title: Development of simple and rapid elution methods for proteins from various affinity beads for their direct MALDI-TOF downstream application.
abstract: Commercially available desalting techniques, necessary for downstream MALDI-TOF analysis of proteins, are often costly or time consuming for large-scale analysis. Here, we present techniques to elute proteins from various affinity resins, free from salt and ready for MALDI mass spectrometry. We showed that 0.1% TFA in 50% acetonitrile or 40% ethanol can be used as salt-free eluents for His-tagged proteins from variety of polyhistidine-affinity resins, while washing of resin beads twice with double-distilled water prior to the elution effectively desalted and recovered wide-range-molecular size proteins than commercially available desalting devices. Modified desalting and elution techniques were also applied for Flag- and Myc-tag affinity resins. The technique was further applied in co-precipitation assay, where the maximum recovery of wide-range molecular size proteins is crucial. Further, results showed that simple washing of the beads with double distilled water followed by elution with acetonitrile effectively desalted and recovered 150 kDa factor H protein of the sheep and its binding partner ~30 kDa BbCRASP-1 in co-precipitation assay. In summary, simple modifications in the desalting and elution strategy save time, labor and cost of the protein preparation for MALDI mass spectrometry; and large-scale protein purifications or co-precipitations can be performed with ease.
authors.affiliation: Laboratory of biomedical microbiology and immunology, University of veterinary medicine and pharmacy, Kosice, Slovakia.
authors.forename: Patrik
authors.initials: P
authors.lastname: Mlynarcik
authors.forename: Elena
authors.initials: E
authors.lastname: Bencurova
authors.forename: Marian
authors.initials: M
authors.lastname: Madar
authors.forename: Rastislav
authors.initials: R
authors.lastname: Mucha
authors.forename: Lucia
authors.initials: L
authors.lastname: Pulzova
authors.forename: Stanislav
authors.initials: S
authors.lastname: Hresko
authors.forename: Mangesh
authors.initials: M
authors.lastname: Bhide
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D010449:Peptide Mapping / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D011506:Proteins