1. Protein identification

2. Peptide Mass Fingerprinting In situ digestion Peptide extraction MALDI-MS Putative Candidates Score 1. Larval serum protein 2 494 2. Larval serum protein 2 precursor 493 3. CG7251-PA 52

3. !SEPARATION electrophoresis ( 1-D, 2-D ) chromatography ( SEC, ion exchange, reversed phase ) !DIGESTION chemical ( BrCN ) enzymatic ( trypsin,, Lys-C, Asp-C ) reduction ( Di-Thio-Threitol,  -Mercapto-Ethanol ) alkylation (IodoAcAcid, IodoAcAmide, Vynil Pyridine) !MALDI MS ANALYSIS protein identification ( peptide mass fingerprinting ) peptide structural information ( post source decay ) EXPERIMENTAL PROCEDURES IN PROTEOMICS !SAMPLE CLEAN UP chromatography (reversed phase) solid phase extraction (Zip Tip)

4. GLSDGEWQLVLNWGKVEA DIPGHGQEVLIRLFKGHP ETLEKFDKFKHLKSEDEM KASEDLKKHGATVLTALG GILKKKGHHEAEIKPLAQ SHATKHKIPVKYLEFISE CIIQVLQSKHPGDFGADA QGAMNKALELFRKDMASN YKELGFQG GLSDGEWQLVLNWGKVEA DIPGHGQEVLIRLFKGHP ETLEKFDKFKHLKSEDEM KASEDLKKHGATVLTALG GILKKKGHHEAEIKPLAQ SHATKHKIPVKYLEFISE CIIQVLQSKHPGDFGADA QGAMNKALELFRKDMASN YKELGFQG Protein sequence databank List all peptide masses Database Search with Peptide Mass Fingerprinting Results Experimental Masses Experimental Masses 450.22 609.37 698.31 1007.54 1199.49 2098.99 GLSDGEWQLVLNWGK SEDEMK ASEDLK YLEFISECIIQVLQSK GHPETLEK HGATVLTALGGILK DMASNYK ELGFQG GLSDGEWQLVLNWGK SEDEMK ASEDLK YLEFISECIIQVLQSK GHPETLEK HGATVLTALGGILK DMASNYK ELGFQG Generate all theoretical peptide 450.22 609.37 664.33 1007.54 1114.44 1183.53 1300.51 1407.64 1526.62 1593.71 2098.99 Theoretical Masses Matches ALB GAD Results GAD High Score ALB Low Score

7. Effect of Mass Accuracy and Mass Tolerance on Peptide Mass Fingerprinting Search Results Search m/z Mass Tolerance Da N° of Hits 15291478 1529.70.1164 1529.730.01 25 1529.7340.001 4 1529.73480.0001 2

8. Effect of multiple Peptide Masses on Protein Identification by Mass Fingerprinting Search m/z Mass Tolerance Da N° of Hits 1529.730.1204 1529.730.1 7 1252.700.1 1529.730.1 1252.700.1 1 1833.880.1

12. Protein Candidates for search C3732785-01D8-294F31B1 69800 sequence searched Ran Est’d Protein Information and Probability % pl kDa ® k Z Sequence Analyse Tools (T) T° gi|7019591|ref|NP037530.1 1 1 1.0e+000 1.61 zinc finger protein 224 28 9.7 84.93 ® [Homo sapiens] 2 2 6.3e-004 0.13 T° gi|11429381|ref|XP002409.1 minichromosome maintenance 17 5.4 80.97 ® deficient (mis5, S. pombe) 6 [Homo sapiens] ProFound Search - Result Summary http://prowl.rockefeller.edu/cgi-bin/ProFound

13. ProFound Search - Input Summary Input Summary http://prowl.rockefeller.edu/cgi-bin/ProFound

25. Error: 300 ppm Error: 100 ppm Effect of Mass Accuracy and Mass Tolerance on Peptide Mass Fingerprinting Search Results