A Chromatography-Guided Co-Fractionation Mass Spectrometry Strategy for Rapid Profiling of Drug-Perturbed Protein Complexes

Protein complexes are central to cellular function and respond rapidly to pharmacological perturbations. Co-fractionation mass spectrometry (CoFrac-MS) is widely employed to analyze protein complexes by analyzing individual chromatographic fractions, but it is labor-intensive and slow. To address these challenges, we introduce a chromatography-guided strategy enabling rapid identification of drug-perturbed protein complexes. It combines cross-linking enhanced reversed phase liquid chromatography cofractionation (XL-CoFrac) for high-resolution separation with ChromaQuant, a custom tool for precise peak quantification and differential analysis (https://hplcfdu.shinyapps.io/ChromaQuant/). Subsequent targeted MS analyses, guided by ChromaQuant, collectively establish the XL-CoFrac-Q-MS workflow. In proof-of-concept studies, we first adopted XL-CoFrac to MCF7 cells and profiled representative protein complexes. ChromaQuant demonstrated exceptional precision, achieving coefficients of variation below 1% and replicate correlations exceeding 0.98. Furthermore, we analyzed RS4;11 leukemia cells treated with increasing concentrations of the BCL-2 inhibitor ABT-199 using the XL-CoFrac-Q-MS workflow. Seven chromatographic peaks that changed consistently with the drug concentration were selected to be identified by this approach. MS analysis of these peaks revealed cross-linked peptides from the BCL-2 associated protein complex. Specially, cross-linking peptides between BCL-2 and FKBP38 may shed light on the mechanisms underlying resistance to ABT-199. Further pathway enrichment analysis provides new insights into the molecular mechanisms driving ABT-199 induced apoptosis. Collectively, the XL-CoFrac-Q-MS strategy holds significant potential for broad applications, including rapid screening of drug targets and elucidation of protein complex dynamics across various pharmacological and pathological conditions.

蛋白质复合物是细胞功能的核心组成部分，且能对药物扰动做出快速响应。共分级质谱（Co-fractionation mass spectrometry, CoFrac-MS）被广泛用于通过分析单个色谱分级组分解析蛋白质复合物，但其操作繁琐且耗时较长。为解决上述问题，本研究提出一种色谱引导策略，可快速识别受药物扰动的蛋白质复合物。该策略将用于高分辨分离的交联增强反相液相色谱共分级（cross-linking enhanced reversed phase liquid chromatography cofractionation，简称XL-CoFrac）与定制化峰定量及差异分析工具ChromaQuant（https://hplcfdu.shinyapps.io/ChromaQuant/）相结合。后续以ChromaQuant为指导的靶向质谱分析，共同构建了XL-CoFrac-Q-MS工作流程。在概念验证研究中，研究团队首先将XL-CoFrac应用于MCF7细胞，并对代表性蛋白质复合物开展了表征分析。ChromaQuant展现出极高的精密度，其变异系数低于1%，重复相关性超过0.98。此外，研究团队借助XL-CoFrac-Q-MS工作流程，分析了经梯度浓度BCL-2抑制剂ABT-199处理的RS4;11白血病细胞。研究团队选取了7个随药物浓度变化趋势一致的色谱峰，通过本方法进行鉴定。对这些色谱峰的质谱分析结果显示，存在来自BCL-2相关复合物的交联肽段。尤为重要的是，BCL-2与FKBP38之间的交联肽段或可为阐明ABT-199耐药机制提供新线索。后续的通路富集分析进一步揭示了ABT-199诱导细胞凋亡的分子机制。综上，XL-CoFrac-Q-MS策略具有广阔的应用前景，可用于快速筛选药物靶点，并解析多种药物及病理条件下的蛋白质复合物动态变化。