Location: Plant, Soil and Nutrition ResearchTitle: A comparative study of lectin affinity based plant n-glycoproteome profiling using tomato fruit as a model
|RUIZ-MAY, ELIEL - Cornell University - New York|
|HUCKO, SIMON - Cornell University - New York|
|ZHANG, SHENG - Cornell University - New York|
|SHERWOOD, ROBERT - Cornell University - New York|
|Thannhauser, Theodore - Ted|
|ROSE, JOCELYN - Cornell University - New York|
Submitted to: Proteomes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/20/2013
Publication Date: 11/6/2014
Citation: Ruiz-May, E., Hucko, S., Howe, K.J., Zhang, S., Sherwood, R.W., Thannhauser, T.W., Rose, J.K. 2014. A comparative study of lectin affinity based plant n-glycoproteome profiling using tomato fruit as a model. Proteomes. 13:566-579.
Interpretive Summary: N-glycosylation is one of the most heterogeneous and common post-translational modifications of eukaryotic proteins and one that affects many aspects of protein targeting, enzymatic properties, stability and intermolecular interactions. There is therefore considerable interest in developing robust and sensitive high throughput analytical pipelines to isolate and structurally characterize N-glycoprotein populations, allowing glycoprotein identification and analysis of the glycosylation site occupancy and N-glycan structure. To this end, lectin affinity chromatography is increasingly popular. Various lectins are known to have different binding affinities for N-glycans and so the selective binding of N-glycoproteins in complex protein extracts to these lectins and their subsequent release allows a critical enrichment step before sequencing and glycan analysis by MS. Here we present a refinement of this approach: multiple lectin affinity chromatograph (MLAC). In this study, we address both the structural basis of the binding of lectins to plant derived N-glycoproteins and the limitations of typical “shotgun based” profiling approaches in identifying and characterizing low abundance N-glycoproteins in complex protein extracts. From these studies, it became evident that the large dynamic range of N-glycoprotein abundance was a significant limiting factor in the structural determination of the tomato and that there was a bias toward the detection of highly abundant N-glycopeptides. We therefore evaluated the use of an in planta recombinant expression strategy combined with affinity purification-mass spectrometry as a means to characterize the N-glycan structures of glycoproteins whose abundance is too low to be readily determined via the usual “shotgun” pipeline, using a tomato xyloglucan-specific endoglucanase inhibitor protein (XEGIP) as a test case.
Technical 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.