protein identification with mascot software (laxmana rao y. and gopalacharyulu p.v.)

Protein Identification with Mascot Software

(Laxmana Rao Y. and Gopalacharyulu P.V.)

Proteomics & Mass Spectrometry

MALDIESI

QTofFTMS . . .

Protein extract fromBiological sample

Protein Identification

Peptide Mass Fingerprinting

Complex Protein Mixture

2D Gel Separation

Purified Protein

Proteolysis Peptide Digest

Mass Spec

337 nm UV laser

cyano-hydroxycinnamic acid

MALDI

0

20

40

60

80

100

m/z

%T

IC

0

20

40

60

80

100

m/z

%T

IC

Theoretical MS Experimental MS

MRNSYRFLASSLSVVVSLLLIPEDVCEKIIGGNEVTPHSRPYMVLLSLDRKTICAGALIAKDWVLTAAHCNLNKRSQVILGAHSITYEEPTKQIMLVKKEFPYPCYDPATREGDLKLLQL

In Silico DigestionProtein Database

LASSLSVVVSLLLIPEDVCEKIIGGNEVTPHSRPYMVLLSLDRTICAGALIAKDWVLTAAHCNLNKRITTTYEEPTKQIMLVKEFPYPCYDPATREGDLKLL

MS/MS Fragment Ion Analysis

Complex Protein Mixture

Proteolysis Peptide Digest

MS/MS

02040

6080

100

m/z

%T

IC

020406080

100

m/z

%T

IC

Experimental Fragmentation

Spectrum

MRNSYRFLASSLSVVVSLLLIPEDVCEKIIGGNEVTPHSRPYMVLLSLDRKTICAGALIAKDWVLTAAHCNLNKRSQVILGAHSITYEEPTKQIMLVKKEFPYPCYDPATREGDLKLLQL

Protein Database

LASSLSVVVSLLCEKIIGGNEVTPHSRPYMVLLSLDRTICAGALIAKDWVLTAAHCNLNKRITTTYEEPTKQIMLVKEFPYPCYDPATREGDLKLL

Theoretical Fragmentation

Spectrum

P YMVLLSLDRPYM VLLSLDRPYMV LLSLDRPYMVL LSLDRPYMVLL SLDRPYMVLLS LDRPYMVLLSL DRPYVLLSLD MRPYMVLLSLD R

HPLC

In Silico Digestion

In Silico Fragmentation

Mascot Scoring

Mascot Score: 120 = 1x10-12

– 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 detailin a live-web demo at the end of thepresentation

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 detailin a live-web demo at the end of thepresentation

Mascot Sequence Query

Mascot Sequence Query: Result

Results will be explained in more detailin a live-web demo at the end of thepresentation