Comparative Mapping of Recombinant Proteins and Glycoproteins by Plasma Desorption and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry
| Autor: | Michael Karas, Anthony Tsarbopoulos, Birendra N. Pramanik, Tattanahalli L. Nagabhushan, Kerstin Strupat, Franz Hillenkamp |
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| Rok vydání: | 1994 |
| Předmět: |
chemistry.chemical_classification
Chromatography Protein mass spectrometry Chemistry Lasers Analytical chemistry Peptide Mass spectrometry Peptide Mapping Mass Spectrometry Recombinant Proteins Analytical Chemistry Surface-enhanced laser desorption/ionization Matrix (chemical analysis) Mice Matrix-assisted laser desorption/ionization Desorption Mass spectrum Animals Humans Glycoproteins |
| Zdroj: | Analytical Chemistry. 66:2062-2070 |
| ISSN: | 1520-6882 0003-2700 |
| Popis: | The mass spectrometric (MS) techniques of 252Cf-plasma desorption (PD) and matrix-assisted laser desorption/ionization (MALDI) are compared in the molecular weight determination and the mapping analysis of several recombinant proteins and glycoproteins. MALDI MS analysis exhibited better sensitivity and mass measurement accuracy and a remarkably short analysis time compared with PD MS analysis. The latter was not successful in the analysis of rhIFN-gamma and the higher mass mammalian cell-derived IL-5 glycoproteins. Mapping of the Escherichia coli-derived rhIFN alpha-2b and rhIL-4 proteins, by direct PD or MALDI MS analysis of the trypsin-generated peptide mixtures provided signals for ca. 95% and 88% of the expected tryptic peptides, respectively. Peptide signals below m/z 1500 were generally more intense in the PD mass spectra, while higher mass signals were more intense in the MALDI mass spectra. Both PD and MALDI MS analyses provided a rapid confirmation of the existing two and three disulfide bonds in the rhIFN alpha-2b and rhIL-4 proteins, respectively. In the mapping of the CHO IL-4 glycoprotein, detection of the trypsin-generated glycopeptides was only possible by MALDI, where their detection was greatly improved by using the super-DHB (sDHB) matrix, a 9:1 mixture of 2,5-dihydroxybenzoic acid (DHB) with 2-hydroxy-5-methoxybenzoic acid. This sDHB matrix also generated significantly enhanced and better resolved MALDI peptide signals, which in turn resulted in a much improved mass measurement accuracy. |
| Databáze: | OpenAIRE |
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