Novel approaches to protein expression analysis

1. Chapter 10 Novel approaches to protein expression analysis presented by shin jin a

2. Contents Introduction The scope of functional proteomics Proteome analysis: the 2-DE based strategy Alternatives to 2-DE for protein expression analysis separation-dependent methods separation-independent methods: towards ‘protein chips’? Conclusions

3. Focus of this chapter • Introduction to some of the technical limitations of the major current approach to protein expression analysis • Describing alternative methods that are actively under investigation • Discussing potential novel approaches for protein expression profiling

4. Introduction - Genome sequencing projects  new approaches to mRNA expression analysis and techniques to analyze protein-protein interaction using the two-hybrid assay - Recent advances in microarray technology  increase speed at which sequence information and differential gene expression data may be gathered  At present, these functional genomics require the direct analysis of the products encoded by the genes and mRNAs – the protein (Functional proteomics)

5. The scope of functional proteomics Functional proteomics: global analysis of protein expression (e.g. analysis of differential protein expression in disease states, knockout of individual genes, drug treatments and changing extracellular conditions) - analysis on the level of protein expression - analysis on the post-translational modifications to which the protein may be subjected • Aanalysis of protein function requires that each of these be examined and the data amalgamated

6. Protein expression analysis and protein characterization by 2-DE 2-DE for protein separation Protein detection Protein identification Matching – pI, Mr, peptide mass fingerprinting, amino acid composition, sequence tag De novo – Tandem MS, microsequencing 2-D maps and databases

7. Technical limitations of 2-DE as a platform technology for protein expression analysis Reproducibility Sensitivity Range of display - Number - Control - ‘Difficult’ proteins Data analysis Skill, effort and time required Lack of automation

8. Alternatives to 2-DE for protein expression analysis • - Separation-dependent methods • :simple separation • :orthogonal separation • Separation-independent methods: towards ‘protein chip’ • :protein recognition molecules • :molecularly imprinted polymers • :detection methods

9. Separation-dependent methods • 1. Simple separation • - fractionation and limited protein separation, facilitated • by the potential of MS • 1-D gel electrophoresis  proteolytic digestion  • analyze by MS • : useful for the rapid and detailed characterization of protein • complexes • (2) Ciphergen’s SELDI MS method

10. 2. Orthogonal separation • - methods combining two or more methods of • fractionation or separation • - liquid chromatography or capillary electrophoresis- • based methods are attractive : readily amenable to • automation  potential for high-throughput environment • Modified High-performance liquid chromatography (HPLC) • using capillary columns: • more miniaturized and automated, reversed phase capillary • HPLC + automatic fraction collection  MALDI-TOF-MS • Edman sequencing

11. (2) Modified IR-MALDI-MS scanning method: separation by HPLC  collect eluate onto PVDF strips  incubation with matrix solution  scanning by IR-MALDI- MS (3) Automated 2D-HPLC method that parallels 2-DE: size exclusion chromatography  automatic separation by reversed-phase liquid chromatography  generation of 2D-chromatogram advantages: high reproducibility of separation, easy automation of sample loading, running and fraction collecting, keeping proteins in solution which make subsequence analysis by MS very straightforward

12. (4) Capillary electrophoresis(CE): extremely effective method of analyzing clinical protein samples (rapid, highly reproducible and don’t require large sample vol. for high-sensitivity detection) - Miniaturization of CE through the use of chips: scale down the process of CE by generating a chip with channel etched into it

13. Separation-independent methods: towards ‘protein chip’? - Alternative way: using the power of ‘Molecular Recognition’ (using the specific recognition between two molecules to isolate, detect and /or identify the target molecule)  “Protein chip” (Separation-dependent techniques: still require large quantities of protein sample, require more than one step, operate in sequential process)

14. - Protein chip: protein version of DNA chip • Key components of protein chip: spatially addressed • molecules recognizing individual protein moieties, • method to detect the interaction of individual proteins • with their recognition molecules

15. - Difficulties in making protein chip mimic DNA chip • ① Fluorescent labeling of proteins has several inherent • difficulties in contrast to that of cDNAs • ② Incorporation of fluorescent moieties into proteins • has unpredictable effect on the hybridization • reactions, which is not the case for nucleic acids • New approaches to generate the recognition molecule and detection method for interaction of proteins with the recognition molecule

16. 1. Protein recognition molecules • - Most obvious molecule for the recognition molecule: • Antibody • - Criteria to use antibody for protein chips: • generation and selection of antibodies to individual proteins • and each of their post-translationally modified forms • ② high specificity • binding of the individual proteins must occur under similar • conditions • unlikely to support development of genome-scale protein chips

17. - Disadvantages to the use of antibody as protein recognition molecule: poor storage property, degradability by any contaminating protease activity - Alternatives for recognition molecules: any group of molecules that can specifically recognize individual proteins w/ appropriate affinity and avidity in immobilized form ① Large libraries of compounds generated by combinatorial chemistry ② Libraries of aptamers: single-stranded oligonucleotides possessing high affinity for conformational biomolecules such as proteins (a few hundred nucleotides in length)

18. 2. Molecularly imprinted polymers - Molecular imprinting: synthesis of artificial recognition sites on a surface by mimicking the shape of the template molecule(protein) in a polymeric film, forming a molecularly imprinted polymer (MIP) - Two methods constructing MIP: ①mixing template molecule with polymer reagents  allowing the matrix to harden  removing the template with a specific solvent ②coating protein onto the mica  cover with disaccharide layer that mimics surface conformation of the protein  remove mica and protein  generate sugar-coated imprint

19. 3. Detection methods - Fluorescence-based methods: ① fiber-optic biosensor: coat capture antibody onto a fiber-optic probe  apply a detection antibody labeled with cyanine dye  laser excitation of the fiber-optic probe to enable quantification ② saturate antibody-coated matrix with fluorescently labeled target antigen  displace target antigen by unlabeled target present in the sample  measure released fluorescent material

20. - Non-fluorescence-based Biosensors: comprises a glass surface onto which a thin layer of metal(usually gold) covered by a matrix, binding of target protein is detected by changes in the refractive index of the matrix ① Quartz crystal microbalance(QCM): bind antibodies to quartz crystals  binding of the target antigen results in small changes in mass on the surface of crystal  changes are measure by detecting changes in resonance frequency via two gold electrodes

21. Conclusions - We have reviewed some of these developments and, in doing so, it has become evident that any future technology for high-throughput protein expression analysis will almost certainly require a multidisciplinary approach and the further development of novel methods • The rapidly growing academic and commercial interest in proteomics seems likely to catalyze such developments