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Community evaluation of glycoproteomics informatics solutions reveals high-performance search strategies for serum glycopeptide analysis.

ABSTRACT: Glycoproteomics is a powerful yet analytically challenging research tool. Software packages aiding the interpretation of complex glycopeptide tandem mass spectra have appeared, but their relative performance remains untested. Conducted through the HUPO Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates solutions for system-wide glycopeptide analysis. The same mass spectrometrybased glycoproteomics datasets from human serum were shared with participants and the relative team performance for N- and O-glycopeptide data analysis was comprehensively established by orthogonal performance tests. Although the results were variable, several high-performance glycoproteomics informatics strategies were identified. Deep analysis of the data revealed key performance-associated search parameters and led to recommendations for improved 'high-coverage' and 'high-accuracy' glycoproteomics search solutions. This study concludes that diverse software packages for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies and specifies key variables that will guide future software developments and assist informatics decision-making in glycoproteomics.

SUBMITTER: Kawahara R

PROVIDER: S-EPMC8566223 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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Community evaluation of glycoproteomics informatics solutions reveals high-performance search strategies for serum glycopeptide analysis.

Kawahara Rebeca R Chernykh Anastasia A Alagesan Kathirvel K Bern Marshall M Cao Weiqian W Chalkley Robert J RJ Cheng Kai K Choo Matthew S MS Edwards Nathan N Goldman Radoslav R Hoffmann Marcus M Hu Yingwei Y Huang Yifan Y Kim Jin Young JY Kletter Doron D Liquet Benoit B Liu Mingqi M Mechref Yehia Y Meng Bo B Neelamegham Sriram S Nguyen-Khuong Terry T Nilsson Jonas J Pap Adam A Park Gun Wook GW Parker Benjamin L BL Pegg Cassandra L CL Penninger Josef M JM Phung Toan K TK Pioch Markus M Rapp Erdmann E Sakalli Enes E Sanda Miloslav M Schulz Benjamin L BL Scott Nichollas E NE Sofronov Georgy G Stadlmann Johannes J Vakhrushev Sergey Y SY Woo Christina M CM Wu Hung-Yi HY Yang Pengyuan P Ying Wantao W Zhang Hui H Zhang Yong Y Zhao Jingfu J Zaia Joseph J Haslam Stuart M SM Palmisano Giuseppe G Yoo Jong Shin JS Larson Göran G Khoo Kai-Hooi KH Medzihradszky Katalin F KF Kolarich Daniel D Packer Nicolle H NH Thaysen-Andersen Morten M

Nature methods 20211101 11

Glycoproteomics is a powerful yet analytically challenging research tool. Software packages aiding the interpretation of complex glycopeptide tandem mass spectra have appeared, but their relative performance remains untested. Conducted through the HUPO Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates solutions for system-wide glycopeptide analysis. The same mass spectrometrybased glycoproteomics datasets from huma ...[more]

PMID: 34725484

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Project description:A fully developed understanding of protein glycosylation requires characterization of the modifying oligosaccharides, elucidation of their covalent attachment sites, and determination of the glycan heterogeneity at specific sites. Considering the complexity inherent to protein glycosylation, establishing these features for even a single protein can present an imposing challenge. To meet the demands of glycoproteomics, the capability to screen far more complex systems of glycosylated proteins must be developed. Although the proteome wide examination of carbohydrate modification has become an area of keen interest, the intricacy of protein glycosylation has frustrated the progress of large-scale, systems oriented research on site-specific protein-glycan relationships. Indeed, the analytical obstacles in this area have been more instrumental in shaping the current glycoproteomic paradigm than have the diverse functional roles and ubiquitous nature of glycans. This report describes the ongoing development and analytically salient features of bead immobilized pronase for glycosylation site footprinting. The present work bears on the ultimate goal of providing analytical tools capable of addressing the diversity of protein glycosylation in a more comprehensive and efficient manner. In particular, this approach has been assessed with respect to reproducibility, sensitivity, and tolerance to sample complexity. The efficiency of pronase immobilization, attainable pronase loading density, and the corresponding effects on glycoprotein digestion rate were also evaluated. In addition to being highly reproducible, the immobilized enzymes retained a high degree of proteolytic activity after repeat usage for up to 6 weeks. This method also afforded a low level of chemical background and provided favorable levels of sensitivity with respect to traditional glycoproteomic strategies. Thus, the application of immobilized pronase shows potential to contribute to the advancement of more comprehensive glycoproteomic research methods that are capable of providing site-specific glycosylation and microheterogeneity information across many proteins.

Dataset Information

Community evaluation of glycoproteomics informatics solutions reveals high-performance search strategies for serum glycopeptide analysis.

Publications

Community evaluation of glycoproteomics informatics solutions reveals high-performance search strategies for serum glycopeptide analysis.

Similar Datasets

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