Coupling 2D SDS−PAGE with CNBr Cleavage and MALDI-TOFMS:  A Strategy Applied to the Identification of Proteins Induced by a Hypochlorous Acid Stress in Escherichia coli

• Published in: Analytical chemistry (Washington) Vol. 70; no. 20; pp. 4433 - 4440
• Main Authors: Dukan, S, Turlin, E, Biville, F, Bolbach, G, Touati, D, Tabet, J. C, Blais, J. C
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 15.10.1998
• Subjects: Bacteria; Bacterial Proteins - analysis; Bacteriology; Biological and medical sciences; Chemistry; Cyanogen Bromide; Electrophoresis, Polyacrylamide Gel - methods; Escherichia coli - chemistry; Escherichia coli - drug effects; Fundamental and applied biological sciences. Psychology; Hypochlorous Acid - pharmacology; Indicators and Reagents; Microbiology; Molecular Weight; Morphology, structure, chemical composition; Proteins; Rosaniline Dyes; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Hypochlorous acid; Gel electrophoresis; Escherichia coli; Laser desorption; Protein; Matrix isolation; Separation method; Investigation method; Photoionization; Time of flight method; Metabolic activation; Bacteria; Qualitative analysis; Acid medium; Mass spectrometry; Enterobacteriaceae
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac980132z

A protocol including 2D SDS−PAGE, electroblotting proteins onto nitrocellulose membranes, and CNBr cleavage, followed by MALDI-MS analysis of intact proteins and peptide fragments and a database search, has been optimized and applied to the rapid identification of the Escherichia coli response to hypochlorous acid. The methodology has proved to be efficient from the point of view of sensitivity (picomole range) and selectivity. In particular, MALDI analysis of proteins and CNBr fragments by directly dissolving the membrane in an acetone solution of matrix, without previous elution, is reliable and reproducible. The accuracy of the MW determination is somewhat reduced compared to that of methods involving elution and purification of proteins and digests; nevertheless, the utilization of large MW windows combined with the pI entry in database searches had allowed, for most of the spots, the selection of only one protein candidate. Finally, 19 proteins exhibiting a response to hypochlorous acid stress have been confirmed or identified on the basis of this protocol.