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Database Searches

Database Searches. Peptide mass fingerprinting. 1554.25 2055.39 1942.44 1755.67 987.55 855.34 677.68. Spectrum processing. digest. MS. Peak list. Protein (gel band or spot). Mass spectrum. peptides. Search. Protein X theoretical digest. Protein Y theoretical digest.

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Database Searches

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  1. Database Searches

  2. Peptide mass fingerprinting 1554.25 2055.39 1942.44 1755.67 987.55 855.34 677.68 Spectrum processing digest MS Peak list Protein (gel band or spot) Mass spectrum peptides Search Protein X theoretical digest Protein Y theoretical digest Protein Z theoretical digest Report HIT SCORE Protein X 1000 Protein Y 50 Protein Z 5

  3. Data processing …before searching • Baseline correct • Noise reduction • Peak deisotope for monoisotopic peaks …what is a monoisotopic peak • Copy to Excel and sort for peak intensity • Search with the top 75 peak ions (peptides)

  4. Generate mass list of all peptides • Computer program used to convert graph data (from mass spectrum) to table data (list of peptide masses)

  5. Simple Mass Spectrum Intensity 681.7 200 400 600 800 1000 1200 1400 1600 Mass (Da)

  6. Database search using computer algorithm PLEASEMAKEMANYKRISPYKREMETREATSTHEYAREGREAT e.g. MASCOT Sequence matches List of masses

  7. Mass list of all peptides in the mass spectrum copied into here www.matrixscience.com

  8. Coverage = 26% We found peptides that make up only ¼ of the entire protein – but this is enough for a confident identification!

  9. QUESTIONS

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