CEEPC/IPM/CMSC - Abstrakt prezentace

(CEEPC/IPM/CMSC 2022 - FrO-12)
Prediction of Intact N-Glycopeptide Retention Time Windows in Hydrophilic Interaction Liquid Chromatography

Tomáš Ječmen 1 *, Petr Kozlík 2

  1. Department of Biochemistry, Faculty of Science, Charles University
  2. Department of Analytical Chemistry, Faculty of Science, Charles University


Analysis of protein glycosylation is challenging due to micro- and macro-heterogeneity of the attached glycans. Mass spectrometry is a key tool for structural characterization of intact glycopeptides. However, to achieve high-confidence identification, information-rich MS/MS spectra are needed. If such spectra are not available for all analyzed glycopeptides, the confidence of their identification can be improved by including orthogonal information, such as those derived from chromatographic parameters into the search engine algorithms. Here, we propose a simple model predicting relative retention time (RRT) windows for glycopeptides in hydrophilic interaction liquid chromatography (HILIC), which is a mode of choice for separation of this type of analytes as they are inadequately resolved by reversed phase chromatography.
We determined chromatographic parameters for 6 differently glycosylated tryptic peptides of 3 plasma proteins – haptoglobin, hemopexin, and sex hormone-binding globulin – separated by HILIC. For all of them, we calculated retention times of different glycoforms attached to the same peptide relative to the respective bi-antennary form, which is typically found in high yield. We showed that the RRT differences between the glycoforms do not depend greatly on the character of the peptide moiety, and based on the observed variance of the RRTs of individual glycoforms we derived an easy-to-use mathematical model. To prove the concept, we used it to accurately predict the retention time windows, within which fetuin glycopeptides are eluted. The model could therefore be included into post-search filtering of the glycopeptide assignments when HILIC-MS approach is selected for site-specific protein glycosylation characterization. [1]

* Korespondující autor: tomas.jecmen@centrum.cz


  1. Kozlik P, Molnarova K, Jecmen T, Krizek T, Bosakova Z. Prediction of Intact N-Glycopeptide Retention Time Windows in Hydrophilic Interaction Liquid Chromatography. Molecules. 2022; 27(12):3723.


This research was funded by the Czech Science Foundation (Grant No 19-18005Y) and by the Ministry of Education, Youth and Sports of the Czech Republic (LTC20078 in frame of the COST Action CA18103 INNOGLY).

Partneři společnosti

LabRules LCMS LabRules GCMS


Bruker HPST Merck Pragolab Amedis EastPort Shimadzu Waters