title: MALDI analysis of proteins after extraction from dissolvable ethylene glycol diacrylate cross-linked polyacrylamide gels.
abstract: Although the extraction of intact proteins from polyacrylamide gels followed by mass spectrometric molecular mass determination has been shown to be efficient, there is room for alternative approaches. Our study evaluates ethylene glycol diacrylate, a cleavable cross-linking agent used for a new type of dissolvable gels. It attains an ester linkage that can be hydrolyzed in alkali conditions. The separation performance of the new gel system was tested by 1D and 2D SDS-PAGE using the outer chloroplast envelope of Pisum sativum as well as a soluble protein fraction of human lymphocytes, respectively. Gel spot staining (CBB), dissolving, and extracting were conducted using a custom-developed workflow. It includes protein extraction with an ammonia-SDS buffer followed by methanol treatment to remove acrylamide filaments. Necessary purification for MALDI-TOF analysis was implemented using methanol-chloroform precipitation and perfusion HPLC. Both cleaning procedures were applied to several standard proteins of different molecular weight as well as 'real' biological samples (8-75 kDa). The protein amounts, which had to be loaded on the gel to detect a peak in MALDI-TOF MS, were in the range of 0.1 to 5 μg, and the required amount increased with increasing mass.
authors.affiliation: Institute of Pharmaceutical Chemistry, Cluster of Excellence "Macromolecular Complexes", Goethe University, Frankfurt am Main, Germany.
authors.forename: Dimitrios G
authors.initials: DG
authors.lastname: Papasotiriou
authors.forename: Stavroula
authors.initials: S
authors.lastname: Markoutsa
authors.forename: Jan
authors.initials: J
authors.lastname: Gorka
authors.forename: Enrico
authors.initials: E
authors.lastname: Schleiff
authors.forename: Michael
authors.initials: M
authors.lastname: Karas
authors.forename: Bjoern
authors.initials: B
authors.lastname: Meyer
mesh_terms: D000180:Acrylic Resins / Q000737:chemistry*; D045744:Cell Line, Tumor; D002736:Chloroplasts / Q000737:chemistry; D003432:Cross-Linking Reagents; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D019855:Ethylene Glycol / Q000737:chemistry*; D006801:Humans; D008956:Models, Chemical; D011506:Proteins / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D000180:Acrylic Resins; D003432:Cross-Linking Reagents; D011506:Proteins; C016680:polyacrylamide gels; D019855:Ethylene Glycol
keywords: Dissolvable gel matrix; Ethylene glycol diacrylate cross-linked polyacrylamide gel; Intact protein extraction; MALDI mass spectrometry; Top down