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Protein Identification with Mascot Software

Protein Identification with Mascot Software. (Laxmana Rao Y. and Gopalacharyulu P.V.). Protein extract from Biological sample. MALDI ESI. QTof FTMS . . . . Protein Identification. Proteomics & Mass Spectrometry. 337 nm UV laser. cyano-hydroxy cinnamic acid. MRNSYRFLASSL

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Protein Identification with Mascot Software

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

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

  3. 337 nm UV laser cyano-hydroxy cinnamic acid MRNSYRFLASSL SVVVSLLLIPED VCEKIIGGNEVT PHSRPYMVLLSL DRKTICAGALIA KDWVLTAAHCNL NKRSQVILGAHS ITYEEPTKQIML VKKEFPYPCYDP ATREGDLKLLQL 100 100 LASSLSVVVSLLLIPEDVCEK IIGGNEVTPHSR PYMVLLSLDR TICAGALIAK DWVLTAAHCNLNKR ITTTYEEPTK QIMLVK EFPYPCYDPATR EGDLKLL 80 80 60 60 %TIC %TIC 40 40 20 20 0 0 m/z m/z Protein Database In Silico Digestion Peptide Mass Fingerprinting Complex Protein Mixture Purified Protein 2D Gel Separation Peptide Digest MALDI Proteolysis Mass Spec Theoretical MS Experimental MS

  4. In Silico Digestion In Silico Fragmentation Protein Database 100 MRNSYRFLASSL SVVVSLLLIPED VCEKIIGGNEVT PHSRPYMVLLSL DRKTICAGALIA KDWVLTAAHCNL NKRSQVILGAHS ITYEEPTKQIML VKKEFPYPCYDP ATREGDLKLLQL 100 80 LASSLSVVVSLLCEK IIGGNEVTPHSR PYMVLLSLDR TICAGALIAK DWVLTAAHCNLNKR ITTTYEEPTK QIMLVK EFPYPCYDPATR EGDLKLL P YMVLLSLDR PYM VLLSLDR PYMV LLSLDR PYMVL LSLDR PYMVLL SLDR PYMVLLS LDR PYMVLLSL DR PYVLLSLD MR PYMVLLSLD R 80 60 %TIC 60 %TIC 40 40 20 20 0 0 m/z m/z MS/MS Fragment Ion Analysis Complex Protein Mixture Peptide Digest Proteolysis HPLC MS/MS Theoretical Fragmentation Spectrum Experimental Fragmentation Spectrum

  5. Mascot Scoring • 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 Mascot Score: 120 = 1x10-12

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

  7. Peptide Mass Fingerprint: Query

  8. MS/MS Ions Search: Input data file

  9. MS/MS Ions Search: Query

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

  11. Mascot Sequence Query

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

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