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

Abstract

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 (eEF1Bc), termed ElfA and B have been identified and phylogenetically confirmed to belong to the eEF1Bc 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 lmol/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.

The Glutathione System of Aspergillus nidulans Involves a Fungus-specific Glutathione S-Transferase

“…4A). A peroxiredoxin homologous to yeast Ahp1p (AN8692.3, 40% identical) as well as elongation factor 1B (AN9304.3), of which the Aspergillus fumigatus ortholog (elfA) reportedly has peroxiredoxin activity (40), were more than 2-fold up-regulated in the DGR1 strain. We previously constructed A. nidulans proteome maps comprising 300 intracellular proteins (35 Table 2).…”

Single-pot derivatisation strategy for enhanced gliotoxin detection by HPLC and MALDI-ToF mass spectrometry

“…NaBH 4 (500 mM) was prepared in HPLC-grade water and used within 30 min of preparation. A. fumigatus strains Af293 (wild-type) and ΔgliZ (a kind gift from Professor Nancy Keller, University of Wisconsin-Madison), a binuclear cluster domain transcription factor-deficient mutant incapable of gliotoxin biosynthesis [23], were cultured as described elsewhere [17,24]. Sporidesmin A was purchased from AgResearch (Hamilton, New Zealand).…”

“…Specimens (0.5 μL), neat or diluted up to 1/200 in 0.1% (v/v) trifluoroacetic acid, for mass determination were layered upon 0.5 μL α-cyano-4-hydroxycinnamic acid (5 mg 200 μL −1 50%(v/v) acetonitrile in 0.1% (v/v) aqueous trifluoroacetic acid), previously deposited onto mass spectrometry slides and allowed to dry prior to delayed extraction, reflectron ToF analysis at 20 kV [24]. Internal calibrants, angiotensin III and adrenocorticotropic hormone fragments 18-39, were used to calibrate spectra.…”

Purification and characterisation of a xylanase from Thermomyces lanuginosus and its functional expression by Pichia pastoris

“…Protein samples for MS analysis were excised from SDS-PAGE gels, digested with trypsin and deposited onto mass spectrometry slides following equal volume mixture with ␣-cyano-4-hydroxycinnaminic acid (2.5% (w/v) in 50% (v/v) acetonitrile with 0.1% (v/v) aqueous trifluoroacetic acid) [25]. Protein identification was carried out by m/z data interrogation of the NCBI nr database using the Mascot search engine.…”