title: A workflow for large-scale empirical identification of cell wall N-linked glycoproteins of tomato (Solanum lycopersicum) fruit by tandem mass spectrometry.
abstract: Glycosylation is a common PTM of plant proteins that impacts a large number of important biological processes. Nevertheless, the impacts of differential site occupancy and the nature of specific glycoforms are obscure. Historically, characterization of glycoproteins has been difficult due to the distinct physicochemical properties of the peptidyl and glycan moieties, the variable and dynamic nature of the glycosylation process, their heterogeneous nature, and the low relative abundance of each glycoform. In this study, we explore a new pipeline developed for large-scale empirical identification of N-linked glycoproteins of tomato fruit as part of our ongoing efforts to characterize the tomato secretome. The workflow presented involves a combination of lectin affinity, tryptic digestion, ion-pairing HILIC, and precursor ion-driven data-dependent MS/MS analysis with a script to facilitate the identification and characterization of occupied N-linked glycosylation sites. A total of 212 glycoproteins were identified in this study, in which 26 glycopeptides from 24 glycoproteins were successfully characterized in just one HILIC fraction. Further precursor ion discovery-based MS/MS and deglycosylation followed by high accuracy and resolution MS analysis were used to confirm the glycosylation sites and determine site occupancy rates. The workflow reported is robust and capable of producing large amounts of empirical data involving N-linked glycosylation sites and their associated glycoforms.
authors.affiliation: Robert W. Holley Center for Agriculture and Health, USDA-ARS, Cornell University, Ithaca, NY 14853, USA. tt34@cornell.edu
authors.forename: Theodore W
authors.initials: TW
authors.lastname: Thannhauser
authors.forename: Miaoqing
authors.initials: M
authors.lastname: Shen
authors.forename: Robert
authors.initials: R
authors.lastname: Sherwood
authors.forename: Kevin
authors.initials: K
authors.lastname: Howe
authors.forename: Tara
authors.initials: T
authors.lastname: Fish
authors.forename: Yong
authors.initials: Y
authors.lastname: Yang
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
authors.forename: Sheng
authors.initials: S
authors.lastname: Zhang
mesh_terms: D002473:Cell Wall / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D003208:Concanavalin A / Q000737:chemistry; D005638:Fruit / Q000737:chemistry*; D006020:Glycopeptides / Q000032:analysis / Q000737:chemistry; D006023:Glycoproteins / Q000032:analysis* / Q000737:chemistry; D018551:Solanum lycopersicum / Q000737:chemistry*; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D010940:Plant Proteins / Q000032:analysis* / Q000737:chemistry; D020543:Proteome / Q000737:chemistry; D012107:Research Design; D012965:Sodium Chloride; D053719:Tandem Mass Spectrometry / Q000379:methods; D014357:Trypsin / Q000737:chemistry
chemical_list: D006020:Glycopeptides; D006023:Glycoproteins; D010446:Peptide Fragments; D010940:Plant Proteins; D020543:Proteome; D003208:Concanavalin A; D012965:Sodium Chloride; D014357:Trypsin
keywords: HILIC; Lectin affinity chromatography; N-linked glycosylation; Plant glycoproteomics; Precursor ion scan