Study of structure‐dependent chromatographic behavior of glycopeptides using reversed phase nanoLC

Ahn

Kozlík³

et al. 2020

Preprint

Self Cite

Carbohydrates form one of the major groups of biological macromolecules in living organisms. Many biological processes including protein folding, stability, immune response, and receptor activation are regulated by glycosylation. Fucosylation of proteins regulates such processes and is associated with various diseases including autoimmunity and cancer. Mass spectrometry efficiently identifies structures of fucosylated glycans or sites of core fucosylated N-glycopeptides but quantification of the glycopeptides remains less explored. We performed experiments that facilitate quantitative analysis of the core fucosylation of proteins with partial structural resolution of the glycans and we present results of the mass spectrometric SWATH-type DIA analysis of relative abundances of the core fucosylated glycoforms of 45 glycopeptides derived from 18 serum proteins in liver disease of different etiologies. Our results show that a combination of soft fragmentation with exoglycosidases is efficient at the assignment and quantification of the core fucosylated N-glycoforms at specific sites of protein attachment. In addition, our results show that disease-associated changes in core fucosylation are peptide-dependent and further differ by branching of the core fucosylated glycans. Further studies are needed to verify whether tri- and tetra-antennary core fucosylated glycopeptides could be used as markers of liver disease progression.

Section: Core Fucosylation and Limited Fragmentationsupporting

confidence: 73%

Analysis of site and structure specific core fucosylation in liver disease progression using exoglycosidase-assisted data-independent LC-MS/MS

Ahn

Kozlík³

et al. 2020

Preprint

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“…However, we detect approximately 13% occupancy with core-1 and core-2 structures at the T678 (Figure 2 and 3) located near the polybasic furin cleavage site between the S1 and S2 subunits which evolved in the SARS-CoV-2 S protein (7). This is relevant because O-glycans proximal to the convertase cleavage sites of protein substrates regulate proteolysis (15) (Figure 4) follow the expected trends of structure dependent reverse phase chromatographic behavior of glycopeptides (16,17).…”

Section: O-glycopeptide Analysismentioning

confidence: 71%

N and O glycosylation of the SARS-CoV-2 spike protein

Morrison

Goldman

2020

Preprint

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AbstractCovid-19 pandemic outbreak is the reason of the current world health crisis. The development of effective antiviral compounds and vaccines requires detailed descriptive studies of the SARS-CoV-2 proteins. The SARS-CoV-2 spike (S) protein mediates virion binding to the human cells through its interaction with the ACE2 cell surface receptor and is one of the prime immunization targets. A functional virion is composed of three S1 and three S2 subunits created by furin cleavage of the spike protein at R682, a polybasic cleavage sites that differs from the SARS-CoV spike protein of 2002. We observe that the spike protein is O-glycosylated on a threonine (T678) near the furin cleavage site occupied by core-1 and core-2 structures. In addition, we have identified eight additional O-glycopeptides on the spike glycoprotein and we confirmed that the spike protein is heavily N-glycosylated. Our recently developed LC-MS/MS methodology allowed us to identify LacdiNAc structural motifs on all occupied N-glycopeptides and polyLacNAc structures on six glycopeptides of the spike protein. In conclusion, our study substantially expands the current knowledge of the spike protein’s glycosylation and enables the investigation of the influence of the O-glycosylation on its proteolytic activation.

“…It has previously been described that different neutral oligosaccharide units of glycopeptides influence the retention time under the RP-LC [4,5] and HILIC [1,14,15] conditions. Fig.…”

Section: Resultsmentioning

confidence: 99%

“…RP-LC is driven mainly by physical-chemical properties of the peptide backbone with additional influence of the glycan moiety [2]. Several papers documented that the addition of neutral monosaccharide units to the glycan decreases retention of the glycopeptides [3–5] and that the addition of sialic acid increases retention [4,6]. HILIC is suitable for the analysis of polar analytes which are inadequately retained in RP-LC [1].…”

Section: Introductionmentioning

confidence: 99%

Nano reversed phase versus nano hydrophilic interaction liquid chromatography on a chip in the analysis of hemopexin glycopeptides

Kozlík

Journal of Chromatography A

Goldman

2017

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Analysis of the glycosylation of proteins is a challenge that requires orthogonal methods to achieve separation of the diverse glycoforms. A combination of reversed phase chromatography with tandem mass spectrometry (RP-LC-MS/MS) is one of the most powerful tools for glycopeptide analysis. In this work, we developed and compared RP-LC and hydrophilic interaction liquid chromatography (HILIC) in nanoscale on a chip combined with MS/MS in order to separate glycoforms of two peptides obtained from the tryptic digest of hemopexin. We observed reduction of the retention time with decreasing polarity of glycans attached to the same peptide backbone in HILIC. The opposite effect was observed for RP-LC. The presence of sialic acids prolonged the retention of glycopeptides in both chromatographic modes. The nanoHILIC method provided higher selectivity based on the composition of glycan, compared to nanoRP-LC but a lower sensitivity. The nanoHILIC method was able to partially separate linkage isomers of fucose (core and outer arm) on bi-antennary glycoform of SWPAVGDCSSALR glycopeptide, which is beneficial in the elucidation of the structure of the fucosylated glycoforms.