Database Searches. Peptide mass fingerprinting digestMS Search HIT SCORE Protein X 1000 Protein Y 50 Protein Z 5 Protein X theoretical digestProtein Y.

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Presentation transcript:

Database Searches

Peptide mass fingerprinting digestMS Search HIT SCORE Protein X 1000 Protein Y 50 Protein Z 5 Protein X theoretical digestProtein Y theoretical digestProtein Z theoretical digest Spectrum processing Report Protein (gel band or spot) peptides Mass spectrum Peak list

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)

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

Mass (Da) Intensity Simple Mass Spectrum

PLEASEMAKEMANYKRISPYKREMETREATSTHEYAREGREAT Database search using computer algorithm List of masses e.g. MASCOT Sequence matches EAT EGR ISPYK EMETR EMANYK PLEASEMAK EATSTHEYAR

Mass list of all peptides in the mass spectrum copied into here

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

QUESTIONS