GeLC‐FAIMS‐MS workflow for in‐depth middle‐down proteomics

Abstract 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.