. Under low energy collision-induced dissociation (CID), cysteine sulfenic methyl ester motif produces a signature methanol loss (-32 Da), allowing its identification from other possible isomeric structures such as S-hydroxylmethyl (-SCH2OH) and methyl sulfoxide (-S(O)-CH3). Since disulfide bond can be selectively cleaved and modified upon methoxy addition, subsequent MS2 CID of the methoxy addition product provides enhanced sequence coverage as demonstrated by the analysis of bovine insulin. More importantly, this reaction does not induce disulfide scrambling, likely due to the fact that radical intermediates are not involved in the process. An approach based on methoxy addition followed by MS3 CID has been developed for assigning disulfide linkage patterns in peptide disulfide regio-isomers. This methodology was successfully applied to characterizing peptide systems having two disulfide bonds and three disulfide linkage isomers: side-by-side, overlapped, and looped-within-a-loop configurations.