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 15NH3

The molecular mass of the galactose-H+ symport protein GalP. as its histidine-taaged 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 underestimation of the true molecular mass due to anomalous electrophoretic migration. Addition of a size-exclusion chromatography step after Ni2+-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 GaIP(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 protem/peptide mass and sequence determinations were in accord with the predictions of amino acid sequence deduced from the DNA sequence of the galP gene. [ring-2-C-13]Histidine was incorporated into GaIP(His)(6) in vivo, and ESI-MS analysis enabled the measurement of a high (8010) and specific incorporation of label into the histidine residues in the protein. MS could also be used to confirm the labelling of the protein by (NH3)-N-15 (93% enrichment) and [F-19] tryptophan (830,0 enrichment). Such MS measurements will serve in the future analysis of the structures of membrane-transport proteins by NMR, and of their topology by indirect techniques.