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Unknown Genome Proteomics

Presentation (Handout) 2015 6 Pages

Chemistry - Organic Chemistry

Excerpt

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

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Details

Pages
6
Year
2015
ISBN (eBook)
9783668293885
File size
965 KB
Language
English
Catalog Number
v337836
Institution / College
University of Constance – Chemie
Grade
Tags
proteomis MS/MS denovo

Author

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Title: Unknown Genome Proteomics