Combination of multistep IMAC enrichment with high-pH reverse phase separation for in-depth phosphoproteomic profiling.

Typical mass spectrometric phosphoproteome studies are complicated by the need for large amounts of starting material and extensive sample preparation to ensure sufficient phosphopeptide identifications. In this paper, we present a novel strategy to perform optimized multistep IMAC enrichment from whole cell lysates followed by high-pH reverse phase fractionation (multi-IMAC-HLB; HLB means hydrophilic-lipophilic-balanced reversed-phase cartridge). The peptide-to-IMAC ratio was optimized to maximize IMAC performance, while multistep IMAC enrichment enabled improved phosphopeptide acquisition. The addition of the HLB step further fractionates the IMAC enriched phosphopeptides while desalting the samples, which dramatically reduces the sample manipulation time and sample loss compared to other popular strategies. We compared the phosphopeptide identification results of the multi-IMAC-HLB method with 3 mg of starting material to the well-established SCX-IMAC method with 15 mg of starting material. We identified 8969 unique phosphopeptides with the multi-IMAC-HLB method, compared to 5519 unique phosphopeptides identified with the SCX-IMAC method, an increase of 62.5%. The increase in the numbers of identified phosphopeptides is due to the increase in the ratio of identified phosphopeptides out of all detected peptides, 70.5% with multi-IMAC-HLB method compared to 32.3% with the SCX-IMAC method. Multi-IMAC-HLB is a robust and efficient method for in-depth phosphoproteomic research.