Protein Identification with Mascot Software (Laxmana Rao Y. and Gopalacharyulu P.V.)

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Presentation transcript:

Protein Identification with Mascot Software (Laxmana Rao Y. and Gopalacharyulu P.V.)

Proteomics & Mass Spectrometry MALDI ESI QTof FTMS... Protein extract from Biological sample Protein Identification

Peptide Mass Fingerprinting C omplex Protein Mixture 2D Gel Separation Purified Protein Proteolysis Peptide Digest Mass Spec 337 nm UV laser cyano-hydroxy cinnamic acid MALDI m/z %TIC m/z %TIC Theoretical MS Experimental MS MRNSYRFLASSL SVVVSLLLIPED VCEKIIGGNEVT PHSRPYMVLLSL DRKTICAGALIA KDWVLTAAHCNL NKRSQVILGAHS ITYEEPTKQIML VKKEFPYPCYDP ATREGDLKLLQL In Silico DigestionProtein Database LASSLSVVVSLLLIPEDVCEK IIGGNEVTPHSR PYMVLLSLDR TICAGALIAK DWVLTAAHCNLNKR ITTTYEEPTK QIMLVK EFPYPCYDPATR EGDLKLL

MS/MS Fragment Ion Analysis Complex Protein Mixture Proteolysis Peptide Digest MS/MS m/z %TIC m/z %TIC Experimental Fragmentation Spectrum MRNSYRFLASSL SVVVSLLLIPED VCEKIIGGNEVT PHSRPYMVLLSL DRKTICAGALIA KDWVLTAAHCNL NKRSQVILGAHS ITYEEPTKQIML VKKEFPYPCYDP ATREGDLKLLQL Protein Database LASSLSVVVSLLCEK IIGGNEVTPHSR PYMVLLSLDR TICAGALIAK DWVLTAAHCNLNKR ITTTYEEPTK QIMLVK EFPYPCYDPATR EGDLKLL Theoretical Fragmentation Spectrum P YMVLLSLDR PYVLLSLD MR PYMVLLSLD R HPLC In Silico Digestion In Silico Fragmentation

Mascot Scoring Mascot Score: 120 = 1x –The Mascot Score is given as S = -10*Log(P), where P is the probability that the observed match is a random event –The significance of that result depends on the size of the database being searched. Mascot shades in green the insignificant hits using a P=0.05 cutoff. In this example, scores less than 74 are insignificant

PMF: Results Results will be explained in more detail in a live-web demo at the end of the presentation

Peptide Mass Fingerprint: Query

MS/MS Ions Search: Input data file

MS/MS Ions Search: Query

MS/MS Ions Search: Results Results will be explained in more detail in a live-web demo at the end of the presentation

Mascot Sequence Query

Mascot Sequence Query: Result Results will be explained in more detail in a live-web demo at the end of the presentation