Http://creativecommons.org/licens es/by-sa/2.0/. Large Scale Approaches to the Study of Protein Levels and Activity Prof:Rui Alves [email protected] 973702406.

http://creativecommons.org/licenses/by-sa/2.0/

Large Scale Approaches to the Study of Protein Levels and Activity

Prof:Rui [email protected]

973702406Dept Ciencies Mediques Basiques,

1st Floor, Room 1.08Website of the

Course:http://web.udl.es/usuaris/pg193845/Courses/Bioinformatics_2007/ Course: http://10.100.14.36/Student_Server/

The proteome

• The proteome– Protein complement of a genome

• Variable– In different cell and tissue types in same organism

– In different growth and developmental stages of organism

• Dynamic– Depends on response of genome to environmental factors

» Disease state

» Drug challenge

» Growth conditions

» Stress

Why Studying Proteins Directly?

•Just because the gene is regulated and expresses, is it translated?

•REMEMBER small RNA regulation and Smolcke’s talk!!

•If so, under what circumstances ?

•Are there qualitative and quantitative aspects to the regulation of the translation under different conditions?

Proteome Wide Studies

• With fully sequenced genomes available one can finally study how gene expression is regulated in the whole genome simultaneously

•The same has become true of the (almost) entire protein complement of a cell:

•PROTEOMICS

What can we study with proteomics?

• Regulation of Translation– Comparing Protein Levels to corresponding gene

levels allows study of how translation is regulated

• Regulation of Protein Modification (activity)– E.g. comparing phosphorylation state of proteins

• Protein Interaction Networks– E.g. finding out how proteins cooperate to achieve

an effect

• Protein-DNA Interaction Networks• ChIP-chip or PBM arrays

How Can We Measure Protein Levels?

• Core technology– 2D-PAGE

• High resolution protein separation and display

– Mass spectrometry (NMR/IRS)• Protein identification

2D-PAGE

Cell Cell

Protein solubilization

Separation by size/charge (IEF)

Iso-Electric Focusing

PAGE

Staining

• Staining– Silver– Coomassie blue– Fluorescent dyes

• Sypro Ruby

– Radioisotopic labelling

Creation of Master Gel Image

•Gel under basal conditions

•Compare Gels generated under other conditions to Master Gel and determine what has changed•http://www.expasy.org/tools/

Identification of Spots

•At this stage, accumulated images of gels in which spots have been identified and/or knowledge of isoelectric point/size can help identify proteins.•http://www.expasy.org/tools/

•What about unidentifed/confusing spots

Mass SpectroscopyExtraction/Proteolysis

• MALDI/TOF-MS– Matrix-Assisted– Laser Desorption /

Ionisation– Time-Of-Flight

analysis

Mass Spectrometry (MS)• Introduce sample to the instrument• Generate ions in the gas phase• Separate ions on the basis of differences in

m(ass)/z(charge) with a mass analyzer • Detect ions

Mass Spectrometry (MS)

Identifying The Spectra

ACTGHRSKAASKAASRLLMN…

Trypsin

ACTGHRS

KAAS

KAAS

RLLMN…

…

Time

m/z

•From Protein sequence/other knowledge we can know how a given protein is to be hydrolized

•From knowledge of the smaller peptides we can calculate their predicted mass/charge ratio and their migration time

•Comparing the real spectra to theoretical/pre-existing spectra can allow for the identification of proteins

•Spectrum comparison can be made using for example Fourier Transforms

•NMR and other forms of spectroscopy can also be used

Algorithmic approaches to “tag” identification

• Cross Correlation (Eng et al. - SEQUEST): comparison between observed and theoretically generated spectra.

• Peptide sequence tags (Mann): extract an unambiguous sequence tag for ID.

• De novo probability-based matching (Perkins, and the proprietary Mascot by Matrix Science): takes statistical significance of fragmentation into account.

Sequence tagging Algorithms

De novo algorithms

Advantages vs. Disadvantages of proteomics

• Determination of MW and aa. Sequence

• Detection of posttranslational modifications

• High-throughput capability

• High capital costs

• Requires sequence databases for accurate analysis

• De novo methods not very mature yet

Other Proteomic Tools FYR

Array-based Proteomics

• Employ two-hybrid assays• Use GFP, FRET, and GST

– GFP = green florescent protein– FRET = florescence resonance energy

transfer– GST = glutathione S-transferase, a well

characterized protein used as a marker protein.

Two-Hybrid Assay

Figure 12-35. Griffiths et. al. Modern Genetic Analysis.

Array-based Proteomics

Phosphoproteomics

Array-based Proteomics

• Offer a high-throughput technique for proteome analysis.

• These small plates are able to hold many different samples at a time.

• Current research is ongoing in an attempt to interface array methodologies with Mass Spectrometry.

Structural Proteomics

• Pioneering work is undergoing by Baumeister et al, which can significantly reduce the amount of painstaking labor in the crystallization of proteins.

• Current techniques are not considered “high throughput” within the structural realm.

• Novel solutions combine current technologies, such as NMR and XRC.

Conclusions

• Proteomics– Enables global screening of complex

samples– Provides qualitative / quantitative evidence

for changes in protein expression in different biological situations

– Identifies targets for further investigation / validation

Proteomics websites