Selective isolation of glycoproteins and glycopeptides for MALDI-TOF MS detection supported by magnetic particles.
Glycosylation is the most common form of posttranslational modification of proteins (50-80%). The isolation, discovery, and subsequent identification of glycosylated peptides and proteins is becoming more and more important in glycoproteomics and diagnosis. MALDI-TOF mass spectrometry is an ideal technique for identifying peptides and proteins and their corresponding modifications. The enrichment of glycosylated peptides and proteins from different sources can be attained by affinity chromatography supported by functionalized magnetic particles. Covalent coating of magnetic beads with Concanavalin A (ConA) and diboronic acid was performed by carbodiimide and poly-glutaraldehyde methods, respectively. The functionalized beads were employed to establish and optimize protocols for the binding and detection of glycosylated peptides and proteins with respect to an automated workflow and the subsequent detection and identification by MALDI-TOF mass spectrometry. For several model proteins, the capture and identification could be demonstrated by SDS-PAGE and MALDI-TOF mass spectrometry. According to the type of glycosylation (high man-nose, hybrid, or complex type) the different proteins were enriched by ConA or boronic acid-functionalized beads.
Sparbier
Katrin
K
Bruker Daltonik GmbH, Leipzig, Germany. ksp@bdal.de
Koch
Sonja
S
Kessler
Irina
I
Wenzel
Thomas
T
Kostrzewa
Markus
M
D000818:Animals; D001709:Biotechnology / Q000379:methods*; D001897:Boronic Acids / Q000737:chemistry; D002417:Cattle; D003208:Concanavalin A / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006020:Glycopeptides / Q000302:isolation & purification*; D006023:Glycoproteins / Q000302:isolation & purification*; D006031:Glycosylation; D006801:Humans; D008280:Magnetics; D013058:Mass Spectrometry; D008863:Microspheres; D012260:Ribonucleases / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001897:Boronic Acids; D006020:Glycopeptides; D006023:Glycoproteins; D003208:Concanavalin A; D012260:Ribonucleases; C029600:ribonuclease B