Plant N-glycan profiling of minute amounts of material.
Development of convenient strategies for identification of plant N-glycan profiles has been driven by the emergence of plants as an expression system for therapeutic proteins. In this article, we reinvestigated qualitative and quantitative aspects of plant N-glycan profiling. The extraction of plant proteins through a phenol/ammonium acetate procedure followed by deglycosylation with peptide N-glycosidase A (PNGase A) and coupling to 2-aminobenzamide provides an oligosaccharide preparation containing reduced amounts of contaminants from plant cell wall polysaccharides. Such a preparation was also suitable for accurate qualitative and quantitative evaluation of the N-glycan content by mass spectrometry. Combining these approaches allows the profiling to be carried out from as low as 500 mg of fresh leaf material. We also demonstrated that collision-induced dissociation (CID) mass spectrometry in negative mode of N-glycans harboring alpha(1,3)- or alpha(1,6)-fucose residue on the proximal GlcNAc leads to specific fragmentation patterns, thereby allowing the discrimination of plant N-glycans from those arising from mammalian contamination.
Séveno
Martial
M
CNRS-FRE 3090, IFRMP 23, Université de Rouen, 76821 Mont Saint Aignan Cédex, France; Medicago Inc., Québec, QC G1V 3V9, Canada.
Cabrera
Gleysin
G
Triguero
Ada
A
Burel
Carole
C
Leprince
Jérome
J
Rihouey
Christophe
C
Vézina
Louis-Philippe
LP
D'Aoust
Marc-André
MA
Rudd
Pauline M
PM
Royle
L
L
Dwek
Raymond A
RA
Harvey
David J
DJ
Lerouge
Patrice
P
Cremata
José A
JA
Bardor
Muriel
M
D002851:Chromatography, High Pressure Liquid; D006020:Glycopeptides / Q000737:chemistry / Q000378:metabolism; D000455:Medicago sativa / Q000737:chemistry; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D018515:Plant Leaves / Q000737:chemistry; D010944:Plants / Q000737:chemistry*; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014026:Nicotiana / Q000737:chemistry; D062367:ortho-Aminobenzoates / Q000737:chemistry
D006020:Glycopeptides; D011134:Polysaccharides; D062367:ortho-Aminobenzoates; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; C000219:anthranilamide