Top-down proteomics (TDP) is a powerful analytical approach for the highly sensitive measurement of intact proteoforms by mass spectrometry. However, its application to high molecular weight proteoforms remains challenging. Middle-down proteomics (MDP) offers a practical solution but requires pre-fractionation of the complex peptide mixture generated by limited digestion to successfully achieve trace-level peptide detection. Here, we present 2D-GeLC-FAIMS-MS, an innovative gel-based sample pre-fractionation workflow for in-depth MDP. This workflow integrates limited Glu-C digestion with a two-dimensional sample fractionation strategy that combines a BAC (N,N'-bis(acryloyl)cystamine)-cross-linked dissolvable polyacrylamide gel electrophoresis (BAC-PAGE) with PEPPI-MS, a highly efficient passive protein extraction method. Samples are first size-fractionated by BAC-PAGE and subsequently subjected to in-gel Glu-C digestion. The resulting middle-down peptides (< 50 kDa) undergo a second fractionation via SDS-PAGE, followed by peptide recovery using PEPPI-MS and LC-FAIMS-MS analysis. The dissolution properties of BAC gels enable efficient sample transfer between the two PAGE steps with minimal loss, ensuring high-resolution pre-fractionation. This novel workflow provides a robust and efficient strategy for the comprehensive characterization of middle-down peptides, facilitating improved sensitivity and depth in proteome analysis.
Keywords: BAC‐PAGE; GeLC‐FAIMS‐MS; PEPPI‐MS; middle‐down proteomics; proteoforms.
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