Proteomic Analysis to Identify Functional Molecules in Drug Resistance Caused by E-Cadherin Knockdown in 3D-Cultured Colorectal Cancer Models PRINCIPAL INVESTIGATOR:

Typical mass spectrometric phosphoproteomestudies are complicated by the need for large amounts of startingmaterial and extensive sample preparation to ensure sufficientphosphopeptide identifications. In this paper, we present a novelstrategy to perform optimized multistep IMAC enrichment fromwhole cell lysates followed by high-pH reverse phase fractionation(multi-IMAC-HLB; HLB means hydrophilic−lipophilic-balancedreversed-phase cartridge). The peptide-to-IMAC ratio wasoptimized to maximize IMAC performance, while multistepIMAC enrichment enabled improved phosphopeptide acquisition.The addition of the HLB step further fractionates the IMACenriched phosphopeptides while desalting the samples, whichdramatically reduces the sample manipulation time and sampleloss compared to other popular strategies. We compared the phosphopeptide identification results of the multi-IMAC-HLBmethod with 3 mg of starting material to the well-established SCX-IMAC method with 15 mg of starting material. We identified8969 unique phosphopeptides with the multi-IMAC-HLB method, compared to 5519 unique phosphopeptides identified withthe SCX-IMAC method, an increase of 62.5%. The increase in the numbers of identified phosphopeptides is due to the increasein 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.