Download Sequence Alignment

2D Gel Analysis
阮雪芬
Department of Life Science @NTU
Aug 6, 2004
Outline


Introduction to Proteomics
Softwares
– Protein primary structure


–
–
–
–
Amino acid and atomic composition
Computing pI and MW
Sequence searching
Sequence alignment
2D Image Matching
Mass Fingerprint



Mascot
MS
MS/MS
– Post-Translational Modification




SignalP
NetPhos
Predotar
YinOYang
Outline


Introduction to Proteomics
Softwares
– Protein primary structure


–
–
–
–
Amino acid and atomic composition
Computing pI and MW
Sequence searching
Sequence alignment
2D Image Matching
Mass Fingerprint



Mascot
MS
MS/MS
– Post-Translational Modification




SignalP
NetPhos
Predotar
YinOYang
What Is Proteomes
?
Proteomes




Gene + Chromosome Genome
Protein +Genome Proteome
Proteomes are dynamics
Proteome changes as a function of:
– time
– development
– extracellular condition
– intracellular condition
Definitions of Proteomics




First coined in 1995 by Wilkins
Be defined as the large-scale characterization
of the entire protein complement of a cell line,
tissue, or organism.
The study of proteomes
Goal:
-To obtain a more global and integrated view
of biology by studying all the proteins of a cell
rather than each one individually.
Proteomics Origins

In 1975, the introduction of the 2D gel by
O’Farrell who began mapping proteins from E.
coli.


The first major technology to emerge for the
identification of proteins was the sequencing of
proteins by Edman degradationpicomole
MS technology has replaced Edman
degradation to identify proteinsfemtomole
How Proteomics Can Help
Drug Development
http://www.sciam.com.tw/read/readshow.asp?FDocNo=63&CL=18
Types of Proteomics and
Their Applications to
Biology
Mechanisms by Which a
Single Gene Can Give Rise to
Multiple Gene Products
In bacteria, 1 or 2 proteins/gene
In yeast, 3 proteins/gene
In human, 3 or more proteins/gene
Two-dimensional Gel
Approach
Nature 2000, 405, 837-846
kDa
150
Image Matching
Increase
of 50%
70
60
Decrease
of 50%
42
Unmatched
spots
Matched
spots
10
pH
3.5
10
Standard Proteome
Analysis by 2DE-MS
Mass Fingerprint
Searching in
http://www.expas
y.ch/tools/peptide
nt.html
Current Opinion in
Chemical Biology 2000,
4:489–494
Koichi Tanaka


2002 Nobel Prize:The Origin of
Macromolecule Ionization by Laser
Irradiation
In 1985 Feb, instead of using Cobalt Ultra
Fine Metal Power (UFMP), he mistakenly
used a glycerin-UFMP
Schematic of Time of
Flight Mass Spectrometry
2002 Nobel lecture by Koichi Tanaka
Time of Flight Mass
Spectrometry
How Does a Mass
Spectrometer Work?
Sample
input
Ionization
Analyzer
Detector
Macromolecule
Desorption/Ionization using
UFMP Glycerin Mixed Matrix
2002 Nobel lecture by Koichi Tanaka
A Simplified Schematic of the
MALDI Ionization Process
Mass Spectrometry
kcw1212-1 14 (1.129) Cn (Cen,4, 50.00, Ht); Sm (SG, 1x2.00); Sb (15,10.00 ); Cm (6:15)
1132.917
100
1: TOF LD+
7.95e3
1791.532
1792.532
1133.923
%
1517.239
1793.535
1518.245
965.893
1134.924
1500.215
966.895
1519.244
1022.925
1023.925
1794.537
1189.963
1190.965 1349.235
1248.008 1350.239
1351.239
1574.283
1576.280 1774.531
1850.563
1851.577
0
2216.861
1848.577
2168.845
2126.732
2217.870
2218.857
2344.987
2552.115
2732.213
2810.387 2866.446
m/z
Peptide Mass
Fingerprinting
The Information Stored in
Genes Is Expressed by a
Multistage Process
DNA 和蛋白質合成的地方
DNA
Proteins
Sugar Chain
Post-translational
Modification



DNA and mRNA provide no information
concerning the activities and post-translational
modifications of proteins.
The number of documented protein co- and
post-translational modifications now exceeds
400 (http: / / abrf.org / index-.cfm/dm.home).
The elucidation of protein post-translational
modifications is perhaps the most important
justification for proteomics as a scientific
endeavor.
Chemical modification


Covalent linkage of chemical group to
amino acid in protein
Several types:
– Acetylation (common on first amino acid)
– Phosphorylation (often involved in
regulation)
– Lipidation (attachment to membrane)
– Glycosylation (common outside cell)
Processing

Removal of part of protein by either:
– Cleavage (protease cuts protein into
smaller pieces)
– Splicing (self-removal and rejoining of
ends)
Outline


Introduction to Proteomics
Softwares
– Protein primary structure


–
–
–
–
Amino acid and atomic composition
Computing pI and MW
Sequence searching
Sequence alignment
2D Image Matching
Mass Fingerprint



Mascot
MS
MS/MS
– Post-Translational Modification




SignalP
NetPhos
Predotar
YinOYang
Primary Structure
Analysis

Object:
To compute the characters of
proteins.
-Amino acid composition
-Atomic composition
-pI
-Molecular weight
Amino Acid & Atomic
Composition
ProtParam
Amino Acid & Atomic
Composition
http://www.expasy.ch/tools/protparam.
Amino Acid & Atomic
Composition
Amino Acid Composition
Atomic Composition
Outline


Introduction to Proteomics
Softwares
– Protein primary structure


–
–
–
–
Amino acid and atomic composition
Computing pI and MW
Sequence searching
Sequence alignment
2D Image Matching
Mass Fingerprint



Mascot
MS
MS/MS
– Post-Translational Modification




SignalP
NetPhos
Predotar
YinOYang
Computing pI and MW
Computing pI and MW
Computing pI and MW
MW
pI
Outline


Introduction to Proteomics
Softwares
– Protein primary structure


–
–
–
–
Amino acid and atomic composition
Computing pI and MW
Sequence searching
Sequence alignment
2D Image Matching
Mass Fingerprint



Mascot
MS
MS/MS
– Post-Translational Modification




SignalP
NetPhos
Predotar
YinOYang
Protein Sequence
Searching
Protein Sequence
Searching
P02571
Protein Sequence
Searching
Outline


Introduction to Proteomics
Softwares
– Protein primary structure


–
–
–
–
Amino acid and atomic composition
Computing pI and MW
Sequence searching
Sequence alignment
2D Image Matching
Mass Fingerprint



Mascot
MS
MS/MS
– Post-Translational Modification




SignalP
NetPhos
Predotar
YinOYang
Sequence Alignment
Sequence Alignment
Input Query
DNA Sequence
Amino Acid Sequence
Blastp
Compares
Against
protein
Sequence
Database
tblastn
Compares
Against
Translated
Nucleotide
Sequence
Database
blastn
Compares
Against
Nucleotide
Sequence
Database
blastx
tblastx
Compares
Against
protein
Sequence
Database
Compares
Against
Translated
Nucleotide
Sequence
Database
Sequence Alignment
http://www.expasy.ch/
Sequence Alignment
Sequence Alignment
Sequence Alignment
Sequence Alignment
Sequence Alignment
Outline


Introduction to Proteomics
Softwares
– Protein primary structure


–
–
–
–
Amino acid and atomic composition
Computing pI and MW
Sequence searching
Sequence alignment
2D Image Matching
Mass Fingerprint



Mascot
MS
MS/MS
– Post-Translational Modification




SignalP
NetPhos
Predotar
YinOYang
www.expasy.ch/ch2d
http://www.expasy.ch/melanie/
Outline


Introduction to Proteomics
Softwares
– Protein primary structure


–
–
–
–
Amino acid and atomic composition
Computing pI and MW
Sequence searching
Sequence alignment
2D Image Matching
Mass Fingerprint

Mascot
– MS
– MS/MS
– Post-Translational Modification




SignalP
NetPhos
Predotar
YinOYang
World Wide Web Tools for
Searching Databases
Site name
URL
Information available
MOWSE
http://srs.hgmp.mrc.ac.uk/cgi-bin/mowse
Peptide mass mapping and sequencing
ProFound
http://prowl.rockefeller.edu/cgibin/ProFound
Peptide mass mapping and sequencing
PeptIdent
http://www.expasy.ch/tools/peptident.
Peptide mass mapping and sequencing
PepSea
http://195.41.108.38/PepSeaIntro.html
Peptide mass mapping and sequencing
MASCOT
http://www.matrixscience.com/
Peptide mass mapping and sequencing
PepFrag
http://www.proteometrics.com/
Peptide mass mapping and sequencing
Protein Prospector
http://prospector.ucsf.edu/
Peptide mass mapping and sequencing
FindMod
http://www.expasy.ch/tools/findmod/
Posttranslational modification
SEAQUEST
http://fields.scripps.edu/sequest/
Uninterpreted MS/MS searching
FASTA Search
Programs
http://fasta.bioch.virginia.edu/
Protein and nucleotide database
searching
Cleaved
Radioactivity of
Phosphopeptides
http://fasta.bioch.virginia.edu/crp
Protein phosphorylation site mapping
http://us.expasy.org/tools
Mascot
http://www.matrixscience.com
Step 1
Step 2
Step 3
Step 3
Step 4
Step 5
Outline


Introduction to Proteomics
Softwares
– Protein primary structure


–
–
–
–
Amino acid and atomic composition
Computing pI and MW
Sequence searching
Sequence alignment
2D Image Matching
Mass Fingerprint

Mascot
– MS
– MS/MS
– Post-Translational Modification




SignalP
NetPhos
Predotar
YinOYang
Step 1
Step 2
Step 3
Step 3
Step 4
Step 5
Step 6
Step 7
Step 8
Step 9
For peptide
mass
fingerprint
data
pI and MW
Species
Outline


Introduction to Proteomics
Softwares
– Protein primary structure


–
–
–
–
Amino acid and atomic composition
Computing pI and MW
Sequence searching
Sequence alignment
2D Image Matching
Mass Fingerprint

Mascot
– MS
– MS/MS
– Post-Translational Modification




SignalP
NetPhos
Predotar
YinOYang
Publicly Available PTM Web
Resources (I)
Proteomics (2004), 4, 1633-1649
Publicly Available PTM Web
Resources (II)
http://us.expasy.org/Exp
asyHunt/
Fasta Sequence
http://us.expasy.org/tools/#ptm
Search Result
Outline


Introduction to Proteomics
Softwares
– Protein primary structure


–
–
–
–
Amino acid and atomic composition
Computing pI and MW
Sequence searching
Sequence alignment
2D Image Matching
Mass Fingerprint

Mascot
– MS
– MS/MS
– Post-Translational Modification




SignalP
NetPhos
Predotar
YinOYang
Outline


Introduction to Proteomics
Softwares
– Protein primary structure


–
–
–
–
Amino acid and atomic composition
Computing pI and MW
Sequence searching
Sequence alignment
2D Image Matching
Mass Fingerprint

Mascot
– MS
– MS/MS
– Post-Translational Modification




SignalP
NetPhos
Predotar
YinOYang
Predotar:
A Tool for N-terminal
Targeting Sequence

http://genoplanteinfo.infobiogen.fr/predotar/
Proteomics (2004) 4, 1581-1590
P33316
sequence
Outline


Introduction to Proteomics
Softwares
– Protein primary structure


–
–
–
–
Amino acid and atomic composition
Computing pI and MW
Sequence searching
Sequence alignment
2D Image Matching
Mass Fingerprint

Mascot
– MS
– MS/MS
– Post-Translational Modification




SignalP
NetPhos
Predotar
YinOYang
http://us.expasy.org/tools/#ptm
YinOyang
http://www.cbs.dtu.dk/services/YinOYang/
Result