title: Intact protein analysis for site-directed mutagenesis overexpression products: plasmid-encoded R67 dihydrofolate reductase.
abstract: Mass spectrometry is currently the method of choice for the analysis of recombinant protein expression products. By combining proteolytic digestion with peptide mapping and tandem mass spectrometry techniques, verification of site-directed mutagenesis products can be obtained. The proteolytic digestion step converts a purified recombinant protein into a mixture that must be reseparated, thus greatly increasing the analysis time associated with the confirmation of site-directed mutagenesis products. Ion/ion reaction chemistry combined with quadrupole ion trap mass spectrometry provides a fast and efficient way to analyze intact proteins for the correct site-directed mutagenesis products, without heavy reliance on the proteolytic digestion step. Analysis of a series of protein variants (I68M, I68Q, Y69F, and Q67Y) from plasmid-encoded R67 dihydrofolate reductase using ion/ion reaction chemistry confirmed the presence of the correct site-directed mutagenesis products. For the I68M mutant, ion/ion separations detected the presence of extensive degradation from the N-terminal end of the protein. In the case of the Q67Y mutant, a mixture of Q67Y and Q67C species was detected by employing tandem mass spectrometry combined with ion/ion reactions. The ion/ion reaction technique was also performed on a partially purified lysate of the Q67Y/C mixture and successfully screened for the presence of both components in a complex mixture. The ion/ion reaction approach achieved the same results as the proteolytic-digestion-based methodology in a much shorter analysis time.
authors.affiliation: Organic and Biological Mass Spectrometry Group, Oak Ridge National Laboratory, Building 5510, Oak Ridge, TN 37831-6365, USA.
authors.forename: Nathan C
authors.initials: NC
authors.lastname: VerBerkmoes
authors.forename: Michael B
authors.initials: MB
authors.lastname: Strader
authors.forename: R Derike
authors.initials: RD
authors.lastname: Smiley
authors.forename: Elizabeth E
authors.initials: EE
authors.lastname: Howell
authors.forename: Gregory B
authors.initials: GB
authors.lastname: Hurst
authors.forename: Robert L
authors.initials: RL
authors.lastname: Hettich
authors.forename: James L
authors.initials: JL
authors.lastname: Stephenson
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics; D001665:Binding Sites; D002845:Chromatography / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry; D010449:Peptide Mapping; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013762:Tetrahydrofolate Dehydrogenase / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
chemical_list: D000596:Amino Acids; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase