A small-scale method for the preparation of plant N-linked glycans from soluble proteins for analysis by MALDI-TOF mass spectrometry.
The use of plants as production hosts for recombinant glycoproteins, which is rapidly developing, requires methods for fast and reliable analysis of plant N-linked glycans. This study describes a simple small-scale method for the preparation of N-linked glycans from soluble plant protein and analysis thereof by matrix assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF MS). Concentration and protease digestion of plant protein as well as deglycosylation is carried out in a single concentrator unit without the need for intermittent purification to minimize adsorptive loss and to facilitate handling. Plant protein is concentrated in a unit with a 5kDa cutoff, and after buffer exchange, pepsin (EC 3.4.23.1) digestion is carried out in the concentrator overnight to obtain peptides as substrates for deglycosylation. Deglycosylation is carried out with peptide-N-glycosidase A (PNGase A; EC 3.5.1.52) for 24h. Released N-glycans are purified using reverse-phase and cation exchange chromatography micro-columns for removal of peptides and desalting. N-Glycans are directly analyzed by MALDI-TOF MS without derivatization. The method for isolation of N-glycans is compatible with secreted proteins from cell culture supernatant as well as with soluble protein extracts from leaf tissue. As little as 5mug of plant glycoprotein is sufficient for N-glycan preparation for MALDI-TOF MS analysis using this method.
Karg
Saskia R
SR
Institute of Microbiology, ETH Zürich, Wolfgang-Pauli Strasse 10, HCI F406, CH-8093 Zürich, Switzerland.
Frey
Alexander D
AD
Ferrara
Claudia
C
Streich
Daniel K
DK
Umaña
Pablo
P
Kallio
Pauli T
PT
D000818:Animals; D002460:Cell Line; D006023:Glycoproteins / Q000302:isolation & purification*; D006031:Glycosylation; D006801:Humans; D009584:Nitrogen; D010940:Plant Proteins / Q000302:isolation & purification*; D011134:Polysaccharides / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014026:Nicotiana / Q000737:chemistry
D006023:Glycoproteins; D010940:Plant Proteins; D011134:Polysaccharides; D009584:Nitrogen