HILIC Peptide Retention Times Predicted Using New Approach

Scientists from the Manitoba Centre for Proteomics and Systems Biology in Winnipeg, Canada produced a new means of predicting peptide retention times for hydrophilic interaction liquid chromatography (HILIC) at acidic pH in formic-acid based eluents. Their findings were published in the Journal of Chromatography A (1).

Biological protein and molecule, 3d rendering. | Image Credit: © Vink Fan - stock.adobe.com

A high-performance liquid chromatography (HPLC) technique used for separating polar and hydrophilic compounds, HILIC is a normal-phase type of separation that uses reversed-phase (RP) type eluents; as such, the technique provides a column with a hydrophilic stationary phase and an eluent with water, buffer, and a high concentration of water-miscible organic solvent (2). Initially characterized in 1990, HILIC demonstrated that cation-exchange and hydrophilic neutral materials can retain hydrophilic compounds at high organic solvent concentrations. This eventually led to various hydrophilic sorbents being introduced for HILIC separation, with these types of sorbents showing the ability to retain very hydrophilic compounds commonly found in the flow through in RP separations.

Read More: New Review Highlights HILIC Evolution for Oligonucleotide Analysis

There have been attempts to use HILIC alongside other techniques, such as mass spectrometry (MS), but they have typically faced issues like low resolution or impractical analysis times due to longer re-equilibration, which proved too much to accept for potential sensitivity improvements afforded by mobile phase composition. Currently, RP-liquid chromatography (RPLC) is the dominant chromatographic mode in bottom-up proteomics for peptide separation, due to its versatility and ability to achieve higher chromatographic resolution. One popular alternative to reduce sample complexity prior to MS analysis is multidimensional liquid chromatography (mD-LC). In the early years of MS-based proteomics, HILIC was viewed as a promising chromatographic mode to use in the first dimension for mD-LC. That said, there is still more to learn regarding the chromatographic behavior of peptides in HILIC.

Read More:Systematic Evaluation of HILIC Stationary Phases for MS Characterization of Oligonucleotides

In this study, peptide separation selectivity was evaluated using three types of sorbents: zwitterionic ion-exchange HILIC (ZIC-HILIC), zwitterionic cationic HILIC (ZIC-cHILIC), and XBridge amide sorbents, with formic acid (pH 2.7) being used as an eluent additive. Sequence-specific retention prediction algorithms were trained using retention datasets of approximately 30,000 peptides for each column. The retention models successfully attained ∼0.98 R²-value and yielded retention coefficients that can be probed to understand peptide-stationary phase interaction. The columns’ overall hydrophilicity decreased when the mobile phase changed from a 4.5 pH to 2.7 when using 0.1% formic acid in the mobile phase. The acidic residues became protonated (the process of a proton being added to an atom, molecule, or ion), and the resulting hydrophilic interaction is dampened at the lower pH, only leaving basic residues as the primary hydrophilic interactors. This leads to peptides of an increasing charge having higher retention.

When comparing the three columns, ZIC-HILIC yielded the highest chromatographic resolution between groups of peptides of different charge. From the position-dependent retention coefficients for ZIC-HILIC at pH 2.7, the scientists found that amino acids at the terminal positions of the peptide modulate the basicity of the N-terminal amino group or the C-terminal Arg/Lys for tryptic peptides. With respect to the separation orthogonality between HILIC and acidic pH RPLC for two dimensional separations, the orthogonality values were lower at pH 2.7 than operating HILIC at pH 4.5 for the first dimension. For future research, the scientists believe it would be attractive to explore higher pH HILIC separations where negatively charged residues would provide major contributions into peptide retention. These would lead to an expanding chromatographic toolbox for peptide separation in analytical scale HPLC and potentially improve uptake of HILIC methods in bottom-up proteomics.

References

(1) Yeung, D.; Spicer, V.; Krokhin, O. V. Peptide Retention Time Prediction for Hydrophilic Interaction Liquid Chromatography at Acidic pH in Formic-Acid Based Eluents. J. Chromatogr. A 2024, 1736, 465355. DOI: 10.1016/j.chroma.2024.465355

(2) Hydrophilic Interaction Liquid Chromatography. MilliporeSigma 2024. https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/analytical-chemistry/small-molecule-hplc/hilic (accessed 2024-10-29)