Peptide retention time prediction in hydrophilic interaction liquid chromatography. Comparison of separation selectivity between bare silica and bonded stationary phases

Peptide separation selectivity of four different HILIC sorbents was compared through the development of sequence-specific retention prediction algorithms for the datasets of 36-40 thousands peptides each. Hydrophilicity of these sorbents at pH 4.5 (peptide retention under acetonitrile:water gradients) increases in the following order: Luna HILIC < XBridge Amide < Atlantis Silica ∼ Luna HILIC Silica. Bare silica phases are characterized by higher retention coefficients for basic residues (Arg, Lys, His), while interactions with neutral HILIC phases is driven by interaction with the charged residues (Asp, Glu, Arg, Lys, His). Such difference is caused by electrostatic repulsion of negatively charged side chains of Asp and Glu from silanol groups on bare silica sorbents. The sequence-specific features characteristic for both bonded and bare-silica supports were established. Variation of retention coefficients at N- and C-termini showed a peptide orientation effect on silica matrices. The negatively charged surface of silica attracts positively charged groups, thus increasing interaction of N-terminal residues. The effect of peptide helicity is also different between two types of sorbents. The contribution of amphipathic helicity was found to be higher for more hydrophilic silica matrices, causing a reduced predictive accuracy of the models: all four Sequence-Specific Retention Calculator HILIC models had R2-values in 0.973-0.98 range. Development of accurate prediction models provides a solid support for retention prediction filtering of false positive identifications in high-throughput proteomics analyses, guided development of 2D LCMS procedures ((HILIC-RP)). Our analysis also shows that all phases possess a high degree of orthogonality to RPLC (formic acid) with Luna HILIC being the best choice in this regard.