title: A comparative study of lectin affinity based plant N-glycoproteome profiling using tomato fruit as a model.
abstract: Lectin affinity chromatography (LAC) can provide a valuable front-end enrichment strategy for the study of N-glycoproteins and has been used to characterize a broad range eukaryotic N-glycoproteomes. Moreover, studies with mammalian systems have suggested that the use of multiple lectins with different affinities can be particularly effective. A multi-lectin approach has also been reported to provide a significant benefit for the analysis of plant N-glycoproteins; however, it has yet to be determined whether certain lectins, or combinations of lectins are optimal for plant N-glycoproteome profiling; or whether specific lectins show preferential association with particular N-glycosylation sites or N-glycan structures. We describe here a comparative study of three mannose-binding lectins, concanavalin A, snowdrop lectin, and lentil lectin, to profile the N-glycoproteome of mature green stage tomato (Solanum lycopersicum) fruit pericarp. Through coupling lectin affinity chromatography with a shotgun proteomics strategy, we identified 448 putative N-glycoproteins, whereas a parallel lectin affinity chromatography plus hydrophilic interaction chromatography analysis revealed 318 putative N-glycosylation sites on 230 N-glycoproteins, of which 100 overlapped with the shotgun analysis, as well as 17 N-glycan structures. The use of multiple lectins substantially increased N-glycoproteome coverage and although there were no discernible differences in the structures of N-glycans, or the charge, isoelectric point (pI) or hydrophobicity of the glycopeptides that differentially bound to each lectin, differences were observed in the amino acid frequency at the -1 and +1 subsites of the N-glycosylation sites. We also demonstrated an alternative and complementary in planta recombinant expression strategy, followed by affinity MS analysis, to identify the putative N-glycan structures of glycoproteins whose abundance is too low to be readily determined by a shotgun approach, and/or combined with deglycosylation for predicted deamidated sites, using a xyloglucan-specific endoglucanase inhibitor protein as an example.
authors.affiliation: Department of Plant Biology, Cornell University, Ithaca, New York 14853;
authors.forename: Eliel
authors.initials: E
authors.lastname: Ruiz-May
authors.forename: Simon
authors.initials: S
authors.lastname: Hucko
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Howe
authors.forename: Sheng
authors.initials: S
authors.lastname: Zhang
authors.forename: Robert W
authors.initials: RW
authors.lastname: Sherwood
authors.forename: Theodore W
authors.initials: TW
authors.lastname: Thannhauser
authors.forename: Jocelyn K C
authors.initials: JK
authors.lastname: Rose
mesh_terms: D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D006023:Glycoproteins / Q000032:analysis / Q000378:metabolism*; D006031:Glycosylation; D018551:Solanum lycopersicum / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D037121:Plant Lectins / Q000032:analysis / Q000378:metabolism*; D010940:Plant Proteins / Q000032:analysis / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000032:analysis / Q000378:metabolism; D040901:Proteomics
chemical_list: D006023:Glycoproteins; D037121:Plant Lectins; D010940:Plant Proteins; D020543:Proteome