In this study, the soluble protein fraction of P. chrysosporium grown under standard conditions was analyzed by using 2D-PAGE approach and a 2-D reference map was constructed. 910 spots could be separated and detected on Coomassie-stained 2-D gels by the help of Delta2D image analysis software. 720 spots could be cut from the master gel and were subjected to MALDI-TOF MS analysis followed by MASCOT search. A total of 517 spots out of 720 were assigned to specific accession numbers from the P. chrysosporium genome database. Further analysis of the data revealed 314 different gene products (distinct ORFs).
The theoretical pI and MW values were plotted against the experimental migration distances. Results indicated the existence of 124 protein spots whose horizontal migration differed significantly from the expected migration according to the calculated pI values and 52 spots with an apparent molecular weight that is significantly different from their theoretical molecular weight. While protein modification could be predicted by these analyses, the main support was the presence of multiple spots of the same gene product. As much as 118 ORFs yielded multiple spots on the master gel, corresponding to 37.5% of the all distinct ORFs identified in this work.
The relative abundance of each of the 517 identified polypeptides was calculated in terms of spot intensity. The majority of the most abundant proteins were found to be housekeeping ones. When the relative distribution of the proteins into four main functional categories was taken into consideration, &ldquo / Metabolism&rdquo / appeared the most important category with a share of 50.6% among identified proteins. However, among the functional classes, &ldquo / Posttranslational modifications, protein turnover, chaperones&rdquo / which is listed under the main category &ldquo / Cellular Processing and Signalling&rdquo / was represented by the highest number (104) of the identified proteins. Only 6 of the proteins listed in this study were assigned to hypothetical proteins.
Out of the 314 identified gene products shown in P. chrysosporium, 29 were predicted to have a signal peptide sequence according to the SignalP algorithm. By making a WoLF PSORT search, subcellular localization of the proteins was predicted. Accordingly, 147 of the proteins were predicted to be located in cytoplasm. The phosphorylated proteins of P. chrysosporium were detected by ProQ phosphoprotein staining of the 2-D gel. 380 out of 910 distinct protein spots (40%) were found to be phosphorylated in exponentially growing cells of P. chrysosporium. Of these spots, 96 could be matched to the identified proteins.
| Identifer | oai:union.ndltd.org:METU/oai:etd.lib.metu.edu.tr:http://etd.lib.metu.edu.tr/upload/12607062/index.pdf |
| Date | 01 January 2006 |
| Creators | Yildirim, Volkan |
| Contributors | Ozcengiz, Gulay |
| Publisher | METU |
| Source Sets | Middle East Technical Univ. |
| Language | English |
| Detected Language | English |
| Type | M.S. Thesis |
| Format | text/pdf |
| Rights | To liberate the content for public access |
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