Combining various strategies to increase the coverage of the plant cell wall glycoproteome

Abstract

Glycoproteomics recently became a very active field, mostly in mammals. The first part of this paper consists of a mini-review on the strategies used in glycoproteomics, namely methods for enrichment in glycoproteins and mass spectrometry (MS) techniques currently used. In a second part, these strategies are applied to the cell wall glycoproteome of etiolated hypocotyls of Arabidopsis thaliana, showing their complementarity. Several sub-glycoproteomes were obtained by: (i) affinity chromatography on concanavaline A (ConA) and analysis of glycoproteins by MALDI-TOF MS; (ii) multidimensional lectin chromatography (using AIL, PNA, ConA and WGA lectins) and subsequent identification of glycoproteins by MALDI-TOF MS and LC-MS/MS; (iii) boronic acid chromatography followed by identification of glycoproteins by MALDI-TOF MS. Altogether, 127 glycoproteins were identified. Most glycoproteins were found to be putative N-glycoproteins and N-glycopeptides were predicted from MS data using the ProTerNyc bioinformatics software.

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

“…Con A precursor in elute may be from the column itself as mature non-glycosylated con A is derived from its glycosylated precursor which upon modification forms the lectin (Chrispeels et al 1986). The presence of con A in the elute has been reported earlier (Ligat et al 2011;Zhang et al 2011;Lastovickova et al 2011).…”

“…Con A affinoblotting confirm the purification of N-glycoproteome. Spots were in-gel digested and analyzed by MALDI-TOF/TOF observed for barley (Lastovickova et al 2011), maize (Silva-Sanchez et al 2014 and Arabidopsis (Zhang et al 2011). Although very faint polypeptides were observed at 28 and 32 kDa however strong signals were observed on the blot which suggests that these polypeptides were probably heavily glycosylated.…”

“…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).…”

Xyloglucan Metabolism Differentially Impacts the Cell Wall Characteristics of the Endosperm and Embryo during Arabidopsis Seed Germination

“…Previous cell wall proteomic studies have also reported the presence of several XYL1 isoforms in vegetative tissues. The XYL1 protein was detected in extracts from stems and etiolated hypocotyls, and the observed mass shifts were predicted to be due to glycosylation at a number of sites (Minic et al, 2007;Albenne et al, 2009;Zhang et al, 2011). Moreover, a pI shift has also been observed due to protein phosphorylation.…”

Exploring the N-glycosylation Pathway in Chlamydomonas reinhardtii Unravels Novel Complex Structures

“…Whereas mammalian N-glycoproteomes have been studied extensively down to tissue-and cell-type-specific levels (13)(14)(15)(16)(17), less information is available regarding the N-glycoproteomes of plants and green algae (18,19). The use of glycoproteomic approaches could help unravel the identity of endogenous glycoproteins from C. reinhardtii.…”