Peptide retention time prediction for immobilized artificial membrane phosphatidylcholine stationary phase: method development and preliminary observations

Abstract

<jats:p>Development of the first peptide retention prediction model for immobilized artificial membrane phosphatidylcholine (IAM.PC) stationary phase is reported. 2D LC-MS/MS analysis of a whole cell lysate of &lt;em&gt;S. cerevisiae&lt;/em&gt; yielded a retention dataset of ~29,500 tryptic peptides; sufficient for confident assignment of retention coefficients which determine the contribution of individual amino acids in peptide retention. Retention data from the first dimension was used for the modeling: IAM.PC DD2 column, pH 7.4 ammonium bicarbonate, and water/acetonitrile gradient. Peptide separation using IAM.PC was compared to a standard C18 phase (Luna C18(2)). There was a significant reduction in peptide retention (~14% acetonitrile on average), indicating that the phosphatidylcholine stationary phase is significantly more hydrophilic. In comparison to the C18 phase, we found a substantial increase in the relative retention contribution for the positively charged Arg and Lys, and the aromatic Tyr, Trp and His residues. We also observed a decrease in retention contribution for the negatively charged Asp and Glu. This indicates an involvement of electrostatic interactions with the glycerophosphate functional groups, and possibly, delocalization effects from a hydrogen bond between the phosphate group and aromatic side chains in the separation mechanism.</jats:p>

Application of biomimetic HPLC to estimate lipophilicity, protein and phospholipid binding of potential peptide therapeutics

“…In silico predictions of reversed phase retention may provide the possibility to assess the lipophilicity of peptides based on the retention coefficients for amino acids [26][27][28]. Gussakovsky et al [29] analysed over 29 thousand tryptic peptides on the IAM.PC.DD2 stationary phase and compared the retention contribution of the amino acids with those from the C-18 columns. They found that amino acids containing positive charges such as arginine and lysine retain stronger on the IAM phase than on the C18 phase.…”

Immunomodulatory Functions of the Human Cathelicidin LL-37 (aa 13–31)-Derived Peptides are Associated with Predicted α-Helical Propensity and Hydrophobic Index

“…For example, the IAM PC (immobilized artificial membrane phosphatidyl choline) column has been suggested for the study of cell permeability for drug candidates [36]. The first study on peptide behavior using the IAM PC column confirmed similarity in the retention of amphipathic helical peptides with C18 phases [37]. However, it was also observed that positively charged amino acids (Lys, Arg, His) provided a higher contribution to retention on an IAM PC compared to C18, due to stronger interaction of lipid-absorbed peptides with the inner components of the zwitterionic layer—negatively charged phosphate groups.…”

Prediction of Analyte Retention Time in Liquid Chromatography

“…To obtain the desired separation, such chromatographic factors need to be properly adjusted. HILIC methods are often developed using a typical univariate approach, − which can be very time-consuming and often leads to a non-optimized method without adequate comprehension of main and interaction effects of method parameters. In contrast, a multivariate approach via DoE tools emphasizes systematic method development through assessment of multiparameter interaction effects using the minimum number of experiments, while also enabling a better comprehension of the method performance.…”