牛頓雜誌

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Introduction to Functional Proteomics

2 Introduction

Protein overview

Protein separation, purification, and analysis Sample preparation

One-dimensional electrophoresis Two-dimensional electrophoresis Image analysis

The type of mass spectrometers MS for protein analysis and identification Protein modifications

Drug vs. proteomic Microarray and SNP

Modification of protein (MS analysis) Application of proteomics

Student presentation

課程大綱

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地球上所有的故事是這樣開始 … 一五○億年前

大 爆 炸

牛頓雜誌 (1994) 第 132 期, p.20

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宇宙放晴 大渾沌 大爆炸

現在的宇宙 10^-34sec

10^-44sec

10 萬年

150 億年

肥皂泡模型

牛頓雜誌 (1994) 第 129 期, p.116

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牛頓雜誌(1991) 第93 期, p.103

基本粒子 基本粒子 原子核 原子核

原子 原子

+ + + + - -

- -

由基本粒子到原子

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小行星碰撞 岩漿海 地殼形成 第一場大雨 天空放晴

46 億年前 38 億年前

牛頓雜誌 (1994) 第 132 期, p.37

地球上的水分是由殞石帶來的 地球只有薄薄一層地殼是冷的

地球早期演進的重要關鍵

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基本小分子 單位小分子單位小分子

Campbell (1999) Biochemistry (3e) p.16

H H C

N

O H

H H H

H H H

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7

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由基本小分子到單位小分子

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9 10

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後基因體時代將進入蛋白質體科學為主的功能性研究,其將導引 我們發現各種蛋白質間之交互作用,提供我們致病途徑及新藥開 發之機會,開發各種治療方法,以提供根本預防策略為最目終目標

Beyond the Genome

Proteins are ultimately (最終) responsible for all biological processes that take place within cells.

Protein dynamics reflect the state of biological system at a given time Detection and identification of post-translational modifications (PTM)

Proteomic and the New biology

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Proteome (蛋白體)

Proteomics (蛋白質體學)

Genome (基因體)

Genomics (基因體學)

Functional Proteomics (功能性蛋白質體學)

Definition (必考)

It is the study of the proteome, the protein complement of the genome/organism.

Which describe the entire collection of genes in an organism.

It is the study of the genome, the gene complement of an organism.

The proteins complement of the genome/organism. (specific time and target)

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"The analysis of the entire protein complement expressed by a genome, or by a cell or tissue type.“

Wasinger VC et al Progress with gene-product mapping of the mollicutes: Mycoplasma genitalium.

Electrophoresis 16 (1995) 1090-1094

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From Genotype to Phenotype

• Genome: DNAs

• Transcriptome: RNAs

• Proteome: Proteins

• Physiome: Metabolites

• Biome: Environment

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The proteome

1. The proteome consist of all of the proteins expressed by a cell under specific conditions or stimulation

2. The proteome of a organism depends ontype, its developmental stage, environment/stimuli, nutritional and metabolic status etc..

3. The genome of a organism is fixed, however, the

proteome is dynamic

4. The proteome is much larger than the genome. Each gene can translate into mutiple isoforms of proteins 5. The proteome is very hard than genome, for low

expressed protein.

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“omics” 體學, 利用大規模且巨觀的研究方法(包含蛋白質身 份鑑定、細胞及分子生物技術、生物活性測試、電腦程式 計算)來研究觀察整個生物體分子層次之生理角色。

“Omics” in life science Genomics

arrangement, identify, discovery of gene

Transcriptomics

Proteomics

Metabolomics

Metabolite in specific organism

“Omics” terms symbolized a redefinition of how we think about biology and the working of living systems

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Current -omics

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狹義定義:在特定一個時間點,在一個特定標的物(組織,細胞,胞 器…),研究所有蛋白質之種類、特性及其含量。

廣義定義:除了狹義定義之,加上嚴謹之實驗(控制組及實驗組),並 結合生理、藥理、分子生物學及生物資訊之技術及知識,探討蛋 白質之交互作用在生物體上可能扮演之角色。

Proteomics (蛋白質體學)

Don’ Don ’t forget that the proteome is t forget that the proteome is dynamic dynamic, changing to , changing to reflect the environment that the cell is in

reflect the environment that the cell is in

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Metabolic Pathway Analysis

Proteomics Segmentation

Protein Structure

Analysis

3-D Protein Structure Computer Modeling

Protein Function

Protein-Protein Interaction Protein Function

Assays

Protein Discovery

Identification / Characterization

Expression Quantitation Differential Display

PTM PTM’s

Protein Expression Protein Complexes

Cross Linking Studies Higher Order Protein Structure

Epitope Mapping Active Site Investigation

Drug Targets

BioMarkers & Diagnostics

Protein Therapeutics

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DNA

mRNA

Proteins

Cell function

Genome

Proteome

Genomics

Proteomics transcription

translation

Modification (phosphorylation, methylation, glycosylation…)

In human, 40,000 gene m-RNA(1,000,000) protein

Functional protein

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Proteome: 1994 Wilkins and Williams Proteomics:

Yates: He defined proteomics as the scientific discipline (教養) and characterizing and analysis the proteins, interaction and modification of an organism.

Gygi and Aebersold: They defined proteomics as the ability to systematicallyevery protein expressed in a

specific target

Wagner: He defined the proteomics is the entire profiled of all the proteins expressed by a specific target under strictly definedconditions at a given

time. ²²

Genomics vs Proteomics

Same within an organism Different within an organism conditionally and functionally

DNA code revealed and shared

Protein content revealed and shared

Linear information Multi dimensional information

Rich Information Content of Proteomics

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Definition of Proteomic (Depending on your asking)

Expression Proteomic

Identifying all the proteins in an organism

Functional Proteomic

Determining how those proteins join force to form network

Structural Proteomic

Outlining the precise three-dimensional structure of the proteins

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Types of Proteomics

• Protein Expression

– Quantitative study of protein expression between samples that differ by some variable

• Structural Proteomics

– Goal is to map out the 3-D structure of proteins and protein complexes

• Functional Proteomics

– To study protein-protein interaction, 3-D structures, cellular localization and PTMS in order to understand the physiological function of the whole set of

proteome.

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If we can measure gene expression, why bother with proteomics?

We can measure DNA → m-RNA → predict protein expression

= actually protein

m-RNA level:

1. Stability

2. efficiencies in translation

Protein level:

1. stability (degradation)

2. turnover (transcription factor, signal transduction, cell cycle…..) 1 gene = one protein

In fact, the definition of a gene is debatable (爭議)..(promoter, pseudogene, gene product, etc)

1 gene=how many proteins?

Why? (function)

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Co- and Post-translational modification

Proteomics and posttranslational modifications

Patterson and Aebersold, Nature Genetics (supp.), 33, 311 (2003)

protein-ligand interactions protein protein--ligandligand

interactions interactions

protein complexes (machines) protein protein complexes complexes (machines) (machines)

protein families (activity or structural)

protein families protein families (activity or structural) (activity or structural) post-translational

modified proteins postpost--translationaltranslational modified proteins modified proteins Eukaryotic cell.

Examples of protein properties are shown,

including the interaction of proteins and protein

modifications.

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Proteomic Analysis of Post-translational Modifications

Post-translational modifications (PTMs)

– Covalent

• proteolytic cleavage

• addition of a modifying group to one or more amino acids

– Determine its activity state, localization, turnover, interactions with other proteins

– Mass spectrometry and other biophysical methods can be used to determine and localize potential PTMs

• However, PTMs are still challenging aspects of

proteomics with current methodologies

29 Mann and Jensen, Nature Biotech. 21, 255 (2003)

The post-translational modifications

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Nam e Site Mo d ification m ass, D m

N-terminal acetylation Terminal NH2- Replaced by CH₃CONH- 42 N-terminal formylation Terminal NH2- Replaced by HCONH- 28 N-terminal myristylation Terminal NH2- Replaced by CH3(CH2)12CONH- 210 N-terminal palmitoylation Terminal NH2- Replaced by CH3(CH2)14CONH- 238 C-terminal amidation Terminal -COOH Replaced by -CONH₂ -1

Disulfide bonds 2 Cys -SH Replaced by -S-S- -2

Glycosylation (N-linked) N-X-S/T Glycosylation (O-linked) S/T

Sulfation -OH of Y Replaced by -OSO₃H 80

Phosphorylation -OH of Y/S/T Replaced by -OPO3H2 80

N-methylation -NH2 of K/R/H/Q Replaced by -NHCH3 14

O-methylesterification -COOH of E/D Replaced by -COOCH₃ 14

Carboxylation -NH₂ of E/D Replaced by -NHOCH₃ 30

Hydroxylation -NH₂ of P/K/D Replaced by -NHOH 16

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One functional protein = one physiological function

Other protein interaction (complex, modification) form different condition

Function A (complex; transcription factor) Function B (modification; phosphorylation) Function C

. .

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33 Difference between protein chemistry and proteomics

Protein Chemistry Proteomic Individual proteins

Complete sequence analysis Emphasis on structure and function

Complex protein (interaction) Partial sequence

Emphasis on identification by database matching System biology

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Why do proteomics?

1. It focuses on the gene product . In organism, only protein produced directly biological function, not mRNA or DNA.

2. mRNA expression analysis (array chip, south blot…) does not always reflect the expression level of protein.

3. Analysis the modification for proteins that are not actuality happen from mRNA or DNA.

4. Biological sample (such as CSF, serum, urine…) are not suitable for mRNA analysis.

5. Analysis the location of proteins.

6. Protein-protein interaction

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The specific aims of PROTEOMICS

The expression of proteins profile in specific target

The modifications of proteins

The response of different insult (diseases, drug response)

Did not predict from gene sequence, and some modification of specific protein under different stimulation/condition

Find out the protein, which may plays an important role of some physiological function

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Applications of Proteomics

Mining (礦業)

Protein-expression profiling

Protein-network mapping

Mapping of protein modifications Identifying all (possible) of the proteins in a sample DNA/gene microarrays only predict

Combine MS data base and software

Identifying of proteins in a particular samples as a function of a particular state of the organism (e.g. differentiation, development, disease)

The information has provide future study.

How the protein interact with each other in living system. The interaction include signal transduction cascades, biosynthetic or degradation

The task of identifying how and where proteins are modified Specific modifications on specific site

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New biological tool-proteomics

NCBI (美國國家醫學圖書館)MEDLINE(生物醫學資料庫), 1995, key in “proteomics” about 10

2005, key in “proteomics” = 3570 2006.10, key in “proteomics”=8960

7.8 10.8

23.8 32.8

44.8 56.8

15.8

0 10 20 30 40 50 60

1999 2000 2001 2002 2003 2004 2005

億美元

產值 蛋白質體學(Proteomics)全球產值趨勢

資料來自工研院

Proteomics related journals

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The challenge of proteomics

Enormous protein, some protein express too lowdifficult to determine.

Variation

Different condition

Data bank

30,000-40,000 gene might produce 3,000,000-40,000,000 proteins Protein expression levels are not predictable from mRNA expression levels

human and orangutan (猩猩) gene has > 99% similarity, but different protein expression result in different appearance

Proteins are uniquely modified and processed in ways which are not apparent from gene sequence

Food, living environment, drug, disease…. Induced protein change Proteome are dynamic and reflect the state of biological systems

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The landmark of proteomics

1950 Smith gel electrophoresis

1975 O’Farrel 2-D electrophorsis

1985 Karas and Hillenkamp develop MALDI; Fenn develop ESI

1990 Human Genome Project

1994 Wilkins and Williams first “proteomics”

1997 First book about “proteomics”

2003 Human genome ok

1990 美國能源部(department of energy)與國家衛生研究院 (National Institutes of Health) human genome project

3,000,000,000 (30億) nucleotide of DNA for completely sequence

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Application of proteomics

1.新藥開發

2.疾病診斷

3.儀器及設備提供

4.技術平台發展

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Proteomics (summary)

Annotation of genomes, i.e. functional annotation – Genome + proteome = annotation (注解) Protein Function

Protein Post-Translational Modification

Protein Localization and Compartmentalization Protein-Protein Interactions

Protein Expression Studies

– Differential gene expression is not the answer

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Protein Production Pathway

mRNA level ≠ expressed protein level nor does it indicate the nature of the functional protein product

Genomic

Sequence mRNA Protein

Product

Functional Protein Product

Transcriptional Regulation

Translational Regulation

Post-Translational Regulation

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Traditional RNA analysis technique : Northern blotting

1. Estimated time to get results: 2-3days 2. Expressed Gene (mRNA) checked: 1-8 species 3. Accuracy: Low to moderate

Electrophoresis Blotting

Probing

Developing Labelling

Labelling R

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New RNA analysis technique : Micro-array (

)

Labelling on sample mRNA as probe cDNA or oligonucleotide spotted on chips

1. Estimated time to get results: 5-7 days 2. Expressed Gene (mRNA) checked: thousands 3. Accuracy: moderate to high

data analysis

Clustered genes

Clustered experiments (High

(High--throughput)throughput)

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Alizadeh et al.

Nature 403 (2000) 503-511

microarray

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Microarray revolutionized biology and medicine research

• One gene at a time before, now tens of thousands simultaneously - PROTEOMICS

• Gene expression

• Gene disease relation

• Gene-gene interaction

• Finding Co-Regulated Genes

• Understanding Gene Regulatory Networks

• Many, many more

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cDNA and Oligonucleotide Microarrays

Type of Microarrays

1. cDNA arrays (Microspotting)

2. Oligonucleotide arrays (Photolithographic synthesis, Ink- jet technology, etc.)

Applications

Gene Expression SNPs

Mutations

Deletions and insertions

Genotyping

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Basic idea of Microarray

• 製造原理

– 將可特徵基因之對偶鹼基序列 – 稱為探針

– 排列放置在微晶片(microchip) 上

• 應用原理

– 將含基因序列之樣品 (sample) 液體到在微 晶片上

– 利用互補鹼基雜交作用(hybridization) 的

原理，由 樣品 與微晶片上基因序列相互

作用的情形摘取所需的資訊

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Basic idea of Microarray

• Construction

– Place array of probes on microchip

• Probe (for example) is oligonucleotide ~25 bases long that characterizes gene or genome

• Each probe has many, many clones

• Chip is about 2cm by 2cm

• Application principle

– Put (liquid)

containing genes on microarray and allow probe and gene

sequences to hybridize and wash away the rest

– Analyze hybridization pattern

 Fabrication via Printing

 DNA sequence stuck to glass substrate

 DNA solution pre- synthesized in the lab

 Fabrication In Situ

 Sequence “built”

 Photolithographic techniques use light to release capping chemicals

 365 nm light allows 20-μm resolution

Fabrication

Fabrication

53 O O O O O

Light (deprotection)

HO HO O O O T T O O O

T T C C O Light

(deprotection)

T T O O O

C A T A T A G C T G T T C C G Mask

Mask

Substrate Substrate

MaskMask

Substrate Substrate

T –T –

C –C –

REPEAT REPEAT

On chip photolithographic synthesis of oligonucleotides

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cDNA microarray schema

cDNA晶片製造原理

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GeneChip for gene expression profile

Specimens Bioinformatics

Labeled Targets

0 0 0

0

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cDNA

Labeled Targets (cDNA or cRNA) mRNA

0 0 0 0

AAAA

Labeled Fragments

0 0

0 0

Specimens

Target Preparation

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Rat Genome Chip

8799 Probe Sets

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Rat-Tox Chip

1031 Probe Sets

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Control Test

RT &

Labeled with Fluor Dyes

cDNA Microarrays

scanning Cy3 Cy5

Bioinformatics

2-Dye Technology of cDNA Microarrays

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Microarray analysis

Operation Principle:

Samples are tagged with flourescent material to show pattern of sample-probe interaction (hybridization) Microarray may have 60K probe

Compare with two sample gene expression profile

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Microarray Processing sequence

From: Shin-Mu Tseng tsengsm@mail.ncku.edu.tw

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63 http://www.bio.davidson.edu/courses/genomics/chip/chip.html

Demonstration

64 One gene 1 protein

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Traditional Protein technique:

peptide sequencing

1. Protein purification: necessary 2. Protein idetified: 1 per purified sample

Cut desired band

Peptide N terminal sequencing Edman degradation

Database searching for homolog

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Protein analysis technique : Western blotting

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Protein analysis technique : Western blotting

2.14 1.12 0.91 1 1 1.98 2.2 2.0 3.14 3.98 0.95

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New protein analysis technique : Protein-array

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New protein analysis technique : Protein-array

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Applications of proteomics to Medicine

• Detection of disease markers in body fluids

• Pharmaceutical studies

• Toxicological studies

• Glycosylation and phosphorylation of proteins and hormons

• Tissue analysis for cancer research

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DNA RNA Protein Cell animal

Sequencing microarray proteomic Cellular assay transgenic

Lab

Disease model Clinical diagnosis

SNP Gene therapy

Clinical diagnosis Recombine DNA Protein expression

Functional assay

Diagnosis (antibody)

Disease therapy Structure modeling for compound screening

industrial circles

screening

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Virtual Screening Virtual

Screening High- Throughput

Drug Screening

Proteomic Proteomic

Clinical Trial

Protein Drug

Vaccine Disease Pathway

Disease Pathway

提供藥靶 (Target) (Target)

Protein Array Chemical Array

Proteomic在新藥開發過程中的角色 Proteomic

Proteomic在新藥開發過程中的角色 在新藥開發過程中的角色

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From proteomics to medicine

Identification of disease marker

Diagnosis, Patient profiling

Identification of drug target

Discovery, validation, clinical trials, patient monitoring

Identification of the interaction between protein- protein and protein-drug

Side effect predict, potency predict..

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Drug discovery procession

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The essential tools of proteomics

Protein separation or collection Data base

Mass spectrometry (MS) Software (for MS data base)

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• Sample generation

• Sample processing

– Gels (1D/2D), columns, other methods

• Mass Spectrometry

– Spectra, machine and componentry types, parameter, processing methods

• In Silico analysis

– Database name + version, partial sequence, search parameters, search hits, accession numbers

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蛋白質體學樣品分析流程

架構:工研院

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Proteomic study step

Step 1: Sample Preparation Step 2: Isoelectric Focusing

Step 3: SDS Polyacrylamide Gel Electrophoresis Step 4: Staining of the Gels

Step 5: Scanning of Gels and Image Analysis Step 6: 2D DIGE or 2D electrophoresis Step 7: Spot Excision

Step 8: Sample Destaining Step 9: In-gel Digestion Step 10: Microscale Purification

Step 11: Chemical Derivatisation of the Peptide Digest Step 12: MS Analysis

Step 13: Calibration of the MALDI-ToF MS Step 14: Preparing for a Database Search Step 15: PMF Database Search Unsuccessful

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The front end of an exciting system

PDQUEST

SAMPLE PREP SAMPLE PREP

MASS SPEC MALDI-TOF MASS SPEC MALDI-TOF SWISS-PROT,

TrEMBL, etc.

HTML Links

MS/MS ESI-MS IMAGING

SPOT CUTTER

ProteinLynx PepSeq

PROTEIN DIGEST STATION PROTEIN DIGEST STATION 2-D GELS

OR BLOTS

WorksBase WorksBase

84 Characterization

Automated Characterization Protein Isolation

Identification of 2-D Separated Proteins

Biological Material

tissue cells body fluids

2-D gel protein spot 2-D gel spot immobilized

on a membrane

de novo sequence sequence

fractionated

peptide peptide mass

fingerprint peptide mix

N-terminal sequence

amino acid composition Amino acid

analysis Edman

degradation blotting

excision Biological

pre-fractionation

Biochemical pre-fractionation

HPLC

Edman

degradationESI MS/MS carboxy- database search

peptidase ESI-MS/MS

MALDI-MS

sequence tag

protein ID digestion

detection imaging

excision analysis software

ProteomeWorks Spot Cutter

PDQuest