17th International Mass Spectrometry Conference :: Prague, 2006
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|Presentation date:||Mon, Aug 28, 2006|
|Presentation time:||14:30 – 16:00|
Sue Francis-McIntyre1, Deana Sugden1, Isabel Riba-Garcia1, Christian Cole1, Sally Barnes2, David B. Archer2, Simon J. Hubbard1, Simon J. Gaskell11 University of Manchester, Manchester, United Kingdom
Correspondence address: Sue McIntyre, University of Manchester, School of Chemistry, Sackville Street, Manchester, M601QD United Kingdom.
Keywords: Electrophoresis; Mass Spectrometry, Liquid Chromatography; Protein Identification; Proteomic.
Novel aspect: Proteomics analysis of filamentous fungi.
Filamentous fungi such as Aspergillus niger are often used in the biotechnology industry for recombinant protein production since they can be manipulated to secrete proteins in large quantities.
The study of cells grown under stress conditions can help us understand the molecular mechanisms underlying protein secretion and the response to stress as the cell tries to maintain essential processes such as protein synthesis. Dithiothreitol (DTT), a reducing agent, can be used to induce stress responses in A.niger.1 Analysis at the transcriptome level shows that the presence of DTT results in the up-regulation of a number of genes including bipA and pdiA, and the induction of the unfolded protein response (UPR).1,2
In order to characterise the changes in protein expression associated with the induction of the stress response we have compared the endo-proteome for DTT-treated and untreated A. niger cultures. Ground mycelia were crushed using glass bead agitation, with proteins being extracted in a detergent based lysis solution before being separated by SDS-PAGE. Extensive areas of interest were excised, digested and analysed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS).
Whilst a number of Aspergillus species such as A. nidulans and A. fumigatus genomes have been publicly sequenced and validated, only limited genome data are available for A. niger. Data were initially searched using Mascot using an in-house A. niger sequence database supplied by Genecor. In an attempt to improve A. niger protein identification data were also searched against a cross species database allowing the comparison of several fungal genomes, to identify homologues from other aspergillus species.
1. H. Al-Sheikh, A. J. Watson, G. A. Lacey, P. J. Punt, D. A. MacKenzie, D. J. Jeenes, T. Pakula, M. Penttila, M. J. Alcocer and D. B. Archer, Mol. Microbiol. 53, 1731 (2004).
2. D. A. MacKenzie, T. Guillemette, H. Al-Sheikh, A. J. Watson, D. J. Jeenes, P. Wongwathanarat, N. S. Dunn-Coleman, N. van Peij and D. B. Archer, Mol. Genet. Genomics 274, 410 (2005).