Location: Plant, Soil and Nutrition ResearchTitle: A workflow for large-scale empirical identification of cell wall N-linked glycoproteins of tomato (Solanum lycopersicum) fruit by tandem mass spectrometry) Author
|Thannhauser, Theodore - Ted|
Submitted to: Electrophoresis
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/24/2013
Publication Date: 4/12/2013
Citation: Thannhauser, T.W., Shen, M., Sherwood, R., Howe, K.J., Fish, T., Yang, Y., Chen, W., Zhang, S. 2013. A workflow for large-scale empirical identification of cell wall N-linked glycoproteins of tomato (Solanum lycopersicum) fruit by tandem mass spectrometry. Electrophoresis. DOI: 10.1002/elps.201200656. Interpretive Summary: Glycosylation represents an important post-translational modification of proteins, affecting many protein functions and cellular activities. It is estimated that 50% of all proteins are glycosylated based on the fact that two-thirds of entries in Swiss-Prot database were found to contain at least one N-glycosylation consensus sequon. Glycoproteins can function as structural elements, as signaling molecules, as recognition markers for the assembly of protein complexes and can affect protein folding, solubility and stability. Thus, understanding the detailed structure of glycoproteins has the potential to provide valuable insights to their global functions. Structural elucidation of glycans, glycopeptides and glycoproteins has been notoriously difficult as glycans are typically highly heterogeneous and chemically complex, containing a wide variety of different intersaccharide linkages, with a wide range of abundance between glycoforms and occupancy of glycosylated sites. Additionally, the hydrophilic nature of glycans and frequent multiple adduct formation has often caused poor retention by reverse phase -chromatography, reduced ionization efficiency and low MS signals. Furthermore, the labile nature of the glycan-peptide bond often causes the neutral loss of individual carbohydrates and few fragmented ions from the core peptide during collisional fragmentation, yielding little or no information for peptide identification, glycol-site determination and full glycan sequence. Here we report a work flow for the complete characterization of glycopetides involving enzymatic digestion, multidimensional liquid chromatorgraph, precursor ion driven discovery-based tandem mass spectrometry and use it to identify and characterize a large number of cell wall glycoproteins isolated from the pericarp tissue of tomato fruit at the mature green stage of development.
Technical Abstract: Glycosylation is a common post-translational modification 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 lycosylation sites and their associated glycoforms.