{"title":"Peptide retention time prediction for hydrophilic interaction liquid chromatography at acidic pH in formic-acid based eluents","authors":"","doi":"10.1016/j.chroma.2024.465355","DOIUrl":null,"url":null,"abstract":"<div><p>Peptide separation selectivity was evaluated for hydrophilic interaction liquid chromatography (HILIC) ZIC-HILIC, ZIC-cHILIC, and XBridge Amide sorbents using formic acid as eluent additive (pH 2.7). Sequence-specific retention prediction algorithms were trained using retention datasets of ∼30,000 peptides for each column. Our retention models were able to attain ∼0.98 R<sup>2</sup>-value and yielded retention coefficients that can be probed to understand peptide-stationary phase interaction. Overall, the hydrophilicity for these columns decreased when the mobile phase changed pH from 4.5 to 2.7, when using 0.1 % formic acid in the mobile phase. The acidic residues became protonated, and the resultant hydrophilic interaction is dampened at the lower pH, leaving only the basic residues as the primary hydrophilic interactors. Hence, peptides of increasing charge have higher retention. In this comparison between the three columns, ZIC-HILIC has the highest chromatographic resolution between groups of peptides of different charge. From the position-dependent retention coefficients for ZIC-HILIC at pH 2.7, we found that the 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. We also demonstrate that ZIC-HILIC was able to distinguish citrullinated and deamidated peptides based on predicted retention values.</p></div>","PeriodicalId":347,"journal":{"name":"Journal of Chromatography A","volume":null,"pages":null},"PeriodicalIF":3.8000,"publicationDate":"2024-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Chromatography A","FirstCategoryId":"1","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0021967324007295","RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0
Abstract
Peptide separation selectivity was evaluated for hydrophilic interaction liquid chromatography (HILIC) ZIC-HILIC, ZIC-cHILIC, and XBridge Amide sorbents using formic acid as eluent additive (pH 2.7). Sequence-specific retention prediction algorithms were trained using retention datasets of ∼30,000 peptides for each column. Our retention models were able to attain ∼0.98 R2-value and yielded retention coefficients that can be probed to understand peptide-stationary phase interaction. Overall, the hydrophilicity for these columns decreased when the mobile phase changed pH from 4.5 to 2.7, when using 0.1 % formic acid in the mobile phase. The acidic residues became protonated, and the resultant hydrophilic interaction is dampened at the lower pH, leaving only the basic residues as the primary hydrophilic interactors. Hence, peptides of increasing charge have higher retention. In this comparison between the three columns, ZIC-HILIC has the highest chromatographic resolution between groups of peptides of different charge. From the position-dependent retention coefficients for ZIC-HILIC at pH 2.7, we found that the 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. We also demonstrate that ZIC-HILIC was able to distinguish citrullinated and deamidated peptides based on predicted retention values.
期刊介绍:
The Journal of Chromatography A provides a forum for the publication of original research and critical reviews on all aspects of fundamental and applied separation science. The scope of the journal includes chromatography and related techniques, electromigration techniques (e.g. electrophoresis, electrochromatography), hyphenated and other multi-dimensional techniques, sample preparation, and detection methods such as mass spectrometry. Contributions consist mainly of research papers dealing with the theory of separation methods, instrumental developments and analytical and preparative applications of general interest.