Proteomic analysis of acrylamide gel separated proteins immobilized on polyvinylidene difluoride membranes following proteolytic digestion in the presence of 80% acetonitrile.
Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) combined with mass spectrometry (MS) is a highly accurate and sensitive means of identifying proteins. We have developed a novel method for digesting proteins on polyvinylidene difluoride (PVDF) membranes for subsequent matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS analysis. After Tricine sodium dodecyl sulfate (SDS)-PAGE, separated proteins were electroblotted onto PVDF membranes in a semidry discontinuous buffer system, visualized by staining with Coomassie Blue, excised, digested with trypsin or lysC in 80% acetonitrile, and then analyzed by MALDI-TOF MS. This method has several advantages over in-gel digestion in terms of sample handling, sensitivity, and time. We identified 105 fmol of Bacillus subtilis SecA and 100 approximately 500 fmol of standard proteins. We also analyzed the submembrane protein fraction solubilized by 1% n-dodecyl-beta-D-maltoside from B. subtilis membranes after separation by 2-D PAGE, and identified 116 protein spots. This method can detect proteins at the 10 approximately 50 fmol level by pooling more than ten identical electroblotted protein spots.
Bunai
Keigo
K
Institute of Biological Sciences, University of Tsukuba, Ibaraki, Japan.
Nozaki
Manabu
M
Hamano
Miyuki
M
Ogane
Shinya
S
Inoue
Taro
T
Nemoto
Tadashi
T
Nakanishi
Hiroshi
H
Yamane
Kunio
K
D000097:Acetonitriles; D000251:Adenosine Triphosphatases / Q000032:analysis; D000818:Animals; D001412:Bacillus subtilis / Q000201:enzymology; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis / Q000737:chemistry; D001426:Bacterial Proteins / Q000032:analysis; D030562:Databases, Protein; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006868:Hydrolysis; D026901:Membrane Transport Proteins / Q000032:analysis; D008567:Membranes, Artificial; D011145:Polyvinyls; D011506:Proteins / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D005465:Fluorocarbon Polymers
D000097:Acetonitriles; D000251:Adenosine Triphosphatases; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D008567:Membranes, Artificial; D011145:Polyvinyls; D011506:Proteins; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers; C032159:acetonitrile