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. Molecular & Cellular

Redox Control of Aphid Resistance through Altered Cell Wall Composition and Nutritional Quality

“…Redox changes in the apoplast are likely to influence a wide range of cell wall-processing enzymes that have N-glycosylation sites, Cys thiols, and disulfide bonds. Posttranslational modifications of these enzyme proteins protect them from proteolytic degradation and provide accurate targeting of N-glycoproteins (Zielinska et al, 2012;Ruiz-May et al, 2014). While callose can be deposited in the cell wall around the plasmodesmata to plug the sieve plates and deter feeding (Will and van Bel, 2006), no significant changes in the expression of callose synthases or endo-1,3-b-glucanases were observed in our studies.…”

The Current Status of Proteomic Studies in Somatic Embryogenesis

“…Furthermore, CPLL may serve to increase the effectiveness of other technologies. For instance, in studies of the tomato pericarp N-glycoproteome, a high dynamic range of identified N-glycoproteins was found even after enrichment with lectin affinity chromatography (Ruiz-May et al 2014). Combining an affinity chromatography approach with CPLL may provide an alternative pipeline to reduce the high dynamic range of proteins with posttranslational modifications.…”

“…This versatile technique utilizes a broad range of ligands covalently linked to different types of matrixes (e.g., Sepharose, agarose, and others) (Zielinska et al 2012;Ruiz-May et al 2014). A wide variety of ligands is now commercially available including: antibodies used to enrich specific antigens, substrates to trap enzymes, ligands to isolate receptors, and lectins to enrich and characterize low abundance glycoproteins (Wilchek and Chaiken 2000;Steen et al 2006;Hage and Matsuda 2015).…”

“…Combining affinity chromatography with powerful mass spectrometers could provide important clues about low abundant glycoproteins with key roles during the induction of somatic embryogenesis and the transdifferentiation of the SE and ZE. Recently, several pipelines including affinity chromatography with lectins with multiple affinities to broad types of sugar structures in glycoproteins have been reported as effective approaches for the enrichment of a comprehensive population of glycopeptides and glycoproteins (Ruiz-May et al 2014;Zielinska et al 2012).…”

Hippophae rhamnoides N-glycoproteome analysis: a small step towards sea buckthorn proteome mining

“…Glycoproteome has been analyzed from Arabidopsis (Lastovickova et al 2011;Zhang et al 2011;Minic et al 2007), barley grain (Lastovickova et al 2011), tomato (Catala et al 2011;Thannhauser et al 2013;Ruiz-May et al 2013), maize (Silva-Sanchez et al 2014), tobacco (Hruba and Tupy 1999), rice (Komatsu et al 2009), Brachypodium (Zhang et al 2014) and cotton (Kumar et al 2013). As glycosylation plays an important role in various biological processes including growth and development (Palmisano et al 2010).…”