Proteomes and proteomics : “P” words in the world of Functional Genomics

 

Introduction

Gene ,Genomes and Gene Products

Nowadays , The genome content are available for many organism . Whereas a genome sequence does not tell the function of the organism but study of protein is essential for show this .

 

Proteome

Definition : Protein complement  expressed by a genome or tissue.

Proteomes are dynamic but  genome is slow evolution, proteomes change as a function of time, development, extracellular conditions and intracellular conditions.

 

Proteomics

Definition : proteomics is the study of proteomes

Proteomics aims to separate, identify and characterise proteins on a large scale,define levels of protein complexes, elucidate protein functions, pathways, and interrelationships.

 

The method to separate proteins

 

Traditional protein chemistry

- column chromatography  is an old methods, separate one proteins at a time and take time for many weeks to months.

- Two dimensional gel electrophoresis .It take an image as a reference map for cell, tissue or protein complex and purify protiens up to thousands at once .The principle of  this method are the first dimension for chage-based separation and the second dimension for mass based separation. And it take a shotter time  than traditional method in only one to three days.

 

The method to identification and characterization proteins

Previous methods eg. Edmab sequencing, antibodies, amino acid composition, co-migration.

Nowadays proteins are identified using mass spectrometry : peptide mass fingerprinting and peptide fragmentation

 

Mass spectrometry

Mass spectrometry precisely measure the mass of molecules.

It have two parts

1. ion source eg. Electrospray ionization (ESI), matrix assisted laser desorption/ionization (MALDI) 

2. measuring apparatus eg. Quadrupoles , time of flight (TOF) detectos, ion traps, combimations of above method.

Common platforms eg, MALDI-TOF, ESI-TOF, ESI/ion trap, (triple) quadrupole TOF

 

 

 

Peptide mass fingerprinting

Principle

 

 

 

-         Protein form 2-D gel  was cut and put in tube.

-         Digested by enzyme eg trypsin, then several unmodified and modified peptides in tube were feed into mass spectrometry and analysis  by matching masses against theoretically  by a computer

 

Automation of protein identification

High – throughput labs now use automated: spot excision, trypsin digestion, MALDI target loading, spectra adquisition, calibration, peak picking and database matching

 

Automated mass spectrometry

Proteomics requires automated such as Spectra acquisition, calibration

Diconvolution, peak picking , data export

Now we have an automate spectrometry offer.

1. MALDI – TOF MS  

- high thoughput (500 per day)

- high degree of automation

2. ESI-TOF machines currently

    Slower and less automated

 

 

 

 

 

 

 

 

 

 

 

 

 

Diagram of Pipeline tool automate analyses below

 

 

 

 

Study protein modification

Because of one gene can give many proteins

 

 

Isoforms on gels are due to protein processing or modifications. Modifications changes protein and peptide mass eg. Methylation , some modification change protein charge and mass eg. Phosphorylation makes protein more acidic.

Many modification can be studied with mass spectrometry. Sometimes, this can be done with peptide mass fingerprinting. But usually this requires fragmenting peptides using MS-MS.

 

 

 

 

 

 

 

Peptide fragmentation and characterization by  MS-MS

Principle

 

      - Protein form 2-D gel  was cut and put in tube.

- Digested by enzyme eg trypsin and take through MS

- Then select peptides to fragment in MS.

- Analyse fragment peptides by MS-MS and  matching  peptides to predictions from tools or databases.

 

Conclusion

Proteomics will

- Define the proteome of a cell or tissue

- Provide means of comparing proteomes to explain phenotypes (eg. Disease vs normal states )

- Provide clues to protein function by difining co-stimulated ans co-regulated proteins

- Be powerful in combination with other technologies such as two- hybrid function assays and gene knockout

- Proteomics will not:

- Replace genome sequencing

- Be as easy as genome sequencing