We have explored the use of electrostatic repulsion hydrophilic interaction chromatography (ERLIC) as an alternative to the gold-standard in shotgun proteomics: reversed-phase (RP) LC for online ESI-MS/MS. Conditions for sample solubilization and initial gradient conditions were optimized to strike a balance between peptide solubility and maximum peptide retention when using mobile phase with high organic solvent concentration. Online ERLIC-MS demonstrated a 57% increase in total peptide identifications compared to RP-MS. We examined the mechanism of this improved performance and found that it stems from ERLICs propensity to retain longer peptides, which can be identified with greater confidence. Online nanoscale ERLIC-MS provides a powerful new tool for enhancing MS-based shotgun proteomic in a broad range of applications.
- mass spectrometry
- online ERLIC
- reversed phase liquid chromatography
- shotgun proteomics