Department of Structural Biology, Leibniz─Forschungsinstitut für Molekulare Pharmakologie (FMP), Robert-Roessle-Str. 10, Berlin 13125, Germany.
Thermo Fisher Scientific, 355 River Oaks Pkwy, San Jose, California 95134, United States.
Anal Chem. 2023 Mar 28;95(12):5248-5255. doi: 10.1021/acs.analchem.2c05141. Epub 2023 Mar 16.
Cross-linking mass spectrometry (XL-MS) is a universal tool for probing structural dynamics and protein-protein interactions and . Although cross-linked peptides are naturally less abundant than their unlinked counterparts, recent experimental advances improved cross-link identification by enriching the cross-linker-modified peptides chemically with the use of enrichable cross-linkers. However, mono-links (, peptides modified with a hydrolyzed cross-linker) still hinder efficient cross-link identification since a large proportion of measurement time is spent on their MS2 acquisition. Currently, cross-links and mono-links cannot be separated by sample preparation techniques or chromatography because they are chemically almost identical. Here, we found that based on the intensity ratios of four diagnostic peaks when using PhoX/tBu-PhoX cross-linkers, cross-links and mono-links can be partially distinguished. Harnessing their characteristic intensity ratios for real-time library search (RTLS)-based triggering of high-resolution MS2 scans increased the number of cross-link identifications from both single protein samples and intact cells. Specifically, RTLS improves cross-link identification from unenriched samples and short gradients, emphasizing its advantages in high-throughput approaches and when instrument time or sample amount is limited.
交联质谱（XL-MS）是一种通用的工具，用于探测结构动力学和蛋白质-蛋白质相互作用。虽然交联肽的丰度自然低于其未交联的对应物，但最近的实验进展通过使用可富集的交联剂对交联剂修饰的肽进行化学富集，提高了交联的鉴定。然而，单键（，用水解交联剂修饰的肽）仍然阻碍了有效的交联鉴定，因为大量的测量时间都花在它们的 MS2 采集上。目前，交联和单键不能通过样品制备技术或色谱法分离，因为它们在化学上几乎是相同的。在这里，我们发现，基于使用 PhoX/tBu-PhoX 交联剂时四个诊断峰的强度比，可以部分区分交联和单键。利用它们的特征强度比进行实时文库搜索（RTLS）触发高分辨率 MS2 扫描，增加了来自单个蛋白质样品和完整细胞的交联鉴定数量。具体来说，RTLS 提高了未富集样品和短梯度的交联鉴定，强调了其在高通量方法中的优势，以及在仪器时间或样品量有限的情况下的优势。
