Analysis of carbohydrates on proteins by offline normal-phase liquid chromatography MALDI-TOF/TOF-MS/MS.
Glycoproteins typically exist as a diverse population of glycoforms, which can consist of a great number of different glycan structures (including structural isomers) that vary in their degree of occupancy at given glycosylation sites. Hence, the detailed characterization of such glycans can be a very demanding task. Liquid chromatography in combination with mass spectrometric techniques can provide a fast and sensitive tool for the analysis of these structurally complex molecules. Here we describe a sensitive method that employs capillary normal-phase HPLC coupled offline to MALDI-TOF/TOF-MS/MS for the detailed characterization of enzymatically released N-glycans. The normal-phase chromatography allows the separation of some isobaric glycan structures and analysis of the glycans by MALDI high-energy CID gives sequence, linkage and branching information.
Tryfona
Theodora
T
Department of Biochemistry, University of Cambridge, Cambridge, UK.
Stephens
Elaine
E
D000096:Acetone / Q000737:chemistry; D000586:Amination; D000818:Animals; D002417:Cattle; D011232:Chemical Precipitation; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid / Q000379:methods*; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D019476:Insect Proteins / Q000737:chemistry / Q000378:metabolism; D019158:N-Acetylneuraminic Acid / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D006023:Glycoproteins; D019476:Insect Proteins; D010940:Plant Proteins; D011134:Polysaccharides; D000096:Acetone; D019158:N-Acetylneuraminic Acid