Retention Time Prediction for Glycopeptides in Reversed Phase Chromatography for Glycoproteomic Applications

ABSTRACT: The Sequence-Specific Retention Calculator algorithm was adapted for the prediction of retention times of N-glycopeptides separated by reversed-phase high performance liquid chromatography (RPLC). Retention time shifts (dHI = HI glyco - HI deglyco, where HI is hydrophobicity index, measured in acetonitrile % units) used for modeling were measured for 602 glycopeptides vs. 123 of their deglycosylated analogs. Our method used a tryptic digestion of 12 purified glycoproteins, glycopeptide enrichment, deglycosylation with PNGaseF and RPLC-MS/MS analysis of combined (deglycosylated and intact) peptide mixtures. On average, glycosylation yields a 0.79% acetonitrile decrease in retention compared to their deglycosylated analogs. These values, however, are drastically different for asialo (-1.37), mono- (-0.47), di- (+0.61) and tri-sialylated (+1.94% acetonitrile) glycans. Peptide retention time shifts upon glycosylation (dHI) vary depending on the number of monosaccharide units, the presence/absence of sialic acid, peptide hydrophobicity and a number of position-dependent features. The latter are mostly driven by competing effects of acidic residues (aspartic acid, sialic acid) on ion-pairing formation and nearest-neighbor effects of hydrophilic glycans. The accuracy of the modified prediction model for glycopeptides approaches that of prediction for non-modified species (R2 0.97 vs. 0.98). However, retention time prediction based on experimental retention values of deglycosylated analog (HI glyco = HI deglyco + dHI, R2=0.995) is much more accurate, thus providing a solid support for glycopeptide identification in complex samples based on mass and retention time.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Bos Taurus (ncbitaxon:9913) Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Oleg Krokhin  

PROVIDER: MSV000084174 | MassIVE | Wed Aug 07 09:42:00 BST 2019

REPOSITORIES: MassIVE