Unknown Genome Proteomics


Presentation (Handout), 2015
6 Pages

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

Abbildung in dieser Leseprobe nicht enthalten

Evaluate borderline hits

Abbildung in dieser Leseprobe nicht enthalten

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

Abbildung in dieser Leseprobe nicht enthalten

MS BLAST of de novo spectra from 55 kDa band of D. salina

Abbildung in dieser Leseprobe nicht enthalten

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

Abbildung in dieser Leseprobe nicht enthalten

Overall identification of 55 unique proteins by MASCOT (match to already known sequences), cross-species identifications, and mere MS BLAST

Abbildung in dieser Leseprobe nicht enthalten

Proteomics-genomics approach: workflow

Abbildung in dieser Leseprobe nicht enthalten

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

Abbildung in dieser Leseprobe nicht enthalten

Edman sequencing

Abbildung in dieser Leseprobe nicht enthalten

Identification of ORF

Abbildung in dieser Leseprobe nicht enthalten

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

[...]

Excerpt out of 6 pages

Details

Title
Unknown Genome Proteomics
College
University of Constance  (Chemie)
Course
Proteomics
Author
Year
2015
Pages
6
Catalog Number
V337836
ISBN (eBook)
9783668293885
File size
965 KB
Language
English
Tags
proteomis, MS/MS, denovo
Quote paper
Manuel Langer (Author), 2015, Unknown Genome Proteomics, Munich, GRIN Verlag, https://www.grin.com/document/337836

Comments

  • No comments yet.
Read the ebook
Title: Unknown Genome Proteomics


Upload papers

Your term paper / thesis:

- Publication as eBook and book
- High royalties for the sales
- Completely free - with ISBN
- It only takes five minutes
- Every paper finds readers

Publish now - it's free