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.