Carboxylate-Modified Magnetic Bead (CMMB)-Based Isopropanol Gradient Peptide Fractionation (CIF) Enables Rapid and Robust Off-Line Peptide Mixture Fractionation in Bottom-Up Proteomics.

Deep proteome coverage in bottom-up proteomics requires peptide-level fractionation to simplify the complex peptide mixture before analysis by tandem mass spectrometry. By decreasing the number of coeluting precursor peptide ions, fractionation effectively reduces the complexity of the sample leading to higher sample coverage and reduced bias toward high-abundance precursors that are preferentially identified in data-dependent acquisition strategies. To achieve this goal, we report a bead-based off-line peptide fractionation method termed CIF or carboxylate-modified magnetic bead-based isopropanol gradient peptide fractionation. CIF is an extension of the SP3 (single-pot solid phase-enhanced sample preparation) strategy and provides an effective but complementary approach to other commonly used fractionation methods including strong cation exchange and reversed phase-based chromatography. We demonstrate that CIF is an effective offline separation strategy capable of increasing the depth of peptide analyte coverage both when used alone or as a second dimension of peptide fractionation in conjunction with high pH reversed phase. These features make it ideally suited for a wide range of proteomic applications including the affinity purification of low-abundance bait proteins.