Parfentev Iwan, Schilbach Sandra, Cramer Patrick, Urlaub Henning
Research group Bioanalytical Mass Spectrometry, Max-Planck-Institute for Biophysical Chemistry, Goettingen, Germany.
Department of Molecular Biology, Max-Planck-Institute for Biophysical Chemistry, Goettingen, Germany.
J Proteomics. 2020 May 30;220:103754. doi: 10.1016/j.jprot.2020.103754. Epub 2020 Mar 19.
Cross-linking mass spectrometry (XL-MS) is steadily expanding its range of applications from purified protein complexes to more complex samples like organelles and even entire cells. One main challenge using non-cleavable cross-linkers is the so-called n problem: With linearly increasing database size, the search space for the identification of two covalently linked peptides per spectrum increases quadratically. Here, we report an alternative search strategy that focuses on only those peptides, which were demonstrated to cross-link under the applied experimental conditions. The performance of a parallel XL-MS experiment using a thiol-cleavable cross-linker enabled the identification of peptides that carried a cleaved cross-link moiety after reduction and hence were involved in cross-linking reactions. Based on these identifications, a peptide database was generated and used for the database search of the actual cross-linking experiment with a non-cleavable cross-linker. This peptide-focused approach was tested on protein complexes with a reported structural model and obtained results corresponded well to a conventional database search. An application of the strategy on in vivo cross-linked Bacillus subtilis and Bacillus cereus cells revealed a five- to tenfold reduction in search time and led to significantly more identifications with the latter species than a search against the entire proteome. SIGNIFICANCE: Instead of considering all theoretically cross-linkable peptides in a proteome, identification and pre-filtering for a subset of cross-link peptide candidates allows for a dramatically decreased search space. Hence, there is less potential for the random accumulation of false positives ultimately leading to a higher sensitivity in the XL-MS experiment. Using the peptide-focused approach, a cross-linking database search can be conducted in a fraction of time while yielding a similar or higher number of identifications, thereby enabling the cross-linking analysis of samples of mammalian proteome complexity.
交联质谱法（XL-MS）的应用范围正在稳步扩大，从纯化的蛋白质复合物到更复杂的样品，如细胞器甚至整个细胞。使用不可裂解交联剂的一个主要挑战是所谓的n问题：随着数据库大小呈线性增加，每个谱图中鉴定两个共价连接肽段的搜索空间呈二次方增长。在此，我们报告了一种替代搜索策略，该策略仅关注那些在应用实验条件下被证明发生交联的肽段。使用可硫醇裂解交联剂的平行XL-MS实验的性能，使得能够鉴定出在还原后带有裂解交联部分的肽段，因此这些肽段参与了交联反应。基于这些鉴定结果，生成了一个肽段数据库，并将其用于使用不可裂解交联剂的实际交联实验的数据库搜索。这种以肽段为重点的方法在具有已报道结构模型的蛋白质复合物上进行了测试，获得的结果与传统数据库搜索结果吻合良好。该策略在体内交联的枯草芽孢杆菌和蜡样芽孢杆菌细胞上的应用表明，搜索时间减少了五到十倍，并且与对整个蛋白质组进行搜索相比，对后一种物种的鉴定显著增多。意义：通过对交联肽候选子集进行鉴定和预筛选，而不是考虑蛋白质组中所有理论上可交联的肽段，可以显著减少搜索空间。因此，随机积累假阳性的可能性降低，最终导致XL-MS实验具有更高的灵敏度。使用以肽段为重点的方法，可以在短时间内进行交联数据库搜索，同时产生相似或更多的鉴定结果，从而能够对具有哺乳动物蛋白质组复杂性的样品进行交联分析。
