Characterization of proteins utilized in the desulfurization of petroleum products by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF/MS) with delayed extraction is utilized in linear, reflected-ion and post-source decay (PSD) modes to directly characterize enzymes being developed for use in a petroleum desulfurization process. The DNA sequence for the genes isolated from Rhodococcus sp. strain IGTS8 that produce three of the four enzymes under study had been previously reported with a discrepancy in residue assignments for one of the enzymes, dsz-C. The use of proteolytic digests followed by MALDI/TOF/MS with delayed extraction in the reflected-ion mode provided sequence-specific information with mass accuracies exceeding 40 ppm over a range of masses and signal-to-noise values. Peptide mapping of >80% of the residues was accomplished for all four proteins. The use of PSD established the true sequence for dsz-C, resolving the discrepancy in the literature. A posttranslational loss of N-terminal methionine was observed for each of the four proteins in linear MALDI/MS and was reconfirmed by peptide mapping for three of the proteins.
Wolf
B P
BP
Department of Chemistry, Texas A&M University, College Station, Texas 77842, USA. bwolf@chemvx.tamu.edu
Sumner
L W
LW
Shields
S J
SJ
Nielsen
K
K
Gray
K A
KA
Russell
D H
DH
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D010450:Endopeptidases; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D050862:Oxidoreductases Acting on Sulfur Group Donors; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D010578:Petroleum / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D001426:Bacterial Proteins
D010450:Endopeptidases; D010088:Oxidoreductases; D050862:Oxidoreductases Acting on Sulfur Group Donors; D010446:Peptide Fragments; D010578:Petroleum; C113832:2-(2-hydroxybiphenyl)-benzenesulfinate desulfinase; D001426:Bacterial Proteins