Protein Analytical Chemistry, Genentech Inc., 1 DNA Way, South San Francisco, California 94080, United States.
Protein Metrics Inc., 20863 Stevens Creek Boulevard #450, Cupertino, California 95014, United States.
J Am Soc Mass Spectrom. 2022 Mar 2;33(3):598-602. doi: 10.1021/jasms.1c00312. Epub 2022 Feb 14.
Detection and characterization of cross-linked peptides of unknown chemical nature and location is challenging. An analytical workflow based on the use of O-labeling tryptic digestion ( 2013, 85, 5900-5908) was previously utilized to identify reduction-resistant scrambled disulfide dipeptides within an IgG that was exposed to light under forced degradation conditions ( 2018, 15, 1598-1606). The analytical workflow denoted as XChem-Finder, while effective, is cumbersome and requires extensive manual effort for detection of O-incorporated peptides and subsequent de novo sequencing of partial peptide sequences to aid in the identification of cross-linked peptides. Here, we provide an automatic workflow using Byos (Protein Metrics Inc.) to facilitate the detection of cross-linked peptides. The LC-MS/MS data files that were subjected to the XChem-Finder workflow that identified the scrambled disulfides were utilized as the test-case data set for the automated O-labeling workflow in Byos. The new workflow resulted in the detection of a photoinduced cross-linked dipeptide with unknown linker chemistry, which was subsequently identified as a cross-linked dipeptide with a novel cysteine-tryptophan (thioether) linkage. This work demonstrates that combining O-labeling tryptic digestion with the Byos workflow enables rapid detection of cross-linked dipeptides.
检测和表征未知化学性质和位置的交联肽具有挑战性。先前使用基于 O 标记胰蛋白酶消化（2013，85，5900-5908）的分析工作流程来鉴定在 IgG 中暴露于强制降解条件下的光下的还原抗性 scrambled 二硫键二肽（2018，15，1598-1606）。该分析工作流程称为 XChem-Finder，虽然有效，但繁琐，并且需要大量的手动工作来检测 O 结合肽，并随后对部分肽序列进行从头测序，以帮助鉴定交联肽。在这里，我们提供了一个使用 Byos（Protein Metrics Inc.）的自动工作流程，以方便检测交联肽。XChem-Finder 工作流程中用于鉴定 scrambled 二硫键的 LC-MS/MS 数据文件被用作 Byos 中自动 O 标记工作流程的测试数据集。新的工作流程导致检测到一种具有未知连接化学的光诱导交联二肽，随后将其鉴定为具有新型半胱氨酸-色氨酸（硫醚）键的交联二肽。这项工作表明，将 O 标记胰蛋白酶消化与 Byos 工作流程相结合，可以快速检测交联二肽。
