engleski

MALDI-TOF MS analysis of N-glycans released from mammillaria gracillis proteins separated by two-dimensional electrophoresis

N-glycosylation of proteins has been intensively investigated during past decades in context of its influence to correct folding, biological activity and stability of proteins. Little information, however, is available about the N-glycoprotein patterns related to cell differentiation, dedifferentiation and transformation in plants. In the present study a general protocol for detection of cellular glycoproteins specific to the different developmental stages of Mammillaria gracillis tissues cultivated in vitro (shoot, callus, hyperhydric regenerant and TW tumour) is presented. This strategy has been developed in order to obtain detailed structural information about changes of N-glycosylation patterns related to specific developmental stage of the tissue. Proteins were separated by 2-D electrophoresis, transferred to a nitrocellulose membrane and treated with Con A to detect N-glycosylated proteins. According to the visual inspection of 2D-ConA-affinoblot more glycoprotein spots were detected in unorganised and partly organised (hyperhydric regenerants) tissues than in the fully organised cactus shoots, while a highest number of glycoproteins was visible in the sample from the TW tumour. In order to explore variations of N-glycoforms in the same protein depending on the organisation stage of the tissue, a selected glycoprotein spot, common for all investigated tissues, was excised from each appropriate gel and in-gel digested by PNGase A. The released glycans were extracted from the gel, purified by the carbon-tip and submitted to MALDI-TOF MS for mass mapping. Seven molecular ions, detected in all investigated tissues, could be considered as a general N-glycosylation outfit of this plant. Molecular ions observed in the MALDI mass spectra from the shoot and callus could be assigned to the typical plant-specific short complex-type N-glycans containing a β (1, 2)-xylose and/or α (1, 3)-fucose, where the callus tissue glycan mixture was more complex than that of the shoot. A significantly higher number of components was found in oligosaccharide mixtures from the hyperhydric regenerant and the TW tumour, respectively, clearly rendering a higher diversity of detected N-glycans as well, like larger structures of mono- and bi-antennary N-linked complex and hybrid glycans. All results obtained in this study indicate that N-glycosylation patterns of the same protein can reflect the organisation level of the plant tissue and therefore studies on N-glycosylation based on in-gel deglycosylation and MALDI-TOF MS provide sensitive and accurate structural data to be directly correlated to the particular morphogenic stage of cactus Mammillaria gracillis tissues in in vitro culture.

N-glycans; differentiation; plant tissue culture; 2D-PAGE; MALDI-TOF MS

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