Download Protein Identification with Mascot Software

Protein Identification with Mascot Software
(Laxmana Rao Y. and Gopalacharyulu P.V.)
Proteomics & Mass Spectrometry
Protein extract from
Biological sample
MALDI
ESI
QTof
FTMS
...
Protein Identification
Peptide Mass Fingerprinting
337 nm UV laser
Complex
Protein
Mixture
cyano-hydroxy
cinnamic acid
2D Gel Purified Proteolysis Peptide
Separation Protein
Digest
MALDI
Mass Spec
100
LASSLSVVVSLLLIPEDVCEK
80
IIGGNEVTPHSR
PYMVLLSLDR
60
TICAGALIAK
40
DWVLTAAHCNLNKR
20
ITTTYEEPTK
0
QIMLVK
EFPYPCYDPATR
EGDLKLL
100
80
Protein Database In Silico Digestion
%TIC
%TIC
MRNSYRFLASSL
SVVVSLLLIPED
VCEKIIGGNEVT
PHSRPYMVLLSL
DRKTICAGALIA
KDWVLTAAHCNL
NKRSQVILGAHS
ITYEEPTKQIML
VKKEFPYPCYDP
ATREGDLKLLQL
60
40
20
m/z
Theoretical MS
0
m/z
Experimental MS
MS/MS Fragment Ion Analysis
Complex
Protein Proteolysis
Mixture
MS/MS
In Silico In Silico
Digestion Fragmentation100
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
40
20
0
100
80
60
40
20
0
%TIC
MRNSYRFLASSL
SVVVSLLLIPED
VCEKIIGGNEVT
PHSRPYMVLLSL
DRKTICAGALIA
KDWVLTAAHCNL
NKRSQVILGAHS
ITYEEPTKQIML
VKKEFPYPCYDP
ATREGDLKLLQL
HPLC
%TIC
Protein Database
Peptide
Digest
m/z
Theoretical
Fragmentation
Spectrum
m/z
Experimental
Fragmentation
Spectrum
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
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