Analysis of major intracellular proteins of Aspergillus fumigatus by MALDI mass spectrometry: identification and characterisation of an elongation factor 1B protein with glutathione transferase activity.
Aspergillus fumigatus is a recognised human pathogen, especially in immunocompromised individuals. The availability of the annotated A. fumigatus genome sequence will significantly accelerate our understanding of this organism. However, limited information is available with respect to the A. fumigatus proteome. Here, both a direct proteomic approach (2D-PAGE and MALDI-MS) and a sub-proteomic strategy involving initial glutathione affinity chromatography have been deployed to identify 54 proteins from A. fumigatus primarily involved in energy metabolism and protein biosynthesis. Furthermore, two novel eukaryotic elongation factor proteins (eEF1Bgamma), termed ElfA and B have been identified and phylogenetically confirmed to belong to the eEF1Bgamma class of GST-like proteins. One of these proteins (ElfA) has been purified to homogeneity, identified as a monomeric enzyme (molecular mass=20 kDa; pI=5.9 and 6.5), and found to exhibit glutathione transferase activity specific activities (mean+/-standard deviation, n=3) of 3.13+/-0.27 and 3.43+/-1.0 micromol/min/mg, using CDNB and ethacrynic acid, respectively. Overall, these data highlight the importance of new approaches to dissect the proteome of, and elucidate novel functions within, A. fumigatus.
Carberry
Stephen
S
National Institute for Cellular Biotechnology, Department of Biology, National University of Ireland-Maynooth, Co. Kildare, Ireland.
Neville
Claire M
CM
Kavanagh
Kevin A
KA
Doyle
Sean
S
D000595:Amino Acid Sequence; D001232:Aspergillus fumigatus / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional; D005656:Fungal Proteins / Q000032:analysis*; D016681:Genome, Fungal; D005982:Glutathione Transferase / Q000032:analysis*; D008969:Molecular Sequence Data; D020648:Peptide Elongation Factor 1 / Q000032:analysis* / Q000302:isolation & purification; D010445:Peptide Elongation Factors / Q000032:analysis* / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D005656:Fungal Proteins; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors; D005982:Glutathione Transferase