MATRIX-ASSISTED LASER-DESORPTION IONIZATION MASS-SPECTROMETRY OF PROTEINS ELECTROBLOTTED AFTER POLYACRYLAMIDE-GEL ELECTROPHORESIS

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) of proteins, electroblotted onto polymer membranes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation is demonstrated. The proteins are desorbed directly from the blot membranes after matrix application. Desorption with 2.94-mu m infrared radiation and succinic acid as matrix was found superior to 355-nm UV desorption using 2,5-dihydroxybenzoic acid as matrix. Several commercially available membranes tested resulted in protein signals after matrix incubation of the membrane. Systematic investigations for five different poly(vinylidene fluoride) (PVDF) membranes showed improved results for membranes exhibiting high specific surfaces. The matrix should be applied immediately after the blotting procedure while the blot is still wet. Lateral resolution of the protein band is preserved after the MALDI preparation procedure. Staining with organic dyes results in broad protein signals shifted in mass due to addition of several dye molecules; staining with colloidal stains such as colloidal gold or India ink somewhat deteriorates the quality of the spectre, but renders the correct protein mass.