A novel, simple and sensitive ligand affinity capture method for detecting molecular interactions by MALDI mass spectrometry.
A simple and sensitive ligand affinity capture method (LAC) was developed to detect biotinylated biomolecules bound to a biotin-avidin base by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI ToF MS). Glass slides covered with a metal film for MALDI MS applications were treated with amino-silane and derivatized with biotin followed by binding of avidin. Washing buffers with high ionic strength increased the specificity of the subsequent binding of biotinylated biomolecules to the avidin layer. A combined thin layer-dried droplet method using alpha-cyano-4-hydroxycinnamic acid (CHCA) in acetone or ethyl acetate resulted in the most intense ions of biotinylated polymyxin B, whereas the matrix conditions did not influence the detection of angiotensin II. Addition of biotinylated biomolecules in the low femtomole to low picomole range resulted in sufficient ion intensity for detection by the LAC method. The LAC concept was extended by binding of biotinylated lipopolysaccharide to the biotin-avidin base followed by preferential capture and specific detection of the binding antagonist polymyxin B.
Jørgensen
Ann Louise Worsøe
AL
Department of Food Science, University of Aarhus, Blichers Allé 20, DK-8830 Tjele, Denmark.
Juul-Madsen
Helle Risdahl
HR
Stagsted
Jan
J
D000345:Affinity Labels / Q000737:chemistry; D000809:Angiotensins / Q000737:chemistry; D001360:Avidin / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D019921:Biotinylation; D003373:Coumaric Acids / Q000737:chemistry; D008070:Lipopolysaccharides / Q000737:chemistry; D008958:Models, Molecular; D011112:Polymyxin B / Q000737:chemistry; D012680:Sensitivity and Specificity; D012821:Silanes / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D000345:Affinity Labels; D000809:Angiotensins; D003373:Coumaric Acids; D008070:Lipopolysaccharides; D012821:Silanes; D001360:Avidin; C007175:alpha-cyano-4-hydroxycinnamate; D001710:Biotin; D011112:Polymyxin B