The Combination of Lectin Affinity Chromatography, Gel Electrophoresis and Mass Spectrometry in the Study of Plant Glycoproteome: Preliminary Insights

Abstract

The study of plant proteome is important because proteins and glycoproteins are involved in many crucial biological processes that could enhance the agronomical and nutritional value of crops and food products. The analysis of model organisms (e.g., Arabidopsis thaliana) is a popular means to aid in understanding many plant features. Proteomics has the potential to contribute to cereal science, mainly through the description of grain quality by elucidating the ways in which the genes are expressed during grain filling under particular environmental conditions. Since barley grain composition (i.e., its protein and glycoprotein analysis) is important for the brewing industry, human and animal nutrition, plant breeding or cultivar identification, the profiles of glycoproteins released from barley were one of the aims of this paper. This work combines affinity chromatography with 1D gel electrophoresis followed by mass spectrometry for the characterization of modified plant proteins. Lectin chromatography with Concanavalin A (ConA) was integrated to the procedure for the selective isolation of N-glycoproteins. For the verification of the universality of a chosen approach, proteins were extracted by deionized water from two different model plant materials-barley (Hordeum vulgare) and A. thaliana. Our attention was focused on the glycoproteins present in both barley grains and grains after the malting procedure and Arabidopsis seedlings. The proteins retained in an affinity column were separated by SDS-PAGE, excised, digested by trypsin and studied by MALDI-TOF MS. SDS gels revealed the differences between ConA non-bound and bound fractions. The majority of proteins were found in non-bound fractions. Several important glycoproteins were confirmed in studied materials: barley grain (serine carboxypeptidase; alphaamylase inhibitor BMAI-1), barley malt (e.g., b-D-xylosidase) and Arabidopsis (b-D-xylosidase, peroxidase, etc.).

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).…”

Cerealn-glycoproteins Enrichment by Lectin Affinity Monolithic Chromatography

“…Lectin affinity chromatography (AC) was used for enrichment of cereal glycoproteins. This work partially took up the previous study of our team (Laštovičková et al 2011) where manually filled columns with ConA have been used for the purification of plant glycoproteins. To improve the efficiency of the affinity enrichment which is crucial in analysis of low abundant glycoproteins, we decided to perform the purification on a high capacity monolithic ConA HPLC column.…”

“…in barley (Soerensen et al 1986;Laštovičková et al 2011), wheat (Breddam et al 1987) or rice (Park et al 2013). The special attention in research is concentrated to analysis of cereal allergenic glycoproteins.…”