从富集的交联肽中选择性去除未水解的单链肽以提高蛋白质复合物分析的覆盖率,Analytical Chemistry

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从富集的交联肽中选择性去除未水解的单链肽以提高蛋白质复合物分析的覆盖率
Analytical Chemistry ( IF 6.7 ) Pub Date : 2022-02-22 , DOI: 10.1021/acs.analchem.1c04927
Yuxin An _{1,

2} , Qun Zhao ₁ , Hang Gao _{1,

2} , Lili Zhao _{1,

2} , Xiao Li ₁ , Xiaodan Zhang ₁ , Zhen Liang ₁ , Lihua Zhang ₁ , Yukui Zhang ₁

Affiliation

化学交联与质谱 (CXMS) 相结合，使蛋白质复合物的全局表征成为可能，具有高通量和准确性。尽管引入了可富集的交联剂以排除常规肽的干扰，但交联肽的鉴定仍然受到大量单联肽的严重抑制。在这项工作中，我们提出了一种称为 MoTE（未水解的单联肽靶向消除）的策略来去除未水解的单联肽，同时丰富交联肽以进行常规肽去除。在该策略中，在交联反应之后，使用胺类生物素试剂阻断交联剂上未反应的反应基团，随后，基于点击化学的可裂解生物素-叠氮化物配体标记交联蛋白以进行富集。依次进行以下交联蛋白质消化、链霉亲和素珠纯化和通过生物素-叠氮化物配体的化学切割释放。在这种情况下，胺生物素封闭的未水解单链肽具有牢不可破的臂，保留在链霉亲和素珠上，无需任何额外步骤即可实现选择性去除。通过将体内交联与我们提出的用于 HeLa 细胞蛋白质复合物分析的 MoTE 策略相结合，在单次 LC-MS 运行中，高可靠性（分数 <E-04）互连肽的数量增加了 43%，并且结构和相互作用映射低丰度和柔性蛋白质的能力大大增强。这些结果表明，MoTE 策略在提高基于 CXMS 的蛋白质复合物分析的覆盖率方面具有巨大潜力。值得注意的是，这也是第一份专注于去除高丰度单链肽的报告。

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Selective Removal of Unhydrolyzed Monolinked Peptides from Enriched Crosslinked Peptides To Improve the Coverage of Protein Complex Analysis

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.

更新日期：2022-02-22

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