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

• Published in: Journal of Chromatography A 2013-04, Vol.1286, p.222-228
• Main Authors: Wouters, Bert, Vanhoutte, Dominique J.D., Aarnoutse, Petra, Visser, Adriaan, Stassen, Catherine, Devreese, Bart, Kok, Wim Th, Schoenmakers, Peter J., Eeltink, Sebastiaan
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; chromatography; condensation; detection limit; Fundamental and applied biological sciences. Psychology; gels; General aspects, investigation methods; lysine; MALDI; mass spectrometry; peptides; Proteins; Proteomics; silver nitrate; Spatial chromatography; Staining; stainless steel; two-dimensional gel electrophoresis
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2013.02.064

► Flat-bed polymer monoliths have the potential to be employed for spatial 2D-LC. ► Classical 2D-PAGE visualization methods for proteins were adapted and applied. ► Staining was achieved in 15min, yielding detection limits comparable to gels. ► MALDI-MS on monoliths showed a significant loss in mass precision and sensitivity. ► Fluorescamine labeling of peptides and proteins proved to be MS-compatible. 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 10–15min 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.