Molecular dissection of membrane-transport proteins: mass spectrometry and sequence determination of the galactose-H+ symport protein, GalP, of Escherichia coli and quantitative assay of the incorporation of [ring-2-13C]histidine and (15)NH(3)

The Biochemical Journal
Henrietta VenterRichard B Herbert

Abstract

The molecular mass of the galactose-H(+) symport protein GalP, as its histidine-tagged derivative GalP(His)(6), has been determined by electrospray MS (ESI-MS) with an error of <0.02%. One methionine residue, predicted to be present from the DNA sequence, was deduced to be absent. This is a significant advance on the estimation of the molecular masses of membrane-transport proteins by SDS/PAGE, where there is a consistent under-estimation of the true molecular mass due to anomalous electrophoretic migration. Addition of a size-exclusion chromatography step after Ni(2+)-nitrilotriacetate affinity purification was essential to obtain GalP(His)(6) suitable for ESI-MS. Controlled trypsin, trypsin+chymotrypsin and CNBr digestion of the protein yielded peptide fragments suitable for ESI-MS and tandem MS analysis, and accurate mass determination of the derived fragments resulted in identification of 82% of the GalP(His)(6) protein. Tandem MS analysis of selected peptides then afforded 49% of the actual amino acid sequence of the protein; the absence of the N-terminal methionine was confirmed. Matrix-assisted laser-desorption ionization MS allowed identification of one peptide that was not detected by ESI-MS. All the protein/peptide ma...Continue Reading

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Citations

Jul 20, 2007·Analytical and Bioanalytical Chemistry·Bernhard GranvoglLutz Andreas Eichacker
May 7, 2008·The Journal of Membrane Biology·Kate BraceyDennis Wray
Sep 11, 2007·Applied Microbiology and Biotechnology·Irene NeophytouDavid Barlow
Nov 19, 2008·The Journal of Biological Chemistry·Vladislav B BergoJohn L Spudich
Jul 6, 2014·Protein Expression and Purification·Saeed SeyedmohammadHenrietta Venter
Nov 22, 2005·The Journal of Biological Chemistry·Yushi MatsumotoTatsushi Mogi
Sep 29, 2004·The Journal of Biological Chemistry·Lutz A EichackerEnrico Schleiff

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