protein identification with mascot software (laxmana rao y. and gopalacharyulu p.v.)
TRANSCRIPT
![Page 1: Protein Identification with Mascot Software (Laxmana Rao Y. and Gopalacharyulu P.V.)](https://reader030.vdocuments.mx/reader030/viewer/2022032705/56649d885503460f94a6d9c9/html5/thumbnails/1.jpg)
Protein Identification with Mascot Software
(Laxmana Rao Y. and Gopalacharyulu P.V.)
![Page 2: Protein Identification with Mascot Software (Laxmana Rao Y. and Gopalacharyulu P.V.)](https://reader030.vdocuments.mx/reader030/viewer/2022032705/56649d885503460f94a6d9c9/html5/thumbnails/2.jpg)
Proteomics & Mass Spectrometry
MALDIESI
QTofFTMS . . .
Protein extract fromBiological sample
Protein Identification
![Page 3: Protein Identification with Mascot Software (Laxmana Rao Y. and Gopalacharyulu P.V.)](https://reader030.vdocuments.mx/reader030/viewer/2022032705/56649d885503460f94a6d9c9/html5/thumbnails/3.jpg)
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
![Page 4: Protein Identification with Mascot Software (Laxmana Rao Y. and Gopalacharyulu P.V.)](https://reader030.vdocuments.mx/reader030/viewer/2022032705/56649d885503460f94a6d9c9/html5/thumbnails/4.jpg)
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
![Page 5: Protein Identification with Mascot Software (Laxmana Rao Y. and Gopalacharyulu P.V.)](https://reader030.vdocuments.mx/reader030/viewer/2022032705/56649d885503460f94a6d9c9/html5/thumbnails/5.jpg)
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
![Page 6: Protein Identification with Mascot Software (Laxmana Rao Y. and Gopalacharyulu P.V.)](https://reader030.vdocuments.mx/reader030/viewer/2022032705/56649d885503460f94a6d9c9/html5/thumbnails/6.jpg)
PMF: Results
Results will be explained in more detailin a live-web demo at the end of thepresentation
![Page 7: Protein Identification with Mascot Software (Laxmana Rao Y. and Gopalacharyulu P.V.)](https://reader030.vdocuments.mx/reader030/viewer/2022032705/56649d885503460f94a6d9c9/html5/thumbnails/7.jpg)
Peptide Mass Fingerprint: Query
![Page 8: Protein Identification with Mascot Software (Laxmana Rao Y. and Gopalacharyulu P.V.)](https://reader030.vdocuments.mx/reader030/viewer/2022032705/56649d885503460f94a6d9c9/html5/thumbnails/8.jpg)
MS/MS Ions Search: Input data file
![Page 9: Protein Identification with Mascot Software (Laxmana Rao Y. and Gopalacharyulu P.V.)](https://reader030.vdocuments.mx/reader030/viewer/2022032705/56649d885503460f94a6d9c9/html5/thumbnails/9.jpg)
MS/MS Ions Search: Query
![Page 10: Protein Identification with Mascot Software (Laxmana Rao Y. and Gopalacharyulu P.V.)](https://reader030.vdocuments.mx/reader030/viewer/2022032705/56649d885503460f94a6d9c9/html5/thumbnails/10.jpg)
MS/MS Ions Search: Results
Results will be explained in more detailin a live-web demo at the end of thepresentation
![Page 11: Protein Identification with Mascot Software (Laxmana Rao Y. and Gopalacharyulu P.V.)](https://reader030.vdocuments.mx/reader030/viewer/2022032705/56649d885503460f94a6d9c9/html5/thumbnails/11.jpg)
Mascot Sequence Query
![Page 12: Protein Identification with Mascot Software (Laxmana Rao Y. and Gopalacharyulu P.V.)](https://reader030.vdocuments.mx/reader030/viewer/2022032705/56649d885503460f94a6d9c9/html5/thumbnails/12.jpg)
Mascot Sequence Query: Result
Results will be explained in more detailin a live-web demo at the end of thepresentation