Rapid Analyses of Proteomes and Interactomes Using an Integrated Solid-Phase Extraction–Liquid Chromatography–MS/MS System

Here,
we explore applications of a LC system using disposable solid-phase
extraction (SPE) cartridges and very short LC–MS/MS gradients
that allows for rapid analyses in less than 10 min analysis time.
The setup consists of an autosampler harboring two sets of 96 STAGE
tips that function as precolumns and a short RP analytical column
running 6.5 min gradients. This system combines efficiently with several
proteomics workflows such as offline prefractionation methods, including
1D gel electrophoresis and strong-cation exchange chromatography.
It also enables the analysis of interactomes obtained by affinity
purification with an analysis time of approximately 1 h. In a typical
shotgun proteomics experiment involving 36 SCX fractions of an AspN
digested cell lysate, we detected over 3600 protein groups with an
analysis time of less than 5.5 h. This innovative fast LC system reduces
proteome analysis time while maintaining sufficient proteomic detail.
This has particular relevance for the use of proteomics within a clinical
environment, where large sample numbers and fast turnover times are
essential.

本研究探索了采用一次性固相萃取（Solid Phase Extraction, SPE）小柱与极短液相色谱-串联质谱（LC-MS/MS）洗脱梯度的液相色谱系统的应用场景，该系统可实现分析时长不足10分钟的快速检测。该系统配置包含一台自动进样器，其内置两组共96个STAGE tip作为预柱，搭配运行6.5分钟洗脱梯度的短反相分析柱。该系统可高效适配多种蛋白质组学工作流程，例如离线预分级方法，包括一维凝胶电泳与强阳离子交换色谱。同时，该系统可实现对亲和纯化获取的相互作用组的分析，单次分析时长约为1小时。在一项针对天冬氨酸蛋白酶AspN消化的细胞裂解液的36个强阳离子交换色谱（Strong Cation Exchange Chromatography, SCX）分级组分的典型鸟枪法蛋白质组学实验中，本研究在总分析时长不足5.5小时的条件下，检测到超过3600个蛋白群组。这款创新性的快速液相色谱系统在缩减蛋白质组分析时长的同时，仍可保留足够的蛋白质组学分析细节。这一特性在临床场景下的蛋白质组学应用中具有特殊价值——临床场景需要处理大量样本且要求快速周转。