title: 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.
abstract: 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-μm 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 24h, 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.
authors.affiliation: Vrije Universiteit Brussel, Department of Chemical Engineering, Pleinlaan 2, B-1050 Brussels, Belgium.
authors.forename: Bert
authors.initials: B
authors.lastname: Wouters
authors.forename: Dominique J D
authors.initials: DJ
authors.lastname: Vanhoutte
authors.forename: Petra
authors.initials: P
authors.lastname: Aarnoutse
authors.forename: Adriaan
authors.initials: A
authors.lastname: Visser
authors.forename: Catherine
authors.initials: C
authors.lastname: Stassen
authors.forename: Bart
authors.initials: B
authors.lastname: Devreese
authors.forename: Wim Th
authors.initials: WT
authors.lastname: Kok
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Schoenmakers
authors.forename: Sebastiaan
authors.initials: S
authors.lastname: Eeltink
mesh_terms: D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D004396:Coloring Agents / Q000737:chemistry; D057230:Limit of Detection; D008689:Methacrylates / Q000737:chemistry; D008970:Molecular Weight; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D004396:Coloring Agents; D008689:Methacrylates; D010455:Peptides; D011108:Polymers; D011506:Proteins