Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for identifying the composition of labeled proteins.
Labeled proteins are extensively used in molecular biology and environmental science. The determination of the composition and label ratio is very important for monitoring the efficiency of their separation and purification. In this paper a novel method based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry was developed for this purpose. The results obtained for three commercial labeled proteins showed that they are mixtures of different conjugates. In some cases, the label ratio obtained by UV spectrometry and MALDI mass spectrometry was strikingly different. For fluorescent labels such as fluorescein isothiocyanate, MALDI mass spectrometry determines the number of covalently bound labels, whereas UV absorption yields both bound and adsorbed labels. For biotinylated proteins, label ratios obtained by the 4-hydroxyazabenzene-2'-carboxylic acid (HABA)-avidin method were found to be much smaller those determined by MALDI mass spectrometry. The HABA-avidin method may therefore not be suitable for the determination of biotin label ratios.
Lu
J
J
Department of Chemistry, ETH Zentrum, Universitästrasse 16, Zürich, CH-8092, Switzerland.
Zenobi
R
R
D001710:Biotin; D003574:Cytochrome c Group / Q000737:chemistry; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000737:chemistry; D005456:Fluorescent Dyes; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D012709:Serum Albumin / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet
D003574:Cytochrome c Group; D005456:Fluorescent Dyes; D007328:Insulin; D011506:Proteins; D012709:Serum Albumin; C031573:insulin, fluorescein-isothiocyanated-; D001710:Biotin; D016650:Fluorescein-5-isothiocyanate