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Intact cell matrix‐assisted laser desorption/ionization mass spectrometry as a tool to screen drugs in vivo for regulation of protein expression
Author(s) -
Kulkarni Mahesh J.,
Vinod V. P.,
Umasankar P. K.,
Patole Milind S.,
Rao Mala
Publication year - 2006
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.2675
Subject(s) - chemistry , affinity chromatography , sepharose , recombinant dna , gel electrophoresis , in vivo , biochemistry , microbiology and biotechnology , arabinose , matrix assisted laser desorption/ionization , polyacrylamide gel electrophoresis , mass spectrometry , chromatography , gene , enzyme , desorption , biology , organic chemistry , adsorption , xylose , fermentation
Here we demonstrate for the first time the application of intact cell matrix‐assisted laser desorption/ionization mass spectrometry (ICM‐MS) to study the regulation of protein expression. This technique can be extended to screen the drugs that inhibit protein synthesis in various diseases. We have used Escherichia coli cells expressing a recombinant glutathione‐S‐transferase (GST) gene under an arabinose‐inducible promoter as a model system. Using ICM‐MS analysis, we have detected a 28 kDa peak corresponding to the production of recombinant GST under the arabinose‐induced condition. Furthermore, recombinant GST protein was purified by a single‐step affinity purification using a glutathione Sepharose 4B affinity column from arabinose‐induced E. coli cells. The purified GST protein was found to be a 28 kDa protein by MALDI analysis suggesting the arabinose‐induced protein is indeed GST. The regulation of protein expression was studied using glucose as an alternative metabolite. The glucose‐mediated regulation of the ara ‐operon was followed using the ICM‐MS technique. All the results obtained from ICM‐MS data were validated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) analysis. The present technique can be extended for in vivo screening of drugs and it holds tremendous potential to discover novel drugs against specific protein expressions in different diseases. Copyright © 2006 John Wiley & Sons, Ltd.