Introduction

1. The Plant Proteomics- the post-genomic eraThe systematic analysis and documentation of all protein species of an organism or a specific type of tissue (Wasinger et al., 1995)

2. Introduction • With the completion of the Arabidopsis genome sequence for this model plant. The consequent application of the high-throughput technologies will lead to dramatic increase in knowledge about complex biological network. • Sequence information in itself is not sufficient to provide significant knowledge of the biology of the organisms. It rather provides a sound basis and framework for further investigations.

3. Biology: depending on selective readout of individual genes from genome- converting into primary transcripts- processed into mRNA-- translated into a protein sequence- post-translational protein modifications (glycosylation and phosphorylation)-- protein cleavage and multi-protein complex formation. All these processes influence the function of protein.

5. Only some of the sequenced genes can be assigned a function with certainty. The approaching the world of protein is more diverse and complex than the genomic repertoire. • Proteomics addresses analytical questions about the abundance and distribution of proteins in the organism, the expression profiles of different tissues and the identification and localization of individual proteins of interest. Furthermore, to elucidate the interactions between proteins and other molecules, and may reveal the functional role of proteins. • The emerging methods from proteomics include: 2-DE and MS methods, protein array, the use of antibody and antibody array, the yeast two hybrid system, etc….

6. The classical method of gene expression studies in term of proteomics is two-dimensional gel electrophoresis (2DE). 2-DE---mass spectrometry (MS) to identify protein ----to generate catalogues of expressed proteins in a cell or tissue of interest. • Faults (limitation): sensitivity of the protein complement to extract preparation, running conditions and gel composition, protein separated by 2-DE are obtained in denatured form and in limited amounts—functional characterization is not possible.

7. A highly parallel approach to classical proteomics is developed as protein array technology in recent years—high-throughput approaches in th efield of recombinant expression (concept of the arrayed cDNA expression libraries, large-scale open reading frame cloning, and others) and of protein purification. The protein array concept enables the connection of recombinant proteins to clones identified by DNA hybridization or sequencing and hence a direct link between the gene catalogue and a functional catalogue is created.

8. In summary, Proteomics is a systematic molecular evaluation of the complete genetic information of plants and the resulting cellular activities from transcription, protein expression and protein-protein interaction. This will be essential for future developments of both the scientific understanding of plant biology and the commercial aspects, such as breeding and agricultural application.

9. By building databases (http://www.mips.de) the genomics data can be interrelated with the emerging proteomic and metabolic data as well as with environmental information. • Two-dimensional gel electrophoresis (2DE) • Protein arrays • Antibodies and screening procedures • In vivo protein interactions: yeast two-hybrid system

10. Two-dimensional gel electrophoresis • n classical proteomics, the chief strategy is to identify as many proteins as possible from different organism to achieve different proteome states.--- to generate large databases of expressed genes. • Two analytical techniques are primarily employed in current proteomic research: two-dimensional (2D) gel electrophoresis for the separation and visualization of protein in crude extracts; Mass spectrometry for the identification and characterization of the separated protein.

11. Two-dimensional gel electrophoresis • 2-DE is based on isoelectric focusing (IEF), by which the proteins are separated according to their pI in pH gradient polyacrylamide gel (first dimension) and SDS-PAGE, by which the proteins are separated according to their molecular weights (second dimension). The large gel (46cm x 30cm) 2-DE technique developed by Klose affords separation and visualization of more than 10000 different protein species from animal tissue in a single experiment (Klose, 1975, 1999).

12. Two-dimensional gel electrophoresis • Visualization of the separated proteins is achieved by different staining techniques. Color density and spot size of the detected spots enable protein quantification. The accuracy is limited due to the low dynamic range of the most staining techniques. The fluorescent dyes for proteins (Patton,2000) may overcome this limitation.

13. Two-dimensional gel electrophoresis • The identification of the large numbers of proteins separated by2DE is most commonly achieved by automated matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI TOF-MS) peptide mapping followed byextensive database searches (Henzel et al., 1993).

14. Two-dimensional gel electrophoresis • Many descriptions of proteomic status of different plant tissues were possible in Arabidopsis: Kamo et al., 1995; Tsugita et al., 1996; Gallardo et al., 2001. Rice : Tsugita et al., 1994; Komatsu et al., 1999; Rakwal and Komatsu, 2000. Maize : Chang et al., 2000. The number of spots resolved in plant proteomic 2D projects so far ranges depending on the chosen tissue and plant species between a few hundred and somewhat below 2000 spots. The resolution has not been improved in plant tissue very much in the past few years due to the fact, that plant tissue is low in protein and there are many compounds from the secondary metabolism which negatively affecet protein extraction.

15. Two-dimensional gel electrophoresis • In addition to holistic analyses of proteomes from different plant species, some research groups concentrate on the identification of proteomes from different subcellular compartments, such as membranes or organelles. The description of the proteomic status was possible in chloroplasts of pea (Peltier et al., 2000; Van Wijk, 2000). • The changes in the analytical aims: Comparing different genotypes and plant lines to measure phylogenetic distance (Zivy et al., 1984….). Later projects made use of N-terminal Edman protein sequence or used amino acid analysis for protein determination. Nowadays, with mass spectrometric methods the identification of proteins goes hand in hand with their separation (Porubleva et al., 2001).

16. Two-dimensional gel electrophoresis • Furthermore, other projects focused on comparison of different mutant lines with wild type plants, in order to obtain functional protein profiles followed by identification of differentially expressed genes.

17. Protein arrays • Recently, protein arrays are emerging as a new tool to profile and functionally characterize recombinant proteins encoded by globally or differentially expressed cDNA clones (reviewed in Walter et al., 2000).

18. Protein arrays • This technology presupposes the cloning of a large number of cDNA clones in an appropriate expression vector by generation of cDNA expression libraries or by the high-throughput sub-cloning of open reading frames (ORF). The latter approach depends on the availability of sequenced genomes and is limited, if the expressed sequence cannot be determined from the genome sequence due to differential splicing and post-translational processing.

19. Protein arrays • Several cDNA expression libraries from different plant tissues were constructed and used for immunoscreening. • The most common application of protein arrays is the detection of immobilized antigens with antibodies.

20. Antibodies and screening procedures • Antibodies are important tools for the functional characterizationof plant systems. Rather than the very costly and time-consuming hybridoma technology, they can now be produced without immunization and without the use of animals by using recombinant immunoglobulin gene libraries cloned in phage or phagemid vectors as an in vitro simulation of immune system.

21. Antibodies and screening procedures • By a combination of phage display and magnetic bead technology, antibody phage display libraries can screened against expression products of plant cDNA libraries. • Phage display selection against arrayed plant proteins may provide new plant-specific antibodies in vitro. Western blotting and immunohistochemistry using these antibodies could then applied to detect and localize proteins of interest in plant cells and tissues.

22. Antibodies and screening procedures • Antibody arrays could provide powerful future tools for th eidentification of differentially expressed plant proteins. In addition, diagnostic arrays for the detection of several plant pathogens can be envisaged.

23. In vivo protein interactions: yeast two-hybrid system • The development of the yeast two-hybrid (Y2H) system was a decisive step towards the convenient identification of such protein-protein interactions.

24. In vivo protein interactions: yeast two-hybrid system • This genetic procedure allows the rapid identification of in vivoprotein-protein interactions and the simple isolation of corresponding nuclei acid sequences encoding the interacting partners. In a typical Y2H screen, a hybrid protein sequence consisting of a DNA-binding domain (DBD) and a protein of interest (bait) is assayed against a (prey) library of proteins expressed as fusions with a transcription factor leads to the activation of a reporter system.

25. In vivo protein interactions: yeast two-hybrid system • Yeast colony arrays and automation were employed to identify systematically protein-protein interactios of all 6000 open reading frames in Saccharomyces cerevisiae and establish a single large network of 2358 interactions among 1548 protein (Schwikowski et al., 2000).

26. The Principles of Analytical Proteomics • 1.Overview of analytical proteomics

28. Extracting proteins from biological samples Extraction buffer-- generally done with the aid of:  Detergents- SDS, CHAPS,cholate,Tween, which help to solubilize membrane proteins and aid their separation from lipid.  Reductants- DTT, mercaptoethanol, thiourea, which reduce disulfide bonds or prevent protein oxidation.  Denaturing agents- urea and acids, which disrupt protein-protein interactions, secondary and tertiary structures by altering solution ionic strength and pH.  Enzymes- DNase and RNase, which digest contaminating nucleic acids, carbohydrates, and lipids

29. Protein separations before digestion • The analytical proteins are separated before they are digested. • The three principal separation approaches used with intact proteins are 1D and 2D-SDS-PAGE and preparative isoelectric focusing (IEF). The mixture may be separated into a relatively small number of fractions (as in 1D-SDS-PAGE and preparative IEF) or into many fractions (as in the many spots in 2D-SDS-PAGE). The fractions then are taken for proteolytic digestion.

30. One-dimensional SDS-PAGE In 1D-SDS-PAGE, the protein sample is dissolved in a loading buffer that usually contains a thiol reductant (mercaptoethanol or DTT) and SDS. The binding of SDS to the protein is in roughly constant proportion to molecular weight. The proteins thus are resolved into bands in order of molecular weight.

32. Two-dimensional SDS-PAGE This is actually a combination of two different types of separations. In the first, the proteins are resolved on the basis of isoelectric point by IEF. In the second, focused proteins then are further resolved by electrophoresis on a polyacrylamide ( by molecular weight).

34. Problems with 2D-SDS-PAGE Despite the superiority of 2D-SDS-PAGE over other methods as a means of resolving complex protein mixtures, the technique presents some problems. 1). the difficulty of performing completely reproducible 2D-SDS-PAGE analysis. It becomes important when one wishes to use 2D-SDS-PAGE to compare two samples by comparing the images of the stained gels. Differences in protein migration in either dimension could be mistaken for differences in levels of certain proteins between two samples. 2). The relative incompatibility of some proteins with the first-dimension IEF step. Many large, hydrophobic proteins simply do not behave well in this type of analysis. It leads to protein precipitation and aggregation, and lead to “smearing” of protein within the IPG strip.

36. The relatively small dynamic range of protein staining as a detection technique. Spot densities reflect about a 100-fold range of protein concentrations, at best. This means that staining of 2D-gels allows the visualization of abundant proteins, whereas less abundant proteins frequently cannot be detected.

39. A generic hybrid approach for 2D proteomics

40. Preparative IEF  This technique is analogous to the first step in 2D-SDS-PAGE. In preparative IEF, the separation is carried out on an IPG strip, in a tube gel, or in solution. In commercially available apparatus, such as the BioRad Rotofor cell, the focusing cell is divided by permeable membranes into a series of chambers.

41. Preparative 1D-SDS-PAGE/ or HPLC

42. Enzymatic digestion and subject to HPLC separations • Enzymatic digestion and subject to HPLC separations For MS analysis, the greater the mass of the protein, the greater the absolute magnitude of the error. This is the reason why anslysis of peptides, rather than proteins, is the approach of choice. The ideal protein digestion approach would cleave protein at certain specific amino acid residues to yield fragments that are most compatible with MS analysis. Specifically, peptide fragments of between about 6-20 amino acids are ideal for MS analysis and database comparisons.

43. c-1. What the proteases are really needed for analytical proteomics are stable, well-characterized enzymes with well-defined specificities. These enzymes must be available in quantity and high purity and must be robust enough for application in a variety of circumstances. Table 1 summarizes the proteases that are most widely used in proteomic analysis and their cleavage characteristics.

45. *trypsin is widely used protease in proteomic analysis. It cleaves proteins at lysine and arginine residues, unless either of these is followed by a proline residue in the C-terminal direction. As a general rule, a 50kDa protein will yield about 30 tryptic peptides. *Glu-C (V8-protease) is an endoproteinase that cleaves at the carboxyl side of glutamate in either ammonium bicarbonate buffer. In a sodium phosphate buffer, however, the enzyme cleaves at both glutamate and aspartate residues. *nonspecific proteases- such as subtilysin, pepsin, proteinase K, and pronase. These enzymes cleave proteins more or less randomly to produce multiple overlapping peptides. *cyanogens bromide is the most widely used of chemical to cleave proteins. It cleaves proteins at methionine residues.

46. In-gel digestions • A commonly used approach to digestion of proteins separated by 1D- or 2D-SDS-PAGE is referred to as “in-gel” digestion. The band or spot of interest is cut from the gel, destained, and then treated with a protease (most commonly trypsin). The enzyme penetrates the gel matrix and digests the protein to peptides, which then are eluated from the gel by washing. This is an indispensable element to 2D-SDS-PAGE proteomics strategy

48. Mass Spectrometers for Protein and Peptide Analysis • Two different types of instruments are used for most proteomics MS work: • the MALDI-TOF instruments and the ESI-tandem MS instrument.

49. How MS instruments work • Mass spectrometers have three essential parts: 1). Source: producing ions from the sample. 2). Mass analyzer: resolving ions basing on their mass/charge (m/z) ratio. 3). Detector: detecting the ions resolved by the mass analyzer.

50. In short, the mass spectrometer converts components of a mixture to ions and then analyzes • them on the basis of their m/z. The data are automatically recorded by the data system and can then be retrieved for manual or computer-assisted interpretation. • *MALDI-matrix-assisted laser desorption ionization, referring to the source, a method of ionization. • *TOF- time of flight, referring to the mass analyzer.