Visualization procedures for proteins and peptides on flat-bed monoliths and their effects on matrix-assisted laser-desorption/ionization time-of-flight mass spectrometric detection
| Autor: | Wim Th. Kok, Bart Devreese, Peter J. Schoenmakers, Catherine Stassen, Dominique J.D. Vanhoutte, Sebastiaan Eeltink, Bert Wouters, Petra Aarnoutse, Adriaan Visser |
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| Přispěvatelé: | Analytical Chemistry and Forensic Science (HIMS, FNWI) |
| Jazyk: | angličtina |
| Rok vydání: | 2013 |
| Předmět: |
Proteomics
Polymers Analytical chemistry Peptide Mass spectrometry Fluorescamine Biochemistry Analytical Chemistry chemistry.chemical_compound Limit of Detection Monolith Coloring Agents Polyacrylamide gel electrophoresis chemistry.chemical_classification Detection limit geography Chromatography geography.geographical_feature_category Organic Chemistry Proteins General Medicine Staining Molecular Weight Matrix-assisted laser desorption/ionization chemistry Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Methacrylates Peptides Chromatography Liquid |
| Zdroj: | Journal of Chromatography A, 1286, 222-228. Elsevier |
| ISSN: | 0021-9673 |
| DOI: | 10.1016/j.chroma.2013.02.064 |
| Popis: | The present study concerns the application of visualization methods, i.e. coomassie-brilliant-blue-R staining (CBB-R), silver-nitrate staining, and fluorescamine labeling, and subsequent MALDI-MS analysis of intact proteins and peptides on the surface of flat-bed monoliths, intended for spatial two-dimensional chromatographic separations. The use of 100-mu m thick macroporous poly(butyl methacrylate-co-ethylene dimethacrylate) flat-bed monoliths renders a fixation step obsolete, so that CBB-R and silver-nitrate staining and destaining could be achieved in 10-15 min as opposed to up to 24 h, as is typical on 2D-PAGE gels. The detection limits remained comparable. The compatibility of the monolithic layer with subsequent MALDI-MS analysis of individual proteins and peptide spots was investigated with regards to mass accuracy, mass precision, resolution, and signal intensity. When comparing results from MALDI-MS analysis of proteins and peptides on a flat-bed monolith to results obtained directly on stainless-steel target plates, significant losses in mass precision, signal intensity, and an increased variation in resolution were observed. In addition, a loss in signal intensity up to two orders of magnitude was observed when using monolithic layers. After CCB-R and silver-nitrate staining and destaining to disrupt the protein-dye complexes no MALDI spectra with significant S/N ratios could be achieved. After fluorescamine labeling heterogeneous signals were observed, which resulted from a distribution in the number of fluorescence-labeled lysine groups and from the presence of labeled derivatives that had undergone condensation reactions. (c) 2013 Elsevier B.V. All rights reserved. |
| Databáze: | OpenAIRE |
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