Protein identification. Peptide Mass Fingerprinting In situ digestion Peptide extraction MALDI-MS Putative Candidates Score 1. Larval serum protein 2.

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Protein identification

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

!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)

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 GLSDGEWQLVLNWGK SEDEMK ASEDLK YLEFISECIIQVLQSK GHPETLEK HGATVLTALGGILK DMASNYK ELGFQG GLSDGEWQLVLNWGK SEDEMK ASEDLK YLEFISECIIQVLQSK GHPETLEK HGATVLTALGGILK DMASNYK ELGFQG Generate all theoretical peptide Theoretical Masses Matches ALB GAD Results GAD High Score ALB Low Score

Effect of Mass Accuracy and Mass Tolerance on Peptide Mass Fingerprinting Search Results Search m/z Mass Tolerance Da N° of Hits

Effect of multiple Peptide Masses on Protein Identification by Mass Fingerprinting Search m/z Mass Tolerance Da N° of Hits

Protein Candidates for search C D8-294F31B sequence searched Ran Est’d Protein Information and Probability % pl kDa ® k Z Sequence Analyse Tools (T) T° gi| |ref|NP e zinc finger protein ® [Homo sapiens] e T° gi| |ref|XP minichromosome maintenance ® deficient (mis5, S. pombe) 6 [Homo sapiens] ProFound Search - Result Summary

ProFound Search - Input Summary Input Summary

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