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