Mapping highly complicated disulfide linkages and free thiols via liquid chromatography–tandem mass spectrometry (LC–MS²) is challenging because of the difficulties in optimizing sample preparation to acquire critical MS data and detecting mispairings. Herein, we report a highly efficient and comprehensive workflow using an on-line UV-induced precolumn reduction tandem mass spectrometry (UV-LC–MS²) coupled with two-stage data analysis and spiked control. UV-LC–MS² features a gradient run of acetonitrile containing a tunable percentage of photoinitiators (acetone/alcohol) that drives the sample to the MS through a UV-flow cell and reverse phase column to separate UV-induced products for subsequent fragmentation via low energy collision-induced dissociation. This allowed the alkylated thiol-containing and UV-reduced cysteine-containing peptides to be identified by a nontargeted database search. Expected or unexpected disulfide/thiol mapping was then carried out based on the search results, and data were derived from partially reduced species by photochemical reaction. Complete assignments of native and scrambled disulfide linkages of insulin, α-lactalbumin, and bovine serum albumin (BSA) as well as the free C34-BSA were demonstrated using none or single enzyme digestion. This workflow was applied to characterize unknown disulfide/thiol patterns of the recombinant cyclophilin 1 monomer (rTvCyP1 mono) from the human pathogen Trichomonas vaginalis. α-Lactalbumin was judiciously chosen as a spiked control to minimize mispairings due to sample preparation. rTvCyP1 was determined to contain a high percentage of thiol (>80%). The rest of rTvCyP1 mono were identified to contain two disulfide/thiol patterns, of which C41–C169 linkage was confirmed to exist as C53–C181 in rTvCyP2, a homologue of rTvCyP1. This platform identifies heterogeneous protein disulfide/thiol patterns in a de-novo fashion with artifact control, opening up an opportunity to characterize crude proteins for many applications.

中文翻译：

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用于映射二硫键的全面工作流程，包括游离硫醇和在线紫外线诱导的预柱还原和尖峰控制的错误检查

通过液相色谱-串联质谱（LC-MS ²）绘制高度复杂的二硫键和游离硫醇的图具有挑战性，因为难以优化样品制备以获取关键的MS数据和检测错配。在此，我们报告了一种高效且全面的工作流程，该工作流程采用在线UV诱导的柱前减少串联质谱（UV-LC-MS ²）结合两阶段数据分析和加标控制。紫外LC-MS ²乙腈的梯度运行包含可调节百分比的光引发剂（丙酮/酒精），可通过紫外流动池和反相柱将样品驱动至质谱，从而分离出紫外光诱导的产物，从而通过低能碰撞诱导的解离作用进行后续裂解。这使得可以通过非目标数据库搜索来鉴定烷基化的含巯基和紫外线降低的半胱氨酸肽。然后根据搜索结果进行了预期或未预期的二硫键/硫醇映射，数据是通过光化学反应从部分还原的物种中获得的。胰岛素，α-乳白蛋白和牛血清白蛋白（BSA）以及游离的C34-BSA的天然和加扰的二硫键的完全分配，都使用或不使用一种酶消化进行了验证。Tv CyP1 mono）来自人类病原体阴道毛滴虫。明智地选择α-乳清蛋白作为加标对照，以最大程度减少由于样品制备而造成的配对错误。r Tv CyP1被确定为包含高百分比的硫醇（> 80％）。r的其余电视CYP1单被鉴定为含有两个二硫键/硫醇图案，其中C41-C169连接被证实在R作为C53-C181存在电视CYP2，r的同源物电视CYP1。该平台识别二硫化物异构蛋白/硫醇图案从头伪像控制方式，开辟了一个机会来表征粗蛋白用于许多应用。