1. Database Searches

2. 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 1554.25 2055.39 1942.44 1755.67 987.55 855.34 677.68 Report Protein (gel band or spot) peptides Mass spectrum Peak list

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. Computer program used to convert graph data (from mass spectrum) to table data (list of peptide masses) Generate mass list of all peptides 1007.467 1023.462 1036.481 1066.493 1082.488 1105.601 1153.524 1172.581 972.5954 973.4202 975.6104 977.4627 982.4603 983.4991 998.4552

5. 2004006008001000120014001600 Mass (Da) Intensity 681.7 Simple Mass Spectrum

6. PLEASEMAKEMANYKRISPYKREMETREATSTHEYAREGREAT 320.3 361.4 607.7 681.7 771.9 992.1 1165.2 Database search using computer algorithm List of masses e.g. MASCOT Sequence matches EAT EGR ISPYK EMETR EMANYK PLEASEMAK EATSTHEYAR

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

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

10. QUESTIONS