Site-Specific N-Glycosylation Characterization of Windmill Palm Tree Peroxidase Using Novel Tools for Analysis of Plant Glycopeptide Mass Spectrometry Data

Author

Margaret R. Baker^†, David L. Tabb^‡, Travers Ching^†, Lisa J. Zimmerman^‡, Ivan Y. Sakharov^§ and Qing X. Li^*†

*E-mail qingl@hawaii.edu. Phone: 808-956-2011. Fax: 808-956-3542.

†Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, Hawaii 96822, United States

‡Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37205, United States

§Department of Chemistry, Lomonosov Moscow State University, Moscow 119991, Russia

Journal of Proteome Research

Vol. 15: Issue. 6: Pages. 2026-2038

Publication Date (Web): May 6, 2016

DOI: 10.1021/acs.jproteome.6b00205

Plant secretory (Class III) peroxidases are redox enzymes that rely on N-glycosylation for full enzyme activity and stability. Peroxidases from palm tree leaves comprise the most stable and active plant peroxidases characterized to date. Herein, site-specific glycosylation and microheterogeneity of windmill palm tree (Trachycarpus fortunei) peroxidase are reported. The workflow developed in this study includes novel tools, written in R, to aid plant glycan identification, pGlycoFilter, for annotation of glycopeptide fragmentation spectra, gPSMvalidator, and for relative quantitation of glycoforms, glycoRQ. Mass spectrometry analysis provided a detailed glycosylation profile at the 13 sites of N-linked glycosylation on windmill palm tree peroxidase. Glycan microheterogeneity was observed at each site. Site Asn211 was the most heterogeneous and contained 30 different glycans. Relative quantitation revealed 90% of each glycosylation site was occupied by three or fewer glycans, and two of the 13 sites were partially unoccupied. Although complex and hybrid glycans were identified, the majority of glycans were paucimannosidic, characteristic of plant vacuolar glycoproteins. Further studies pertaining to the glycan structure–activity relationships in plant peroxidases can benefit from the work outlined here.

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http://pubs.acs.org/doi/abs/10.1021/acs.jproteome.6b00205