Comparison of Peptide Fractionation on Different SCX Micro Columns for Proteome and Phosphoproteome Analysis

Abstract

StageTips are widely used for desalting and purification of peptides for various proteomic analysis. However, StageTips are not commonly used for peptide fractionation especially for phosphopeptides fractionation. In the previous study, we found that a degree of peptide separation on C18-SCX StageTip was greatly improved by a novel peptide fractionation method using acid gradient. Our method is simple, reproducible, MS-friendly and applicable for both peptide and phosphopeptide fractionation for in-depth proteome and phosphoproteome analysis. In this study, we further evaluated the disks or resins used for StageTips or similar micro column system from the point of the view of the identified number of peptides or phosphopeptides. We found that C18-SCX Stagetip and SDB-SCX Stagetip showed nearly same performance, whereas SDB-RPS Stagetip showed poor performance compared with SDB-SCX Stagetip. Furthermore, we confirmed that peptides are eluted from not only SCX disks but also SCX resins by TFA-based elution method. Our fractionation method is very simple, flexible in scale, low-cost (less than $0.1/sample) and does not need any special equipment and techniques. Furthermore our method has a potential to be applied to other fields using SCX chromatography.