High-efficiency protein extraction from polyacrylamide gels for molecular mass measurement by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry.
A simple and fast method of protein extraction from Coomassie Brilliant Blue (CBB)-stained polyacrylamide gels suited for molecular mass measurement of proteins by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) is reported. Proteins in CBB-stained gel pieces were extracted by a 10-min soaking in 0.1 M NaOH at 25 degrees C. The recovery of this one-step extraction method was 34-73% for proteins <67 kDa. CBB adduction to proteins during mass spectrometric analysis was avoided by a destaining step before the alkaline extraction. The molecular mass values of the extracted proteins coincided with those of purified proteins within +/-0.01-0.10% deviation for all the proteins <36 kDa. Because of the high extraction recovery, mass measurement was possible for the proteins extracted from CBB-stained gels with loaded protein quantities as little as 34 ng for cytochrome c, alpha-lactalbumin, myoglobin, beta-lactoglobulin, trypsinogen, and carbonic anhydrase (12.4-29.0 kDa), 340 ng for glyceraldehyde-3-phosphate dehydrogenase (35.6 kDa) and albumin (66.3 kDa). This method provides a highly efficient approach to utilize CBB-stained one- or two-dimensional gels for whole protein analysis using MALDI-TOF-MS.
Jin
Ya
Y
Department of Chemistry, Faculty of Science, Ehime University, Matsuyama-City, 790-8577, Japan.
Manabe
Takashi
T
D002256:Carbonic Anhydrases / Q000302:isolation & purification; D045304:Cytochromes c / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007768:Lactalbumin / Q000302:isolation & purification; D007782:Lactoglobulins / Q000302:isolation & purification; D008970:Molecular Weight; D009211:Myoglobin / Q000302:isolation & purification; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification*; D012394:Rosaniline Dyes; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013696:Temperature; D014362:Trypsinogen / Q000302:isolation & purification
D007782:Lactoglobulins; D009211:Myoglobin; D011506:Proteins; D012394:Rosaniline Dyes; D014362:Trypsinogen; D045304:Cytochromes c; D007768:Lactalbumin; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D002256:Carbonic Anhydrases; C004692:coomassie Brilliant Blue