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Proteomic analysis of acrylamide gel separated proteins immobilized on polyvinylidene difluoride membranes following proteolytic digestion in the presence of 80% acetonitrile
Author(s) -
Bunai Keigo,
Nozaki Manabu,
Hamano Miyuki,
Ogane Shinya,
Inoue Taro,
Nemoto Tadashi,
Nakanishi Hiroshi,
Yamane Kunio
Publication year - 2003
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200300529
Subject(s) - chromatography , chemistry , gel electrophoresis , coomassie brilliant blue , trypsin , membrane , sodium dodecyl sulfate , mass spectrometry , matrix assisted laser desorption/ionization , electrophoresis , polyacrylamide gel electrophoresis , tricine , halobacterium salinarum , biochemistry , staining , desorption , bacteriorhodopsin , biology , enzyme , genetics , organic chemistry , adsorption
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∼500 fmol of standard proteins. We also analyzed the submembrane protein fraction solubilized by 1% n ‐dodecyl‐β‐ 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∼50 fmol level by pooling more than ten identical electroblotted protein spots.