Proteomics Data Analysis

Sophie Harvey, Ph.D

Summer Mass Spectrometry workshop 2018
 Proteomics data analysis

MS/MS
?
peptides identify
protein

rank

MS Peptide MW
Found in Selected
compare
120000

Databases
100000

80000

NDALYFPT...

Relative Intensity
Relative Intensity
60000

SWDLTAL...
40000

200 400 600 800 1000 1200 1400 1600

m/z

20000

PTDLDVSY... 0

theoretical spectra
200 400 600 800 1000 1200 1400 1600

m/z
 Database Searching

 Mascot
 Proteome discoverer
 Database Searching

Mascot
•16 node cluster for high-speed data processing

Protein sequences are digested and fragmented In Silico which



produces an enormous peak list

Raw MS/MS data is converted to a peak list and compared against

the In Silico peak list.

Critical point of database searching

•The enzyme must work properly (no non-specific cleavage and missed
cuts)
•The mass accuracy limits must be set appropriately
•Any modifications must be accounted for (modified cysteine)
•The database must contain the protein
Mascot search parameters
Mascot search parameters
Mascot search parameters
Mascot search results
Mascot report builder
 Mascot report builder

The Exponentially Modified Protein Abundance Index (emPAI)

Protein Mol. Weight

Protein Mascot
Score
# of spectra matched
Protein Name # of peptides matched
Mascot protein results
Mascot protein results
Mascot protein results
Mascot peptide MS/MS
Exporting results
 Proteome Discoverer
 Extracts relevant MS/MS spectra from the raw file and determines the precursor charge state and the quality
of the fragmentation spectrum.

 The Sequest search algorithm correlates experimental MS/MS spectra through comparisons to theoretical in-
silico peptide candidates derived from protein databases.

 The proprietary cross-correlation identification algorithm at the core of Sequest uses a sophisticated scoring
system to help assess results.

 Sequest looks for characteristic spectral patterns and then critically evaluates the equivalence of experimental
and theoretical MS/MS spectra.
Proteome Discoverer processing workflow
Proteome Discoverer processing workflow
Proteome Discoverer processing workflow
Proteome Discoverer processing workflow
Proteome Discoverer consensus workflow
Proteome Discoverer consensus workflow
Proteome Discoverer consensus workflow
Proteome Discoverer consensus workflow
Proteome Discoverer consensus workflow
Proteome Discoverer consensus workflow
Proteome Discoverer consensus workflow
Proteome Discoverer consensus workflow
Proteome Discoverer full workflow
Proteome Discoverer results view
Proteome Discoverer filtering
Proteome Discoverer sequence coverage
Proteome Discoverer peptide list
Proteome Discoverer MS/MS spectra
 Combining search results and comparing
data
 Scaffold
Scaffold for combining and comparing data

Conditions

Bio-Replicates

# of Spectra

P-Value
Scaffold performing simple statistics
Scaffold comparing between conditions
 Thank you!

• Liwen Zhang
• Arpad Somoqyi
• Vicki Wysocki

http://www.ccic.ohio-state.edu/MSP