Microwave-assisted protein staining: mass spectrometry compatible methods for rapid protein visualisation.
The effects of microwave irradiation on the staining of electrophoresed and electroblotted proteins have been assessed using currently available detection methods. Although the absorption of microwave radiation was found to be uneven, band intensity following microwave-assisted protein staining (MAPS) was comparable and in some cases exceeded the intensity of the bands visualised by the original staining methods. It was found that microwave treatment drastically reduced the duration of the staining protocols for visualisation of the proteins separated by both one- and two-dimensional electrophoresis. Application of MAPS methods did not affect peptide mass fingerprinting analysis by mass spectrometry and subsequent identification of the protein by database searching. The peptide mass maps corresponding to the proteins visualised using both the conventional and MAPS methods did not show significant difference in signal/noise ratio. Moreover, it appeared that microwave treatment of the gels resulted in the increased recovery of the peptides following in-gel trypsin digestion. Briefly, microwave-assisted protein staining methods were rapid, compatible with mass spectrometry and were equally effective on thin (0.75-mm) and thick (1.5-mm) gels (such as those used in 2D electrophoresis).
Nesatyy
Victor J
VJ
National Research Council of Canada, Institute for Marine Biosciences, Halifax, Nova Scotia, B3H 3Z1, Canada. victor.nesati@nrc.ca
Dacanay
Andrew
A
Kelly
John F
JF
Ross
Neil W
NW
D016172:DNA Fingerprinting; D016208:Databases, Factual; D004586:Electrophoresis; D004591:Electrophoresis, Polyacrylamide Gel; D017979:Gold Colloid / Q000032:analysis; D006868:Hydrolysis; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D008567:Membranes, Artificial; D008872:Microwaves; D008970:Molecular Weight; D010455:Peptides / Q000032:analysis; D011145:Polyvinyls; D011506:Proteins / Q000737:chemistry*; D012394:Rosaniline Dyes; D016622:Silver Staining; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin / Q000737:chemistry; D005465:Fluorocarbon Polymers
D017979:Gold Colloid; D007202:Indicators and Reagents; D008567:Membranes, Artificial; D010455:Peptides; D011145:Polyvinyls; D011506:Proteins; D012394:Rosaniline Dyes; D014357:Trypsin; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers; C048139:Coomassie blue