阮雪芬 Department of Life Science @NTU Aug 6, 2004 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

Image Matching kDa 150 Increase of 50% 70 Decrease of 50% 60 42 Unmatched spots Matched spots 10 pH 3.5 10

Standard Proteome Analysis by 2DE-MS Mass Fingerprint Searching in http://www.expasy.ch/tools/peptident.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 Analyzer Detector Ionization

Macromolecule Desorption/Ionization using UFMP Glycerin Mixed Matrix 2002 Nobel lecture by Koichi Tanaka 2002 Nobel lecture

A Simplified Schematic of the MALDI Ionization Process

Mass Spectrometry

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.html

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

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 Amino Acid Sequence DNA Sequence blastn blastx tblastx Blastp tblastn Compares Against protein Sequence Database Compares Against Translated Nucleotide Sequence Database Compares Against protein Sequence Database Compares Against Translated Nucleotide Sequence Database Compares Against 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/cgi-bin/ProFound PeptIdent http://www.expasy.ch/tools/peptident. PepSea http://195.41.108.38/PepSeaIntro.html MASCOT http://www.matrixscience.com/ PepFrag http://www.proteometrics.com/ Protein Prospector http://prospector.ucsf.edu/ 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

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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

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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/ExpasyHunt/

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://genoplante-info.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