Efficacy and compatibility with mass spectrometry of methods for elution of proteins from sodium dodecyl sulfate-polyacrylamide gels and polyvinyldifluoride membranes.
The resolving power of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) combined with isoelectric focusing in two-dimensional gel electrophoresis has made it one of the most important techniques for resolving complex mixtures, and it is of great importance for proteome mapping projects. As a result of this, methods for postelectrophoretic protein characterization are of great interest as exemplified by in situ protease digestion combined with mass spectrometry (MS), which is the method of choice for identification of proteins. In this study we have developed and compared methods for recovering intact proteins from polyacrylamide gels and electroblotting membranes to define efficient methods compatible with MS. These methods complement in situ digestion protocols and allow determination of the molecular mass of whole proteins separated by SDS-PAGE. Passive elution of proteins from SDS-PAGE gels was efficient only in the presence of SDS, whereas electroelution was achieved using buffers without SDS. Surface-enhanced laser desorption/ionization MS (SELDI-MS) analysis of proteins eluted in the presence of SDS was possible using ion exchange ProteinChip arrays for concentration of sample and removal of SDS. Comparison of different electroblotting methods verified that the different membranes and buffers were equally efficient for transfer of proteins in the range 20-100 kDa. Elution from polyvinyldifluoride membranes was most efficient using either concentrated solutions of trifluoroacetic acid (TFA) or combinations of 8M urea and 1% Triton X-100, 1% Tween 20, or 40% isopropanol. The same result was obtained using nitrocellulose membranes, except that these were incompatible with organic solvent and TFA. Elution by TFA was compatible with matrix-assisted laser desorption/ionization MS (MALDI-MS) but was complicated by a high degree of trifluoroacetylation of the proteins. Alternatively, elution by 8M urea+1% Triton X-100, 1% Tween 20, or 40% isopropanol was compatible with both SELDI-MS and MALDI-MS. Eluted proteins were identified in MS experiments by intact mass determination, by peptide mapping, and by MS/MS analysis.
Jørgensen
Charlotte Svaerke
CS
Department of Research and Development, Statens Serum Institut, DK 2300 Copenhagen, Denmark.
Jagd
Mitchell
M
Sørensen
Birgitte Kjaer
BK
McGuire
Jim
J
Barkholt
Vibeke
V
Højrup
Peter
P
Houen
Gunnar
G
D000180:Acrylic Resins / Q000737:chemistry*; D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D008567:Membranes, Artificial; D010902:Pituitary Gland / Q000737:chemistry; D010920:Placenta / Q000737:chemistry; D011145:Polyvinyls / Q000737:chemistry*; D011247:Pregnancy; D011506:Proteins / Q000737:chemistry*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D005465:Fluorocarbon Polymers
D000180:Acrylic Resins; D008567:Membranes, Artificial; D011145:Polyvinyls; D011506:Proteins; D012967:Sodium Dodecyl Sulfate; C016680:polyacrylamide gels; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers