Towards characterization of the glycoproteome of tomato (Solanum lycopersicum) fruit using Concanavalin A lectin affinity chromatography and LC‐MALDI‐MS/MS analysis

Abstract

The isolation and analysis of glycoproteins by coupling lectin affinity chromatography with MS has emerged as a powerful strategy to study the glycoproteome of mammalian cells. However, this approach has not been used extensively for the analysis of plant glycoproteins. As with all eukaryotes, N-glycosylation is a common post-translational modification for plant proteins traveling through the secretory pathway. Many such proteins are destined for the cell wall, or apoplast, where they play important roles in processes such as modifying cell wall structure, sugar metabolism, signaling, and defense against pathogens. Here, we describe a strategy to enrich for and identify secreted plant proteins based on affinity chromatography using the lectin Concanavalin A and two-dimensional liquid chromatography, together with matrix-assisted laser desorption/ionization MS analysis. The value of this approach is illustrated through the characterization of glycoproteins that are expressed in ripe tomato (Solanum lycopersicum) fruit, a developmental stage that is fundamentally linked with significant changes in cell wall structure and composition. This glycoprotein trap strategy allowed the isolation of a sub-proteome with an extremely high proportion of proteins that are predicted to be resident in the cell wall or secretory pathway, and the identification of new putative cell wall proteins.

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

“…These proteins were identified by MALDI-TOF/TOF as putative ATPDIL1-4 (Arabidopsis thaliana protein disulfide isomerase-like 1-4) electron transporter (spot 1), protein disulphide isomerase (PDI) (spot 10), calreticulin 1 (CRT1) (spot 11), glycosyl hydrolase family 38 protein (GH 38) (spot 26), phantastica (spot 28), maturase k (spot 36), ATXR6 (Arabidopsis trithorax related protein 6) (spot 39), cysteine protease inhibitor (spot 43), con A precursor (spot 46) and an unnamed protein product (spot 47) ( Table 1). Out of these four proteins, GH 38 family protein (Catala et al 2011), PDI (Catala et al 2011), CRT1 (Komatsu et al 2009) and con A precursor (Sheldon and Bowles 1992) are reported to be glycosylated earlier. The identified proteins belong to vital physiological processes like protein targeting, redox homeostasis, organ identity, glycan metabolism and stress.…”

“…It contains thioredoxin (TRX) domain and acts as catalyst of disulfide bond formation in the oxidizing environment of endoplasmic reticulum stabilizing the tertiary and quaternary structure of protein (Gupta and Tuteja 2011). The presence of N-glycosylated form is reported in ripe tomato fruit by Catala et al (2011). PDI is involved in oxidation or reduction of disulfide bridges, cell adhesion, have chaperone and anti-chaperone activity and also act as a primary acceptor of peptides translocated into the ER (Noiva 1999;Onda 2013).…”

“…Glycosyl hydrolase family 38 protein (spot 26) which is a class II a-D-mannosidase involved in the synthesis of glycoproteins has been reported in the N-glycoproteome of xylem sap in B. oleracea (Ligat et al 2011), tomato fruit (Catala et al 2011) and mature stem of Arabidopsis (Minic et al 2007). These are localized in the Golgi apparatus, cytoplasm and vacuole (Kumar et al 2013).…”

“…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).…”

Silicon Era of Carbon-Based Life: Application of Genomics and Bioinformatics in Crop Stress Research

“…Twenty-two highly oxidized proteins involved in a wide range of biological processes were identified in ozone-treated rice using this method [127]. Phosphoproteome [128], glycoproteome, and secretome [129][130][131][132] are sub-categories of proteomics that require special staining and enrichment techniques. Post-translational modifications involved in gene expression regulations will be discussed in the Epigenomics section below.…”