CAS Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian, Liaoning 116023, China.
University of Chinese Academy of Sciences, Beijing 100039, China.
Anal Chem. 2022 Mar 8;94(9):3904-3913. doi: 10.1021/acs.analchem.1c04927. Epub 2022 Feb 22.
Chemical crosslinking combined with mass spectrometry (CXMS) has allowed the global characterization of protein complexes with high throughput and accuracy. Although enrichable crosslinkers have been introduced to exclude the interference of regular peptides, the crosslinked peptide identification is still severely inhibited by a large amount of monolinked peptides. In this work, we proposed a strategy called MoTE (unhydrolyzed Monolinked peptide Targeting Elimination) to remove the unhydrolyzed monolinked peptides, while enriching crosslinked peptides for regular peptide removal. In this strategy, followed by the crosslinking reaction, an amine biotin reagent was used to block the unreacted reactive groups on the crosslinker, and subsequently, the crosslinked proteins were tagged by a cleavable biotin-azide ligand based on click chemistry for enrichment. The following crosslinked protein digestion, purification by streptavidin beads, and release by chemical cleavage of the biotin-azide ligand were sequentially performed. In this case, the amine biotin-blocked unhydrolyzed monolinked peptides with the unbreakable arm remained on the streptavidin beads, which realized selective removal without any additional steps. By combining in vivo crosslinking with our proposed MoTE strategy for protein complex analysis of the HeLa cell, the number of high reliability (score <E-04) interlinked peptides increased 43% in a single LC-MS run, and the structural and interaction mapping capacity for low-abundance and flexible proteins were greatly enhanced. These results demonstrated that the MoTE strategy has great potential to improve the coverage of CXMS-based protein complex analysis. Notably, it was also the first report focused on removing the highly abundant monolinked peptides.
化学交联结合质谱（CXMS）技术允许高通量和高准确度地对蛋白质复合物进行全面的表征。虽然已经引入了可富集的交联剂来排除常规肽的干扰，但交联肽的鉴定仍然受到大量单键肽的严重抑制。在这项工作中，我们提出了一种称为 MoTE（未水解的单键肽靶向消除）的策略，用于去除未水解的单键肽，同时富集交联肽以去除常规肽。在该策略中，交联反应后，使用胺基生物素试剂封闭交联剂上未反应的反应基团，随后，通过点击化学基于可切割生物素-叠氮化物配体对交联蛋白进行标记以进行富集。接下来进行交联蛋白的消化、链霉亲和素珠的纯化以及生物素-叠氮化物配体的化学裂解释放。在这种情况下，带有不可断裂臂的胺基生物素封闭的未水解的单键肽仍保留在链霉亲和素珠上，实现了无需任何额外步骤的选择性去除。通过将体内交联与我们提出的 MoTE 策略相结合，用于 HeLa 细胞的蛋白质复合物分析，在单个 LC-MS 运行中，高可靠性（得分<E-04）交联肽的数量增加了 43%，并且对低丰度和灵活蛋白质的结构和相互作用映射能力得到了极大增强。这些结果表明，MoTE 策略具有提高基于 CXMS 的蛋白质复合物分析的覆盖范围的巨大潜力。值得注意的是，这也是第一个专注于去除高丰度单键肽的报告。
