GeLC-FAIMS-MS workflow for in-depth middle-down proteomics.

Middle-down proteomics (MDP) is an analytical approach in which protein samples are digested with proteases such as Glu-C to generate large peptides (>3 kDa) that are analyzed by mass spectrometry (MS). This method is useful for characterizing high-molecular-weight proteins that are difficult to detect by top-down proteomics (TDP), in which intact proteins are analyzed by MS. In this study, we applied GeLC-FAIMS-MS, a multidimensional separation workflow that combines gel-based prefractionation with LC-FAIMS MS, for deep MDP. Middle-down peptides generated by optimized limited Glu-C digestion conditions were first size-fractionated by polyacrylamide gel electrophoresis, followed by C4 reversed-phase liquid chromatography separation and additional ion mobility fractionation, resulting in a significant increase in peptide length detectable by MS. In addition to global analysis, the GeLC-FAIMS-MS concept can also be applied to targeted MDP, where only proteins in the desired molecular weight range are gel-fractionated and their Glu-C digestion products are analyzed, as demonstrated by targeted analysis of integrins in exosomes. In-depth MDP achieved by global and targeted GeLC-FAIMS-MS supports the exploration of proteoform information not covered by conventional TDP by increasing the number of detectable protein groups or post-translational modifications (PTMs) and improving the sequence coverage.