Biology subjects

Sanda, M.

Publications and source records attributed to Sanda, M..

4 recordsLinked to original sources

bioRxiv2021

Community Evaluation of Glycoproteomics Informatics Solutions Reveals High-Performance Search Strategies of Glycopeptide Data

Glycoproteome profiling (glycoproteomics) is a powerful yet analytically challenging research tool. The complex tandem mass spectra generated from glycopeptide mixtures require sophisticated analysis pipelines for structural determination. Diverse software aiding the process have appeared, but their relative performance remains untested. Conducted through the HUPO Human Proteome Project - Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates the performance of informatics solutions for system-wide glycopeptide analysis. Mass spectrometry-based glycoproteomics datasets from human serum were shared with all teams. The relative team performance for N- and O-glycopeptide data analysis was comprehensively established and validated through orthogonal performance tests. Excitingly, several high-performance glycoproteomics informatics solutions were identified. While the study illustrated that significant informatics challenges remain, as indicated by a high discordance between annotated glycopeptides, lists of high-confidence (consensus) glycopeptides were compiled from the standardised team reports. Deep analysis of the performance data revealed key performance-associated search variables and led to recommendations for improved "high coverage" and "high accuracy" glycoproteomics search strategies. This study concludes that diverse software for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies, and specifies key variables that may guide future software developments and assist informatics decision-making in glycoproteomics.

biochemistry↗

bioRxiv2020

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

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.

biochemistry↗

bioRxiv2020

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

Covid-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 motives 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 proteins glycosylation and enables the investigation of the influence of the O-glycosylation on its proteolytic activation.

biochemistry↗

bioRxiv2020

PD-L1 glycosylation and its impact on binding to clinical antibodies

Immune checkpoint inhibitors, including PD-L1/PD-1, are key regulators of immune response and promising targets in cancer immunotherapy. N-glycosylation of PD-L1 affects its interaction with PD-1 but little is known about the distribution of glycoforms at its four NXS/T sequons. We optimized LC-MS/MS methods using collision energy modulation for the site-specific resolution of specific glycan motifs. Using these methods, we demonstrate that PD-L1 expressed on the surface of breast cancer cells carries mostly complex glycans with high proportion of polyLacNAc structures at the N219 sequon. PD-L1 from whole cell lysate contained, in addition, large proportion of high mannose glycans at all sites. Contrary to the full-length protein, the secreted form of PD-L1 expressed in breast cancer or HEK293 cells demonstrated minimum N219 occupancy and low contribution of the polyLacNAc structures. Molecular modeling of PD-L1/PD-1 interaction with N-glycans suggests that glycans at the N219 site of PD-L1 and N74 and N116 of PD-1 are involved in glycan-glycan interactions, but the impact of this potential interaction on the protein function remains at this point unknown. In addition, the interaction of PD-L1 with clinical antibodies is also affected by glycosylation. In conclusion, our study demonstrates that PD-L1 expressed in the MDA-MB-231 breast cancer cells carries polyLacNAc glycans mostly at the N219 sequon which displays the highest variability in occupancy and is most likely to directly influence the interaction with PD-1.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cancer biology↗