Bioinformatics for Proteomics

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Bioinformatics for Proteomics Shu-Hui Chen
(???) Department of Chemistry National Cheng Kung
University
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What is Proteomics ?
Systematic analysis of All
protein sequences All protein
expression pattern All protein
interactions This involves
Protein identification Protein
quantification Functional
characterization of all proteins
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MS-based Bioinformatics

• MS instrument is so far not sensitive enough to
  resolve proteins in a biological system solely
  based on signals measured.
• MS, however, is able to acquire sufficient data
  for mapping a protein from the database using new
  computer algorithms to analyze the data.
• MS data could be used for Protein Quantification

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The tools of Proteomics
Traditional protein chemistry assay methods
struggle to establish Identity and
quantification Identity requires
Specificity of measurement (Precision)
Mass Spectrometry
MS-based data acquisition algorithm
Database dependent identification-A reference for
comparison Protein sequence
databases Search algorithms
Database-independent
identification-No reference
De-novo sequencing
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MS-based Protein Identification and Quantification
? Mass Mapping Peptide Sequencing
Protein Quantification
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Mass Spectrometry Protein identified by database
mapping
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http//www.expasy.org/tools/
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www.uniprot.org
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Automated Database Search
Number 1 match tumor necrosis factor type 1
receptor associated protein TRAP-1 (Mr) 76030.27
Total coverage 33.4
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History for MS Searching
MOWSE
1993
By Pappin and Bleasby
SEQUEST
1994
By Yates and Eng
MOWSE?
1996
Molecular Weight Search
1997
MOWSE?
1998
MASCOT
By Matrix science
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Scoring algorithm
Final score -10LOG(P), where P is absolute
probability that the observed match is a random
event E value (expected value) describes the
number of hits one can expect to see by chance
when searching a database of a particular size.
A value of zero indicates that no matches would
be expected by chance.
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MS-based Protein Identification and Quantification

• Mass Mapping
• Peptide Sequencing
• Protein Quantification

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Tandem Mass Spectrometry- MS/MS
MS/MS acquisition is controlled by software
setting
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Nomenclature used for CID peptide
fragmentation- Low Energy (eV)- Q, TOF, FT
Bioanalytical Chemistry Mikkelsen, S.R.,
published by John Wiley Sons, Inc.
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Protein Identification by Database Search
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PRIDE website
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http//www.ebi.ac.uk/pride
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Database Independent Sequencing- De Novo
Sequencing
http//www.bioinfor.com/peaks/tutorials/denovo.htm
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Sequence Tag Approach for Peptide Sequencing
Bioanalytical Chemistry Mikkelsen, S.R.,
published by John Wiley Sons, Inc.
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MS-based Protein Identification and Quantification

• Mass Mapping
• Peptide Sequencing
• Protein Quantification

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MS-based Quantification Methods Using Stable
Isotope Labeling
X.J. Li Institute for systems biology, Seattle
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Exercises
1. Choose one protein you are interested in. 2.
Go to the Bioinformatics portal website
http//www.expasy.org/tools/, link to
Uniprot and search for your protein. 3. List
accession number, molecular weight, and aminoacid
sequence of your protein. Briefly describe
the protein function and its subcellular
localization. 4. Link to PaxDb proteomic
database, show the abundance of the protein. 5.
Link to PRIDE or MaxQB proteomic database, list
the peptides (sequences) that have been
identified by MS for this protein. Include
Mascot peptide score for each identified
sequence. If same peptides were identified
by multiple times, list the highest score.