The presentation deals with the problem of traditional proteomics like strong sequence polymorphism or poorly conserved protein families.
The worklow of this technique is displayed in detail and involves MS/MS analysis as well as data base research.
Workflow similarity-driven proteomics
- Searches with highest discriminating power executed first
- Low resolution MS/MS spectra can be employed
- Filtering pose an essential step
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Evaluate borderline hits
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Borderline hits have only a few matching peptides in the MASCOT search
Cross-species identification:
- de novo interpretation of borderline peptide
- MS BLAST search
- Get related database entries
Filtering against background library
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MS BLAST of de novo spectra from 55 kDa band of D. salina
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MS BLAST of de novo spectra after background filtering
- Background spectra recorded with blank LC-MS/MS runs
- Background proteins mainly keratins, trypsins, and serine proteases
- De novo interpretation of spectra leads to redundant sequences
- MASCOT very stringent and therefore not affected
- MS BLAST leads to multiple hits -> unfavourable
Protein Identification of D. Salina
Identification of 10 membrane proteins of D. Salina
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Overall identification of 55 unique proteins by MASCOT (match to already known sequences), cross-species identifications, and mere MS BLAST
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Proteomics-genomics approach: workflow
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Workflow for investigation of proteins induced by the sulfate-reducing bacterium D. phosphitoxidans in the presence of phosphite
- No gene splicing
- Usage of a combination of proteomics and genetics methods
- N-terminal Edman sequencing followed by IPCR of degenerated primers
- FTICR-MS for protein identification from ORF candidates
2D-Gel electrophoresis of soluble fraction
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Edman sequencing
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Identification of ORF
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Bold: identified sequences, underlined: cleavage sites
− DNA regions upstream and downstream evaluated
-> unequivocally determination of the ORF
− MALDI-FTICR approves a single protein with length of 317 aa
− BlastP reveals high similarity to UDP-glucose 4-epimerase of Methanosarcina acetivorans − Putative epimerase/dehydratase identified!
Summary
- High amount of unknown peptides can be identified simultaneosly
- In some cases no high resolution MS needed
- Single proteins with related ORF can be determined
- No previous knowledge about the genome of the organism of interest necessary
References
1. Waridel, P. et al., Proteomics 2007, 7, 2318-2329
2. Simeonova, D. D. et al., Mol. Cell Proteomics, 2009, 8(1):122-31
3. Manea, M., Mastercourse Proteomanalytik, UniversitÄt Konstanz, 2015
[...]
- Arbeit zitieren
- Manuel Langer (Autor:in), 2015, Unknown Genome Proteomics, München, GRIN Verlag, https://www.grin.com/document/337836
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