Combination of Multistep IMAC Enrichment with High-pH Reverse Phase Separation for In-Depth Phosphoproteomic Profiling

Abstract

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.

Comparing Multistep Immobilized Metal Affinity Chromatography and Multistep TiO2 Methods for Phosphopeptide Enrichment

“…20 . Using the 100:1 (mg/μl) peptide-to-IMAC ratio optimized previously 19 , 3 mg of desalted, lyophilized whole-cell digest were resuspended in 360μL of IMAC binding buffer and incubated with 30 μl of bead slurry for 60min with vigorous shaking. After washing with 360 μl of binding buffer three times, phosphopeptides were eluted by shaking 5min in 40μL of 50 mM K 2 HPO 4 /NH 4 OH, pH10.…”

Chemical cytometry of thiols using capillary zone electrophoresis-laser induced fluorescence and TMPAB-o-M, an improved fluorogenic reagent

“…25, 26 The cells were resuspended in DPBS and transferred into 500 µL Eppendorf tubes (1.5×10 5 /100 µL for each tube).. NEM was used to change the thiol concentrations in the cells. 7 2 µL of different concentrations of NEM was added followed by incubation for 5 min at room temperature.…”

Liquid phase based separation systems for depletion, prefractionation, and enrichment of proteins in biological fluids and matrices for in‐depth proteomics analysis—An update covering the period 2011–2014

“…Yue et al [61] developed a novel strategy to perform optimized multistep IMAC enrichment from whole cell lysates followed by high-pH reversed phase fractionation (multi-IMAC-HLB; HLB means hydrophilic–lipophilic-balanced reversed-phase cartridge). Using the multi-IMAC-HLB method with 3 mg of starting material, 8969 unique phosphopeptides could be readily identified, while using the well-established SCX-IMAC method with 15 mg of starting material, 5519 unique phosphopeptides were identified.…”