MALDI-TOF mass spectrometry characterization of recombinant hydrophobic mutants containing the GCN4 basic region/leucine zipper motif.
We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to characterize hydrophobic, alanine-rich mutants of the basic region/leucine zipper (bZIP) protein GCN4. These bacterially expressed proteins were generated to probe how small, alpha-helical proteins bind specific DNA sites. Enzymatic digestion mapping combined with MALDI-TOF MS characterization of protein fragments allowed us to resolve mass discrepancies between the expected and observed molecular mass measurements. Changes in mass were attributed to posttranslational modifications (PTMs) by proteolytic cleavage of the initiating methionine residue, carbamylation at the amino terminus, oxidation of histidine side chains, and oxidative addition of beta-mercaptoethanol (BME) at the cysteine side chain. Proteins can undergo a wide variety of co-translational modifications and PTMs during growth, isolation, and purification. Such changes in mass can only be detected by a high-resolution technique such as MALDI, which in conjunction with enzymatic digestion mapping, becomes a powerful methodology for characterization of protein structure.
Bird
Gregory H
GH
Department of Chemistry, Faculty of Arts and Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Shin
Jumi A
JA
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D051790:G-Box Binding Factors; D016350:Leucine Zippers / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics
D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D051790:G-Box Binding Factors; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D011494:Protein Kinases