1. Bioinformatics for Proteomics
Shu-Hui Chen (陳淑慧)
Department of Chemistry
National Cheng Kung University

2. 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

3. 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

4. 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

5. MS-based Protein Identification and Quantification
 Mass Mapping
Peptide Sequencing
Protein Quantification

6. Protein identified by database mapping
Mass Spectrometry
Protein identified by database mapping

16. Automated Database Search
Number 1 match: tumor necrosis factor type 1 receptor
associated protein TRAP-1 (Mr):
Total coverage: 33.4%

17. History for MS Searching
MOWSE
1993
By Pappin and Bleasby
SEQUEST
1994
By Yates and Eng
MOWSEⅡ
1996
Molecular Weight Search
1997
MOWSEⅢ
1998
MASCOT
By Matrix science

27. Scoring algorithm Final score= -10*LOG(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.

31. MS-based Protein Identification and Quantification
Mass Mapping
Peptide Sequencing
Protein Quantification

32. Tandem Mass Spectrometry- MS/MS
MS/MS acquisition is controlled by software setting

33. Nomenclature used for CID peptide fragmentation-
Low Energy (eV)- Q, TOF, FT
“Bioanalytical Chemistry” Mikkelsen, S.R.,
published by John Wiley & Sons, Inc.

36. Protein Identification by Database Search

46. PRIDE website

53. Database Independent Sequencing- De Novo Sequencing

54. Sequence Tag Approach for Peptide Sequencing
“Bioanalytical Chemistry” Mikkelsen, S.R.,
published by John Wiley & Sons, Inc.

55. MS-based Protein Identification and Quantification
Mass Mapping
Peptide Sequencing
Protein Quantification

56. MS-based Quantification Methods Using Stable Isotope Labeling
X.J. Li Institute for systems biology, Seattle

58. Exercises 1. Choose one protein you are interested in.
2. Go to the Bioinformatics portal website
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.