MS-compatible hydrophobic interaction chromatography of intact proteins using long-ligand stationary phases

Hydrophobic interaction chromatography (HIC) is essential for the separation of proteins under native, nondenaturing conditions; however, traditional methods rely on high concentrations of nonvolatile kosmotropic salts that are incompatible with direct mass spectrometry (MS) coupling. This study evaluates a series of amide-based HIC stationary phases with extended ligands from propyl (C3) to decyl (C10) to overcome this bottleneck. Utilizing UHPLC hardware with titanium frits to mitigate detrimental metal-analyte interactions, we examined retention behavior for the NIST IgG1 monoclonal antibody and a diverse set of structurally heterogeneous top-down proteomic (TDP) targets ranging from 9.1 to 68 kDa. Drawing on the salt-exclusion principle, we demonstrate that increasing ligand length enables stationary phases to reach critical hydrophobicity at significantly lower salt concentrations, effectively offsetting the weaker retention forces of volatile, MS-friendly salts. While industry-standard propyl (C3) ligands failed completely to retain proteins at 0.5 M ammonium acetate, the decyl (C10) phase provided baseline resolution of the complex six-protein mixture. Furthermore, reaching critical hydrophobicity at lower salt levels relaxes the suppression of weak secondary interactions, such as hydrogen bonding via the ligand's amide-based carbonyl groups, which contributes to enhanced selectivity. Distinct retention dynamics were observed: while mAb retention plateaus at the octyl (C8) length, complex intact proteins like Protein G exhibit a near-linear retention increase across the entire series. Native HIC-LC-MS analysis of Protein G revealed consistent charge state distributions across short (C3) and long (C10) ligand conditions, indicating preservation of native-like structure even under increasing organic modifier during elution. These results demonstrate that long-ligand HIC phases maintain native protein structure while providing the necessary retention for direct HIC-MS coupling and the integration of HIC into multidimensional workflows for the identification of low-abundance proteoforms.