IR-MALDI-mass analysis of electroblotted proteins directly from the membrane: comparison of different membranes, application to on-membrane digestion, and protein identification by database searching.
A systematic membrane study investigating different neutral, cationic derivatized, and hydrophilic PVDF membranes for their suitability to carry out on-membrane tryptic digestions and to obtain infrared-matrix-assisted laser desorption/ionization (IR-MALDI) mass information on the proteolytic fragments directly from the membrane was performed. Clearly, the Immobilon CD membrane (Millipore) showed the most reproducible results over a protein mass range from 12 to 66 kDa. Typical protein load to SDS-PAGE was in the 1-2 micrograms range. The protein amount used for enzymatic treatment was estimated to be in the low picomole range. Now both the intact protein mass and the masses of the specific proteolytic fragments are available directly from the membrane. Protein databases can be searched via search algorithms on the Internet using the information on the intact protein mass and the masses, e.g., of its tryptic fragments. Investigations were performed to search for neutral, enzyme-compatible IR matrixes which allow the enzymatic treatment (on-membrane digestion) while the membrane is matrix-incubated. Thiourea could be tolerated during enzymatic cleavage in solution in concentrations of 15 g/L and resulted in high-quality spectra of intact protein signals and turned, therefore, out to be the most promising candidate.
Schleuder
D
D
Institute for Medical Physics and Biophysics, University of Münster, Germany.
Hillenkamp
F
F
Strupat
K
K
D000465:Algorithms; D000595:Amino Acid Sequence; D016208:Databases, Factual; D004591:Electrophoresis, Polyacrylamide Gel; D004798:Enzymes / Q000737:chemistry; D016247:Information Storage and Retrieval; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000032:analysis / Q000737:chemistry; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D017421:Sequence Analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D019802:Succinic Acid / Q000737:chemistry; D013499:Surface Properties; D014357:Trypsin / Q000737:chemistry; D014508:Urea / Q000737:chemistry
D004798:Enzymes; D008567:Membranes, Artificial; D009211:Myoglobin; D011506:Proteins; D014508:Urea; D019802:Succinic Acid; D014357:Trypsin