Rapid and Label-Free Structural Proteomics by One-Step Swift Trypsin LiP-MS

We developed a rapid, surface-targeted limited proteolysis workflow, Swift Trypsin LiP-MS (STLiP-MS), to profile proteome-wide structural changes under near-native conditions. STLiP-MS uses a trypsin-immobilized spin column and centrifugation-controlled contact to achieve instantaneous, highly reproducible limited digestion, followed by reduction/alkylation and desalting. Proof-of-concept analyses were performed on HEK293T cell lysates (± phosphatase inhibitor PhosSTOP) and on a purified GPCR complex (A2A-BRIL with IgG). Peptides were separated on a 75-µm ID C18 column (50 °C) using an UltiMate 3000 RSLCnano and analyzed on an Orbitrap HF-X. Both DDA (top-50 HCD, 70-min gradient) and DIA (variable windows, 70-min gradient) runs were acquired. DIA data were processed with DIA-NN (library-free/in-silico; UniProt human, 2024-03), and DDA of purified samples with PEAKS Studio 12.5.我们开发了一种快速、靶向表面的有限蛋白水解流程——Swift Trypsin LiP-MS（STLiP-MS），用于在近天然条件下绘制全蛋白质组的结构变化图谱。STLiP-MS采用固定化胰蛋白酶的离心柱，并通过离心控制反应接触时间，实现瞬时、高重现性的有限酶解，随后进行还原烷基化与脱盐处理。本研究以HEK293T细胞裂解液（添加/不添加磷酸酶抑制剂PhosSTOP）以及纯化的G蛋白偶联受体（G protein-coupled receptor, GPCR）复合物（携带IgG的A2A-BRIL）为对象进行概念验证分析。肽段经UltiMate 3000 RSLCnano液相色谱系统，在75 μm内径的C18色谱柱（柱温50 ℃）上进行分离，随后通过Orbitrap HF-X质谱仪进行分析。本研究同时采集了数据依赖性采集（data-dependent acquisition, DDA，采用Top-50 HCD模式，梯度时长70 min）与数据非依赖性采集（data-independent acquisition, DIA，采用可变窗口模式，梯度时长70 min）的质谱数据。DIA数据通过DIA-NN软件进行处理（采用无库/虚拟库策略，数据库为2024年3月更新的人类UniProt数据库），纯化样品的DDA数据则使用PEAKS Studio 12.5软件进行分析。