CHAPTER 1 INTRODUCTION

1.1 OVERVIEW Proteomics is something new in the field of biotechnology. It is basically the study of the proteome, the collective body of proteins made a persons cells and tissues.

Since it is proteins, and to a much lesser extent, other types of biological molecules that are directly involved in both normal and disease-associated biochemical processes, a more complete understanding of the disease may be gained by directly looking at the proteins present within a diseased cell or tissue and this is achieved through the study of the proteome, Proteomics. For, Proteomics, we need 2-D electrophoresis equipment ot separate the proteins, mass spectrometry to identify them and x-ray crystallography to know more of the structure and function of the proteins. These equipments are essential in the study of proteomics. Genomics has provided a vast amount of information linking gene activity with disease. It is now recognized that gene sequence information and pattern of gene activity in a cell do not provide a complete and accurate profile of a abundance or its final structure and stateo factivity. The day of spotlight of the human genome is now coming to an end. Researchers are now concentrating on the human proteome, the collective body of all the proteins made by a cells and tissues. The genome- the full set of information in the body-contains only the recipes for making proteins; it is the proteins that constitute the bricks and mortar of cells and that do most of the work. Moreover it is the proteins that distinguish the various types of cells: although all cells have essentially the same genome, they can vary in which genes are active and thus in which proteins are made. Likewise diseased cells often produce proteins that healthy cells dont and vice versa. Proteome research permits the discovery of new protein markers for diagnostic purposes and of novel molecular targets for drug discovery. All living things contain proteins. The structure of a cell is largely built of proteins. Proteins are complex, threedimensional substances composed of one or more long, folded polypeptide chains. These chains, in turn, consist of small chemical units called amino acids. There are twenty kinds of amino acids involved in protein production, and any number of them may be linked in any

order to form the polypeptide chain. The order of the amino acids in the polypeptide chain is decided by the information contained in DNA structure of the genes. Following this translation, most proteins are chemically changed through post-translation modification (PTM), mainly through the addition of carbohydrate and phosphate groups. Such modification plays an important role in modulating the function of many proteins but the genes do not code it. As a consequence, the information from a single gene can encode as many as fifty different protein species. .

1.2 PROTEOMICS:A DESCRIPTION OF METHODLOGY The exact definition of proteomics varies depending on whom you ask, but most of the scientists agree that it can be broken into three main activities: identifying all the proteins made in a given cell, tissue or organism; determining how these proteins join forces to form networks akin to electrical circuits; and outlining the precise three-dimensional structure of the proteins in an effort to find their heels-that is, where drugs might turn their activity on or off. Though the task seems straightforward, it is not as simple as it seems. The critical pathway of

proteome research includes: Sample collection Protein separation. Protein identification Protein characterization Bioinformatics These are the major steps involved in the proteome science studies. Each of these are described in detail. sample collection, handling and storage. Access to relevant body fluid and tissue samples is fundamental to proteome research. Proteome sciences has collected a large bank of clinical samples for its cancer, neurological disease, cardiovascular disease, transplant rejection and diabetes research areas through its ongoing access to the leading hospitals where its collaborative scientists practice. All clinical samples are pathology-authenticated and are accompanied by details medical records to allow the correlation of proteome changes with disease pathology protein separation .The sample we have collected will have many proteins included in it. We need to separate these in order to study them. One of the main technologies used is two-dimensional gel electrophoresis. Electrophoresis is a technique used in laboratory that results in separation of charged particles and proteins in general are charged particles. Electrophoresis may be in general defined as the movement of a solid phase (the proteins in sample) with respect to a liquid (the buffer solution). The main function of the buffer is to carry the current and to keep the pH of the solution constant during migration. A solid substance called the medium supports buffer solution. Here
2

we use a gel as the substrate. A common material is agarose which is prepared from common seaweed. Purified agarose is in powdered form, and insoluble in water at room temperature, but is soluble in boiling water. When it starts to cool, it undergoes polymerization. The polymers crosslink and form the gel. If more agarose is added, the gel will become more firm. While solution is still hot, we pour it into a mould called casting tray so that it will assume the shape we want. For setting, gel body is immersed in deionised water. Deionised water, an insulator, prevents massive heat generation. Much higher voltage, such as 280 volts can be applied to derive rapid sample migration. Scientists add a mixture of proteins to an edge of the gel. An electric field is applied across the gel. The gel is in the form of a mesh network. In twodimensional electrophoresis, separation is done according to mass in one direction an according to electric field in the perpendicular direction. Each protein has an individual mass and charge. So they will separate out as individual dots in the gel. Researchers can then isolate each of these proteins for further analysis.

1.3 PROTEIN ISOLATION:

Protein purification is a series of processes intended to isolate a single type of protein from a complex mixture. Protein purification is vital for the characterization of the function, structure and interactions of the protein of interest. The starting material is usually a biological tissue or a microbial culture. The various steps in the purification process may free the protein from a matrix that confines it, separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps may exploit differences in (for example) protein size, physico-chemical properties, binding affinity and biological activity. Choice of a starting material is key to the design of a purification process. In a plant or animal, a particular protein usually isn't distributed homogeneously throughout the body; different organs or tissues have higher or lower concentrations of the protein. Use of only the tissues or organs with the highest concentration decreases the volumes needed to produce a given amount of purified protein. If the protein is present in low abundance, or if it has a high value, scientists may use technology to develop cells that will produce large quantities of the desired protein . Recombinant expression allows the protein to be tagged, e.g. by a his tag, to facilitate purification, which means that the purification can be done in fewer steps. In addition, recombinant expression usually starts with a higher fraction of the desired protein than is present in a natural source. An analytical purification generally utilizes three properties to separate proteins. First, proteins may be purified according to their isoelectric points by running them through a pH graded gel or an ion exchange column. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps may exploit differences in (for example) protein size, physico-chemical properties, binding affinity and biological activity. Choice of a starting material is key to the design of a purification process. In a plant or animal, a particular protein usually isn't distributed homogeneously throughout the body; different organs or tissues have higher or lower concentrations of the protein. Use of only the tissues or organs with the highest concentration decreases the volumes needed to produce a given amount of purified protein. If the protein is present in low abundance, or if it has a high value, scientists may use technology to develop cells that will produce large quantities of the desired protein . Recombinant expression allows the protein to be tagged, e.g. by a his tag, to facilitate purification, which means that the purification can be done in fewer steps. In addition, recombinant expression usually starts with a higher fraction of the desired protein than is present in a natural source. An analytical purification generally utilizes three properties to separate proteins. First, proteins may be purified according to their isoelectric points by running them through a pH graded gel or an ion exchange column.

CHAPTER 2 HISTORY/BACKGROUND

If the 1990s were the decade of genomics, the first ten years of the new century are set to become the decade of proteomics. For the first time, the technologies of pro- teomics make it possible to generate quantitative protein expression data on a scale and sensitivity comparable to that achieved at the genetic level. This advance has major implications for our understanding of cellular organisation in health and disease, and for pharmaceutical and agricultural biotechnology. Indeed, proteomics is already yielding important findings across a wide range of applications. This article reviews new concepts, innovative technologies and biological applications in proteome research less to manufacture and run than CRTs because the active material used is plastic. Proteomics is a recent member of the omics family that has gained rapid momentum at the turn of the century, particularly in the area of therapeutics. The proteomics repertoire of tools promises a wide range of revolutionary hardware, processes, techniques and strategies that have potential application in various stages of the drug discovery process, from target identification and validation to lead optimization and, subsequently, to toxicological profiling in preclinical and clinical settings. Although there is no doubt that the successful completion of the human genome project in 2003 and the enormous genomic data being generated will help speed up the discovery of new drug targets, experts believe that information on the protein level is needed to bolster rapid drug development because proteins are ultimately the key workhorses in our body. Furthermore, comparative analysis of the information produced by genomic studies and information regarding protein expression has led to theconclusion that genetic messages often fail to correlate with protein abundance and, more importantly, protein function. The rule of one geneone polypeptide no longer holds true either because one gene can encode multiple forms of a protein owing to alternative splicing and post-translational modifications. In addition, the function of a protein is dictated by its three-dimensional (3D) structure, which may change when it interacts with another or with other components inside cellular tissues. Despite these intricacies, proteins are keys to unlocking the nanometer keyholes to understand biological functions and the way these functions vary between health and diseased states. The more keys we can find, the more doors we can open. In addition, most drug targets are proteins; therefore, it is important to focus drug discovery efforts at this level. Because of the great excitement surrounding proteomics, there has been a noticeable emphasis for pharmaceutical and its biotechnological and academic partners to expend tremendous resources to refine and review their strategies to make drug development more efficient and successful. In recent years, businesses have also flocked to the thriving field of proteomics, with more than 100 companies now offering proteomics-related technologies, tools, data and services in the hope of gaining a competitive advantage and finding the best targets for successful drug candidates and diagnostic markers.1 According to the Cambridge Health tech Institutes (CHI) Drug Discovery and Development Deals Dat abase (http://www.discoverydeals.com), the number of business deals clinched in proteomics saw an

upward trend of 62%, with Incyte Genomics, Oxford Glyco Sciences (OGS), Proteome Systems and Applied Bio systems making it to the top 10 dealmakers list in 200001. On top of that, proteomics companies have attracted more than $530 million in venture capital from 1999 to 2001 through a wide array of alliances and collaborations and through public offerings of stock.1 2.1 WHAT IS PROTEOMICS? The word proteome is derived from PROTEins expressed by a genOME, and it refers to all the proteins produced by an organism, much like the genome is the entire set of genes. The human body may contain more than 2 million different proteins, each having different functions. As the main components of the physiological pathways of the cells, proteins serve vital functions in the body such as:

catalyzing various biochemical reactions, e.g. enzymes; acting as messengers, e.g. neurotransmitters; acting as control elements that regulate cell reproduction; influencing growth and development of various tissues, e.g. trophic factors; transporting oxygen in the blood, e.g. hemoglobin; and defending the body against disease, e.g. antibodies

. 2.2 BIOLOGY BEHIND PROTEOMICS The emergence of systems biology is bringing forth a new set of challenges for advancing science and technology. Defining ways of studying biological systems on a global level, integrating large and disparate data types, and dealing with the infrastructural changes necessary to carry out systems biology, are just a few of the extraordinary tasks of this growing discipline. Despite these challenges, the impact of systems biology will be far-reaching, and significant progress has already been made. Moving forward, the issue of how to use systems biology to improve the health of individuals must be a priority. It is becoming increasingly apparent that the field of systems biology and one of its important disciplines, proteomics, will have a major role in creating a predictive, preventative, and personalized approach to medicine. In this review, we define systems biology, discuss the current capabilities of proteomics and highlight some of the necessary milestones for moving systems biology and proteomics into mainstream health care.

CHAPTER3 TECHNICAL OVERVIEW

The rapid evolution of proteomics has continued during then past year, with a series of innovations in the core technologies of two-dimensional electrophoresis and mass spectrometry, and a diversity of productive research programmes. Wellannotated proteomics databases are now emerging in a number of fields to provide a platform for systematic research, with particularly promising progress in clinical applications such nas cardiology and oncology. Large-scale quantitative research, comparable in power and sensitivity to that achieved for gene expression, is thus becoming a reality at the protein levelThe ultimate objectives of proteomics go beyond the simple cataloguing of the proteins that cells express in health and disease states. The eventual goal is to elucidate the organisation and dynamics of the metabolic, signalling and regulatory networks through which the life of the cell is transacted. Moreover, proteomics seeks to understand how these networks become dysfunctional in disease, and to predict how their function can be manipulated through interventions such as drugs and genetic manipulations. These are ambitious goals that will require technologies of increased sensitivity and new concepts before they can be fully realised, but the task of mapping regulatory networks and diagnosing cell states is progressing. In microbes, VanBogelen et al. [1] from ParkeDavis/University of Michigan have identified proteomic signatures of specificThe distinction between quantitative regulation and structural proteomics is useful but it should be emphasised that most proteomics projects combine elements of bothm approaches and rely on both 2DE and MS. Both these core technologies have seen significant advances during the past year. There has been gradual progress on the vexed problem of isolating highly hydrophobic membrane proteins using 2DE through the continued development of sample preparation protocols based on zwitterionic detergents [6] and organic solvents [7], but this remains an important and unsolved problem. The challenge of obtaining samples of just a single cell type from a tissue has been successfully addressed using a laser capture microdissection system, in which a tissue sample is attached to a film and specific cells of interest detached using a laser beamrefer to a tags basic functionality (i.e., it either has a memory or an on -board power), while generation refers to the tag specifications major release or version number. The number of proteomics projects addressing basic biological questions is now very large and continues to grow. In part, this reflects the broadening usage of the term proteomics to embrace not only large-scale biotechnological research but also any project in which proteins are systematically studied by 2DE, MS or other techniques. One of the most significant themes for general biology to emerge out of proteomics has been a deepening insight into the nature of the relationship between genes and proteins. In the mouse proteome project of Klose and coworkers described above [18], hundreds of mouse genes were mapped to chromosomal locations on the basis of protein polymorphisms. It emerged from these studies that many

protein modifications are related to specific genes, such that a protein should be considered as the phenotype not of one but of many genes [20]. Equally, a single genetic mutation may affect many proteins. Ultimately, studies of this kind will lead to a more sophisticated understanding of the relationship between genes and cell function in health and disease. The combination of immune affinity techniques with 2DE and subsequent MS continues to be a major workhorse for studies of specific subsets of a cells proteome. Numerous studies of basic cellular mechanisms are in progress using this approach. For example, immunoblotting using anti phosphotyrosine and anti phosphoserine antibodies was used to extract over 500 phosphorylated proteins from mouse fibroblasts, of which at least 100 were found to undergo alterations in phosphorylation following stimulation with platelet derived growth factor (PDGF) [21]. These studies have revealed new putative signalling pathways downstream of the PDGF b receptor and are applicable to other signal transduction pathways. As a second example, immune precipitation was used to extract the chaperonin GroEL from Escherichia coli, in complex with over 300 newly translated polypeptides, which were subsequently separated by 2DE [22]. The technique enabled the function of GroEL in mediating protein folding to be investigated and is a typical example of how proteomics techniques can be used to target and study a specific subset of cellular components. Finally in this section, it is worth pointing out that proteomics is a burgeoning field in plant biology .

2.1 Working of project

The word proteome is derived from proteins expressed by a genome, and it refers to all the proteins produced by an organism, much like the genome is the entire set of genes. The human body may contain more than 2 million different proteins, each having different functions. As the main components of the physiological pathways of the cells

Proteomics is the large-scale study of proteins, particularly their structure and functions. Proteins are vital parts of living organisms, as they are the main components of the physiological metabolic pathway of cell. The term "proteomics" was first coined in 1997[ to make an analogy with genomic, the study of the genes. The word "proteome" is a blend of "protein" and "genome", and was coined by macromic in 1994 while working on the concept as a PhD student. The proteomis the entire complement of proteins, including the modifications made to a particular set of proteins, produced by an organism or systemcan modulate the voltage sensed at the Transceiver according to the bit pattern it wishes to transmit.

CHAPTER 4 TECHNOLOGY

4.1 PROTEOM ANALYSIS

Proteomic technologies will play an important role in drug discovery, diagnostics and molecular medicine because is the link between genes, proteins and disease. As researchers study defective proteins that cause particular diseases, their findings will help develop new drugs that either alter the shape of a defective protein or mimic a missing one. Already, many of the best-selling drugs today either act by targeting proteins or are proteins themselves. Advances in proteomics may help scientists eventually create medications that are
10

personalized for different individuals to be more effective and have fewer side effects. Current research is looking at protein families linked to diseases including cancer, diabetes and heart disease. Identifying unique patterns of protein expression, or biomarkers, associated with specific diseases is one of the most promising areas of clinical proteomics. One of the first biomarkers used in disease diagnosis was prostate-specific antigen (PSA). Today, serum PSA levels are commonly used in diagnosing prostate cancer in men. Unfortunately, many single protein biomarkers have proven to be unreliable. Researchers are now developing diagnostic tests that simultaneously analyze the expression of multiple proteins in hopes of improving the specificity and sensitivity of these types of assays corresponding rows and columns. These schemes pattern the anode and cathode into perpendicular rows and columns and apply a data signal to the columns while addressing the sequentially. As the number of rows in the display increases, each pixel must be red brightness by a factor of the number or row times the desired brightness, which can exceed 20000cd/m2.the current required to achieve this brightness, levels limits this architecture to relatively small screen sizes. Philips Flat Display systems (Sunnyvale, CA) and Dupont Displays have demonstrated full-colour passive matrix displays. In active matrix architecture, a thin film polysilicon transistor on the substrate address each pixel individually. Active matrix displays are not limited by current consideration. Seiko- Epson, Tosibha (Tokyo,Japan), and Samsung (Seoul, Korea) have now demonstrated full colour active matrix.

4.2 WORKING PRINCIPLE

11

Proteomics studies are also being made using liquid chromatography. Just as 2D separation generates the high separation efficiency in gels, several dimensions are required using LC. In most cases, at least two dimensions are being applied to complex biological samples where most often electrostatic separation mechanisms are being combined with hydrophobic separations. The great advantage using LC is that the interface in-between the first and the second dimension can be made online, as presented in Fig. 6. In this approach, a continuous real-time protein analysis can be made. While one sample is being analyzed in the set-up presented in Fig. 6, the second column (1st dimension) is conditioned. By the time the separation is completed in the first column, the next fraction is introduced into column 2, while column 1 is equilibrated, made ready for the next sample to be introduced. Figure 7 shows the analysis process whereby the separations are made using coupled column LC described in Fig. 6 and the resulting MS spectra generated using both electrospray and MALDI ionization principles [22,23]. The database registration and query path of the annotation procedure is an important part of the bioinformatic work within proteomics. The peptide sequence identities are made where a scoring of the protein identity is made, reflecting the statistical significance whereby the identity is determined and the likelihood that it is the correct protein identityinterrogator). 4.3 PROTEOMICS TECHNOLOGY Amino Acid Composition Array-based Proteomics 2D PAGE Mass Spectrometry Structural Proteomics Informatics

4.3.1 MASS SPECTROMETRY Mass spectrometry is a method of choice for analytical characterization of potential drug molecules and protein identification. Protein structural information, such as peptide molecular weight, amino acid sequence composition, type and location of post-translational modification, could be obtained by MS analysis. Two most widely used MS are: (i) matrix-assisted laser desorption ionization timeof- flight mass spectrometry (MALDI-TOF-MS), which generates ions from solid phase samples (crystallization of samples within a matrix) and measures their peptide mass in a flight tube; and (ii) electrospray ionization mass spectrometry (ESI-MS), which generates ions from liquid samples (fine sprays of highly charged droplets from a strong electric field) and measures their mass using either quadrupole or time of flight detector.17 Mass spectrometry analyses of proteolytic digests provide insights to important protein attributes for protein identification. Two techniques are generally used. The first technique is
12

peptide mass fingerprinting, which compares the masses obtained from spectrometric techniques against theoretical fingerprints from non-redundant protein databases, whereas the second, MS/MS, subjects selected tryptic digest derived from parent protein to secondary fragmentation to obtain a peptide sequence tag that is compared against protein databases for protein identification. 18,19 Recently, there have been marked improvements in MS performances, including higher sensitivity, resolution and mass accuracy, owing to improved instrumental design. Newcomers, such as ion trap, quadruple time-of-flight (Q-TOF) and fourier transform ion cyclotron resonance (FTICR) MS are increasingly being used for protein biomarker discovery to gauge disease progression and drug action. Affinity based MS methods can be used to study ligand target interaction for lead compound discovery and optimization. Industry leaders, such as Applied Biosystems and Bruker Daltonics, are now offering TOF/TOF MS instruments; ProteomeWorks System (a global alliance of Micromass-Waters and Bio-Rad Laboratories)are advancing their latest entries, including M@LDI HT, Q-TOF micro and Q-TOF Ultima,1 and Thermo Electron Corporation has just introduced the Finnigan LXQ linear ion trap mass spectrometer, which enables rapid compound detection, confident structural identification and fast screening for drugs, their metabolites and degradation products. 4.3.2 CHROMATOGRAPHIC TECHNIQUES An alternative approach to 2DGE is LC, a non-gel-based proteinseparation technique. Singleand multidimensional LC can be directly interfaced with the MS, enabling automated analysis of large amounts of data for subsequent protein identification. Shotgun proteomics,which resembles shotgun genome sequencing, is facilitated by theuse of multidimensional protein identification technology (MudPIT).Multidimensional protein identification technology incorporates multidimensional high-pressure liquid chromatography (HPLC),tandem mass spectrometry (MS/MS) and database searching by the SEQUEST algorithm (http://www.thermo.com). This technology shows advantages over gel-based techniques in terms of speed, sensitivity, scope of analysis and dynamic range.16 Shotgun proteomicshas come to preeminence only recently and relatively few descriptionsof its used in human therapeutic intervention and diagnostics have been mentioned. Nonetheless, this approach has been used in the analysis of body fluids for the detection and development of novel biomarkers for human diseases. In our laboratories, we have applied this approach to the analysis of human sweat, tears and saliva, in relation to different disease states and pathological conditions. Protein components in human sweat, for example, have been evaluated in the past several decades by traditional gel electrophoresis. Among the major components identified previously were proteases, lysozymes and immunoglobulins. In our investigations, sweat samples from healthy volunteers were collected, dialysed and concentrated. The protein components were then digested to polypeptides by trypsin and analysed by two-dimensional LC tandem mass spectrometry. We found more than 300 different types of proteins in human sweat with two or more confident peptides matches and these included the immunoglobulins (IgG; IgE), dermcidin precursor (antimicrobial peptides), lysozymes, protease inhibitors and other proteins that may play important innate immune functions or antimicrobial roles. In the near future, we would expect this approach to become more widely used by scientists in academia and industries. In fact, Celera has adopted this methodology for proteome-wide analyses.
13

4.4 2D-GEL ELECTROPHORESIS The 2D electrophoresis was performed with Amersham Biosciences IPGphor IEF and Ettan Dalt six electrophoresis units using the protocol suggested by the company of Amersham Pharmacia. Briefly, 250 g ( 3 l) of serum sample was mixed into 340 l rehydration solution containing 8 M urea, 4% CHAPS, 1mM PMSF, 20 mM DTT and 0.5% IPG buffer. Rehydration step was carried out with precast 18 cm IPG strips for more than 10 hours under a low voltage of 30 V. IEF was run following a step-wise voltage increase procedure: 500 V and 1000 V for 1 hour each and 5000-8000 V for about 10 hours with a total of 64K Vh. After IEF, the strips were subjected to two step equilibration in the equilibration buffers containing 6 M urea, 30% lycerol, 2% SDS and 50 mM Tris-HCl (pH 6.8) with 1% DTT (w/v) for the first step and 2.5% IAA w/v) for the second step. The strips were then transferred onto the seconddimensional SDS-PAGE which was run on 1.5 mm thick 12.5% polyacrylamide gels at 10C.

4.4.1 SILVER STAINING

The gels were fixed in 40% ethanol and 10% acetic acid in water overnight, and then incubated in a buffer solution containing 30% ethanol, 4.1% sodium acetate and 0.2% sodium thiosulfate for 30 minutes. After washing three times in water for 5 minutes each, the gel were stained in 0.1% silver nitrate solution containing 0.02% formaldehyde for 40 minutes.Development was performed for 15 minutes in a solution consisting of 2.5% sodium carbonate and 0.01% formaldehyde. EDTA solution (1.46%) was used to stop the development and the stained gels were then washed three times in water for 5 minutes each.

4.4.2 IMAGE ACQUISITION AND ANALYSIS

The stained gels were scanned in an ImageScanner (Amersham) operated by a software, LabScan 3.00, from Amersham Pharmacia Biotech. Intensity calibration was done with an intensity step wedge prior to gel image capture. Image analysis was carried out using the ImageMaster 2D Elite software 4.01 from Amersham Pharmacia. Image spots were initially

14

detected, matched and then manually edited. Ten gel images of normal serum samples were averaged and set as the reference for comparison. Each spot intensity volume was processed by background subtraction and total spot volume normalization, the resulting spot volume percentage was used for comparison. Only those significantly different spots (2-fold increase or decrease) were selected for analysis with mass spectrometry.

4.4.3 TRYPTIC IN-GEL DIGESTION.

Protein spots were excised and transferred into siliconized 1.5 ml Eppendorf tubes. Gel chips ere de-stained in an 1:1 mixture solution of 30 mM potassium ferricyanide and 100 Mm sodium thiosulfate and then equilibrated in 50 mM ammonium bicarbonate to get pH 8.0. After hydrated with acetonitrile and dried in a SpeedVac, the gels were rehydrated in a minimal volume of trypsin solution (10 g/ml in 25 mM NH4HCO3) and incubated at 37C overnight. The supernatant was directly applied onto the sample plate with equal amount of matrix. If necessary, the in-gel digests were extracted subsequently with 50% and 80% 7 acetonitrile, and then concentrated and de-salted by Zip tips prior to applying on the sample plate.

4.4.4 MALDI-TOF MASS ANALYSIS AND PROTEIN IDENTIFICATION.

Tryptic peptide mass spectra were obtained using a Voyage-DE STR MALDI-TOF mass spectrometer (Applied Biosystems). The instrument setting was reflector mode with 175 ns delay extraction time, 60-65% grid voltage, and 20k accelerating voltage. 250 Laser shots per spectrum were used to acquire the spectra with mass range from 600 to 2500 Daltons. The trypsin autolytic fragment peaks (906.5049, 1153.5741 and 2163.0570) serve as internal standards for mass calibration. Protein identification was performed by searching in NCBInr protein datebase using MS-Fit (http://prospector.ucsf.edu/). The criteria for searching were set with 50 ppm or better mass accuracy, at least 4 matching peptide masses and molecular weight and pI matching estimated values from gels. Post-source decay MS/MS measurement and MS-

15

Tag (http://prospector.ucsf.edu/) searching were also performed to confirm the results from the

MS-it.

4.4.5 PROTEIN SEPARATION Two groups of HBV-infected serum samples together with control samples were applied to 2DPAGE and proteins visualized by silver staining. 2D gels were run three times for each sample to minimize gel-to-gel variation. Figure 1A shows three representative gel images for normal, HBV-infected low and high necroinflammatory score serum (LNS and HNS), respectively. igure 1B is an enlarged master LNS gel displaying the common features of human serum proteins. Overall, the gel has a very similar pattern to the plasma map in the SWISS-2D database (http://www.expasy.ch/ch2d/) except lacking fibrinogen. More than 1000 spots were detected in a gel ranging from 6k to 200k Da of molecular mass with p Is between 4 and 10. Many trains of spots represent those proteins that have primary structure with different degrees
16

of glycosylation and/or phosphorylation (isoforms), resulting in a progressive change in the pI and molecular weight. Spot volume comparison was made between three types of samples with assistance of the ImageMaster program. Significant and constant differences were found in at least seven areas. 4.4.6 PROTEIN IDENTIFICATION The protein spots that have significant differences were cut out and subjected to trypsin digestion, MALDI-TOF mass spectra measurement and database searching. Where appropriate, protein identifications were confirmed by comparing spot locations and patterns to those in the SWISS database plasma map (Table 2). Table 3 summarizes the identified proteins in the seven areas and their alterations among normal, LNS and HNS serum samples.Overall, the expressions of three proteins (groups) are suppressed and five proteins (groups) are enhanced in the sera of HBV-infected patients. One of the remarkable changes is shown in Haptoglobin 2 chain, chain and cleaved chain present their own characteristic train patterns in 2D gels, featuring with three, seven and six detectable isoforms respectively. Compared to that in normal samples, haptoglobin overall slightly increased or had no change in the LNS serum samples but was significantly suppressed in the HNS patients (Table 3). In some cases (30%) the protein was diminished to undetectable level. In contrast, a protein cluster highlighted in circle in the Area 1 of Figure 2 gradually increases its expression level from undetectable in the control to partially visible in LNS and then to fully appearance in HNS. Figure 3 displays another dramatic change occurring in apo-lipoprotein A-I (apoA-I) region (Area 3 in Figure 1B). A normal serum sample has three main apoA-I protein spots (isoforms 2, 1 and 0 from left to right)[15] with one to three very weak cleaved fragment spots immediately below the main spots. In the HBV-infected serum samples, the entire pattern of apoA-I profile was altered. Isoform 2 was significantly up-regulated, isoform 0 was evidently down-regulated, while isoform 1 remained unchange (Table 3). In addition to the three major spots, the weak fragment spots were significantly enhanced as well as a number of new spots appeared in the low molecular weight area (Fig. 3). These new spots were identified to have the primary sequence of apoA-I and they probably belong to the cleaved fragments of apoA-I or its other isoforms. It is also worth noting that overall the main apoA-I protein spots decrease their volumes in both LNS and HNS samples and the cleaved fragments of apoA-I or other isoforms appear in higher volumes in LNS than those in HNS samples (Table 3). One more apoA-I fragment was observed in the even low molecular weight Area 4 (Fig.3).This spot appeared in both LNS and HNS samples but not in the normal serum samples. Similarly in the same Area 4, a spot of apoA-IV was identified in both HBV-infected samples but was undetectable in the control. The only one detectable spot in the Area 4 of normal samples was identified to be transthyretin. The expression level of this transthyretin isoform was apparently decreased in the serum of HBV carriers (Table 3). Figure 4 shows the protein alterations in Areas 5 and 6 in Figure 1B. Three spots in Area 5 and two spots in Area 6 were markedly intensified in both LNS and HNS samples (Table 3). These spots are identified to have the same primary sequence as 1antitrypsin. This protein normally appears in a group of spots representing various phenotypes in the 2D gel ranging from pI 5.0-5.2, MW 54kDa (http://www.expasy.ch/ch2d/) (Fig. 1B). No significant difference in the level of the normal 1-antitrypsin was found between control and HBV serum samples. However, 1-antitrypsin level in the lower molecular weight areas (MW

17

40- 41kDa) is greatly enhanced in HBV samples . The increased expression level of the protein in the Area 6 is 2-fold more in HNS than that in LNS samples. Spots in Area 7 were identified as DNA topoisomerase II (Topo II) (Fig. 5). Only one spot of this protein was detected in the normal serum whereas a group of five spots developed in HBV samples. As a result, the protein level was elevated about 20 fold in total in the HBV serum (Table 3). Given the fact that intact TopoII has a molecular weight of 180kDa, these spots in Area 7 are probably the low-molecular mass fragments.

18

19

The protein spots that have significant differences were cut out and subjected to trypsin digestion, MALDI-TOF mass spectra measurement and database searching. Where appropriate, protein identifications were confirmed by comparing spot locations and patterns to those in the SWISS database plasma map summarizes the identified proteins in the seven areas and their alterations among normal, LNS and HNS serum samples. Overall, the expressions of three proteins (groups) are suppressed and five proteins (groups) are enhanced in the sera of HBVinfected patients. One of the remarkable changes is shown in Figure 2 (Areas 1 & 2 in Figure 1B) concerning haptoglobin. Haptoglobin 2 chain, chain and cleaved chain present their own characteristic train patterns in 2D gels, featuring with three, seven and six detectable isoforms respectively. Compared to that in normal samples, haptoglobin overall slightly increased or had no change in the LNS serum samples but was significantly suppressed in the HNS patients (Table 3). In some cases (30%) the protein was diminished to undetectable level. In contrast, a protein cluster highlighted in circle in the Area 1 of Figure 2 gradually increases its expression level from undetectable in the control to partially visible in LNS and then to fully appearance in HNS. Figure 3 displays another dramatic change occurring in apo-lipoprotein A-I (apoA-I) region (Area 3 in Figure 1B). A normal serum sample has three main apoA-I protein spots (isoforms 2, 1 and 0 from left to right)[15] with one to three very weak cleaved fragment spots immediately below the main spots. In the HBV-infected serum samples, the entire pattern of apoA-I profile was altered. Isoform 2 was significantly up-regulated, isoform 0 was evidently down-regulated, while isoform 1 remained unchange (Table 3). In addition to the three major spots, the weak fragment spots were significantly enhanced as well as a number of new spots appeared in the low molecular weight area (Fig. 3). These new spots were identified to have the primary sequence of apoA-I and they probably belong to the cleaved fragments of apoA-I or its other isoforms. It is also worth noting that overall the main apoA-I protein spots decrease their volumes in both LNS and HNS samples and the cleaved fragments of apoA-I or other isoforms appear in higher volumes in LNS than those in HNS samples (Table 3). One more apoA-I fragment was observed in the even low molecular weight Area 4 (Fig.3). This spot appeared in both LNS and HNS samples but not in the normal serum samples. Similarly in the same Area 4, a spot of apoA-IV was identified in both HBV-infected samples but was undetectable in the control. The only one detectable spot in the Area 4 of normal samples was identified to be transthyretin.

20

21

There seem to be a difference in-between m-RNA regulation and the actual synthesis of proteins [14,15]. Figure 1 illustrates the link in-between the DNA, RNA, and protein. While the genome is identical in all bodies, the protein levels will vary in both cell type and time. The kinetics within proteomics studies is of vital importance and might be the missing link to why the correlative regulation factors of m-RNA and protein are found to be different. Modification of the protein structure is a part in the metabolism of the cells natural life cycle. A large number of post-translational modifications (PTMs) do occur, such as phosphorylation events. These intracellular mechanisms are of vital importance as theyhave a direct link to the biological activity that takes place within the cell. However, most of these PTM activities will not impose a biological effect, such as, for instance, a signaling event. Diseased-linked alterations are most commonly observed in the tissue where morphological changes occur. The degree of severity of the disease will in many cases be linked to, for instance, the decline in organ function and capacity. Figure 2 shows the sample handling steps that often are undertaken in clinical studies from which the proteomics expression profiling is performed. The tissue as such can be analyzed directly by solubilization where a total expression read-out will be generated from the tissue sample as shown in Fig. 1A. There is also a possibility of isolating cells from the tissue by using favorable cultivation conditions and thereby incubating these target cells under proliferative conditions (see Fig. 2B). The human cells will increase in number, and this makes the protein study easier in terms of sample availability (see Fig. 2C). It is of mandatory importance that viability controls of the cells are made, as well as control assays, which ensures the phenotype. Primary human cells as cell lines is another alternative to making in vitro studies with subsequent expression analysisThe enormous challenge that the proteomics field holds, and where the holy grail still needs to be found and defined, can be summarized as simple as not sufficient sensitivity. Figure 3 illustrates the daily struggle and challenge that the research area is working on and developing, where the major limitation is the lack of a protein PCR technology. This has certainly put much pressure on both academic and industrial groups to find new analytical technologies and methodologies that can circumvent this limitation. The large dynamic range, which reaches up to nine orders of magnitude, is also not a trivial challenge to meet since it holds a high degree of sample composition variation.

22

23

4.5 AREA OF PROTEOMICS

4.5.1 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 XRCPort 2 emits the high-order address byte during fetchesfrom external program memory and during accesses toexternal data memory that use 16-bit addresses (MOVX @DPTR). In this application, it uses strong internal pull-ups when emitting 1s. During accesses to external data memorythat use 8bit addresses (MOVX @ RI), Port 2 emits the contents of the P2 Special Function Register.Port 2 also receives the high-order address bits and somecontrol signals during Flash programming and verification. 4.5.2 INFORMATICS: Significant improvements are needed in: Data presentation standards and formatting Software infrastructure ISB - have created many powerful software packages that interpret data from different techniques. EBI and HUPO have come together to promote uniform data storage and analysis. The proteomics community has, over the course of the past four years, become slightly less proprietary. Ron Beavis of U. Manitoba has developed x! tandem, an opensource search algorithm as an alternative to SEQUEST.Development of novel software for both analysis and strategies [for biologists ] to manage the data are two fronts that I can see as opportunities for folks with a CS background. 4.5.3 CLINICAL PROTEOMICS: This area of proteomics focuses on accelerating drug development for diseases through the systematic identification of potential drug targets. Currently, cancer is diagnosed and treated when it is too late: metastasis has already occurred and the success of therapeutic modalities is very limited. Detecting cancers when they are in the earliest

24

stages (even in the premalignant state) ultimately translates into higher cure rates. Nowhere is this dilemma more apparent than for ovarian cancer. More than two-thirds of ovarian cancer cases are detected at an advanced stage, when the ovarian cancer cells have spread away from the ovary surface and disseminated throughout the peritoneal cavity .Although the disease at this stage is advanced, it rarely produces specific or diagnostic symptoms. Consequently, ovarian cancer is usually treated when it is at an advanced stage .The resulting 5-y survival rate is 3540% for late-stage patients even with the best of treatment. Conversely, when ovarian cancer is detected early (stage I), conventional therapy produces a high rate (95%) for 5-y survival .The lack of a specific symptom in early-stage ovarian cancer may provide a new approach for the discovery of early cancer biomarkers. For this reason, ovarian cancer has been a major focus of marker discovery. How could this be accomplished Hopefully, we will have more specific information, instead of raw genes, that will make those complex differential equations much simpler in the coming years.

4.6 ISOTOPE-CODED AFFINITY TAG: It is a chemical modification strategy that enables the rapid and accurate quantification of protein activity and concurrent sequence identification of proteins in a complex mixture. The ICAT approach involves selective conjugation of cysteine thiol groups with two identical reagents isotopically different in mass (one light and one heavy linker) and biotinylated affinity tags, followed by proteolytic digestion and quantitative analysis of the peptideconjugates by LC/MS. The isotopic difference allows relative abundance measurement of the same peptide ions from two different samples (normal vs diseased or control vs treated) by comparing signal intensities in MS mode and subsequent MS/MS peptide sequencing results for protein identification in altered expression levels.21 Isotope-coded affinity tag has tremendous advantageous in the analysis of membrane proteins and low abundant proteins, which are likely to be promising targets for therapeutic and diagnostic development. The approach also offers high-throughput analysis and is highly automated and reproducible. However, a major drawback of the earlier versions of the technology is that the protein must contain cysteine residues in its structure. Because of this requirement, many important proteins, including those with post-translational modifications, are overlooked. Several groups have since described alternative labelling strategies that target lysine and tryptophan residues or peptide N- or Ctermini. These include a non-isotopic variation called the mass-coded abundance tagging (MCAT) strategy developed in 2002.22 In this technique, O-methyl isourea is used to guanidinate the C-terminal lysine residues of peptides in one sample, whereas the other sample remains unmodified; the samples of each are combined and the relative abundance of each can
25

be determined by mass spectrometry. The ICAT technology has been commercialized by Applied Biosystems (a business unit of Applera).1 They have now improved the system to include other isotopic labelling variants, for example, a set of four stable isotopic labels released under the brand name ITRAQ, that can be used for multiplexed protein profiling up to four different samples. These reagents use labels that bind covalently to lysine side chains and N-termini of any peptide and a reporter group specific to each tag. The reporter groups generate fragment ions that appear in the low-mass region of the spectrum where other fragment ions are not generally found and, thus, the protein abundance of samples differentially labelled with each reagent can be compared by looking at the ratio of the peak areas of each reporter group. Like any other discovery science method, the ICAT method is, by itself, not sufficient to address an entire biological process. Prefractionation methods to reduce the complexity of the generated peptides, ICAT technology in combination with some form of MS instrument, as well as improved bioinformatics tools for automated data collection and interpretation23 will undoubtedly provide us with a comprehensive map of protein networks and their biological functions in cells and tissues under conditions of health and disease.

4.7 PROTEIN CHIP: Protein chips, protein biochips or protein microarrays are microproteomic technologies for studying protein interaction and function. Two basic formats of protein arrays have been generated, the forward phase array (FPA) and the reverse phase array (RPA).24 In the FPA, a biochip surface is arrayed with a specific substrate (bait), such as antibodies, proteins, peptides, nucleic acids or other small molecules, and is used to select specific bound analyte to the bait molecule from a heterogeneous mixture of sample analytes. A large amount of patient sample is required for processing such an array. In the RPA, the whole repertoire of a patients protein representingthe diseased state of individual tissue cell populations is arrayed directly onto a spot on a biochip surface.2426 Many patient specimens could be deposited onto the same biochip surface, with each substrate being probed with a distinct antibody. This technology uses only a very small amount of crude sample (e.g. cell lysates, urine or serum) from the patient.24,26 Protein chips are viewed as the most promising tools for proteome-wide analysis. This technology enables thousands of proteins to be analysed en masse (a more rapid profiling approach compared with 2DGE) and it enables screening for specific types of posttranslational modifications. Thus, proteins chips may be designed to study receptorligand interactions, enzyme activity and downstream signalling events, proteinprotein and protein antibody interactions as well as to serve as the basis of a serodiagnostic chip.27 Commercial players, for instance Ciphergen Biosystems and Phylos, are developing a wide range of protein arrays. Ciphergen has developed ProteinChip Arrays, which use chromatographic surfaces derivatized with different affinity matrices to capture proteins of interest.26 These proteins are then purified and analysed through a surface-enhanced laser desorption/ionization time-offlight (SELDI-TOF) mass spectrometer.28 Based on the comparisons of protein mass profiles from any two samples from different biological and pathological conditions, potential biomarkers or disease-related protein targets could be identified. We have used this rapid profiling chip array system for a variety of studie, each exploiting the key strengths and advantages of this technology:

26

it being amendable to analyse small and usually limited quantities of biological samples. (ii) the ability to detect and evaluate proteins without the need for tagging, labelling or processing. (iii) the sensitivity of the system with the ability to detect fmol concentrations of proteins thus making it amendable for analysis even in large epidemiological settings. One such example is the use of the Ciphergen protein chip arrays to identify plasma markers in individuals exposed to high levels of environmental aflatoxin, which could lead to liver cancer. Samples obtained from finger pricks and in an epidemiological setting were evaluated on the protein chip arrays. Our data show that the plasma protein profiles of individuals with high chronic environmental aflatoxin exposure could be differentiated from individuals coming from environments with minimal or low exposure. Protein chips, protein biochips or protein microarrays are microproteomic technologies for studying protein interaction and function. Two basic formats of protein arrays have been generated, the forward phase array (FPA) and the reverse phase array (RPA).24 In the FPA, a biochip surface is arrayed with a specific substrate (bait), such as antibodies, proteins, peptides, nucleic acids or other small molecules, and is used to select specific bound analyte to the bait molecule from a heterogeneous mixture of sample analytes. A large amount of patient sample is required for processing such an array. In the RPA, the whole repertoire of a patients protein representingthe diseased state of individual tissue cell populations is arrayed directly onto a spot on a biochip surface.2426 Many patient specimens could be deposited onto the same biochip surface, with each substrate being probed with a distinct antibody. This technology uses only a very small amount of crude sample (e.g. cell lysates, urine or serum) from the patient.24,26 Protein chips are viewed as the most promising tools for proteome-wide analysis. This technology enables thousands of proteins to be analysed en masse (a more rapid profiling approach compared with 2DGE) and it enables screening for specific types of post-translational modifications. Thus, proteins chips may be designed to study receptorligand interactions, enzyme activity and downstream signalling events, proteinprotein and proteinantibody interactions as well as to serve as the basis of a serodiagnostic chip.27 Commercial players, for instance Ciphergen Biosystems and Phylos, are developing a wide range of protein arrays. Ciphergen has developed ProteinChip Arrays, which use chromatographic surfaces derivatized with different affinity matrices to capture proteins of interest.26 These proteins are then purified and analysed through a surfaceenhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometer.28 Based on the comparisons of protein mass profiles from any two samples from different biological and pathological conditions, potential biomarkers or disease-related protein targets could be identified. We have used this rapid profiling chip array system for a variety of studie, each exploiting the key strengths and advantages of this technology: (i) it being amendable to analyse small and usually limited quantities of biological samples. (ii) the ability to detect and evaluate proteins without the need for tagging, labelling or processing. (iii) the sensitivity of the system with the ability to detect fmol concentrations of proteins thus making it amendable for analysis even in large epidemiological settings. One such example is the use of the Ciphergen protein chip arrays to identify plasma markers in individuals exposed to high levels of environmental aflatoxin, which could lead to liver cancer. Samples obtained from finger pricks and in an epidemiological setting were evaluated on the
27

(i)

protein chip arrays. Our data show that the plasma protein profiles of individuals with high chronic environmental aflatoxin exposure could be differentiated from individuals coming from environments with minimal or low exposure.

28

29

30

Proteomics has broad applications and it aims to: (i) catalogue and characterize the full protein complement in the genome; (ii) compare the levels of protein expression under different conditions (control vs pathological conditions); (iii) identify and characterize protein modifications, such as phosphorylation, acetylation, glycosylation etc.; (iv) identify protein localization and compartmentalization at a given time; and (v) understand how proteins interact with each other and with other biological molecules and chemicals in cellular tissues. However, these combined studies will not yield comprehensive proteome map of an organism because cells are in constant flux, and so is protein expression, in response to age, disease and trauma. Proteomics is a promising approach in the identification of protein targets and the elucidation of fundamental cell and disease biology, biochemical processes and pathways involved in disease process and, thus, plays a significant role in drug development. Disease involves alterations in protein expression levels. Hence, by studying the change in the abundance of proteins within a cell over time or between different cellular states (normal vs diseased states), proteomics provide insights into the pathophysiological basis of protein target identification and validation for disease intervention and treatment. Specific biomarkers identified from proteomics may be used as protein signatures to screen new chemical entities for target organ toxicity in preclinical trials and later on in development during clinical trials,2 ensuring their usefulness in the diagnosis and prognosis of diseases. The use of proteomics to study toxicity at the protein level is called pharmacoproteomics.3 By holistically examining the entire protein
31

profile of cellular tissues treated with drugs or drug candidates, proteomics establishes a comprehensive protein interaction map (interactomes)4 related to disease pathways, hence optimizing the drug development process. Figure summarizes some of the intervening stages in the drug discovery and development process that proteomics can play a major role in. Today, considerable effort is being devoted to the development of new tools and methodologies, complementary to the existing strategies for high-throughput analysis of the total protein repertoire of biological samples. These new, highly sensitive and high-throughput technologies are poised to unravel the mystery of proteins and add a new dimension to large-scale drug discovery. In this regard, the myriad of proteomic approaches currently on the market and areasavailable in the market of 16 column and one Row and more than one Row displays of ongoing research could broadly be categorized into three basic categories: profiling approach, functional approach and structural approach.

4.8 TECHNOLOGIES: INNOVATIONS IN 2DE AND MS

The distinction between quantitative regulation and structural proteomics is useful but it should be emphasised that most proteomics projects combine elements of both approaches and rely on both 2DE and MS. Both these core technologies have seen significant advances during the past year. There has been gradual progress on the vexed problem of isolating highly hydrophobic membrane proteins using 2DE through the continued development of sample preparation protocols based on zwitterionic detergents [6] and organic solvents [7], but this remains an important and unsolved problem. The challenge of obtaining samples of just a single cell type from a tissue has been successfully addressed using a laser capture microdissection system, in which a tissue sample is attached to a film and specific cells of interest detached using a laser beam [8]. The use of MS in proteomics continues to grow in power and versatility with a number of notable recent innovations. A number of groups have introduced novel proteintagging methodologies that improve the power and sensitivity of MS [9,10]. Aebersold and coworkers [10] have, for example, developed a method for labelling peptides with isotope-coded affinity tags (ICATs), which do not only support enhanced analysis of complex peptide mixtures but also allow accurate quantification of differences in the level of expression of proteins, a capability that MS-based proteomics did not previously have. The coupling of MS with a software tool, FindMod, has increased the power of this approach to identify posttranslational modifications [11]. Techniques for the characterisation of protein complexes have also been introduced, including a system based on liquid chromatography/tandem MS [12] and the development of a novel tandem affinity purification (TAP) tag for the rapid purification of complexes from cell samples [13]. Other developments in sampling protocols have recently allowed direct analysis by MS of the peptides from single secretory vesicles and it is anticipated that other organelles will be amenable to analysis by the same technique [14]. It is of course through the integration of 2DE- and MSbased approaches that proteomics achieves its greatest power. A representative example of the degree of integration now possible is the system developed by Hochstrassers group [15] in which whole 2DE gels are subject to in situ digests, electrotransferred onto membranes and directly scanned by MS, leading to the automated generation of an annotated (i.e. the proteins are

32

characterised and identified) 2D map. Developments of this kind, which are also being pioneered within specialist proteomics companies, are now enabling proteomics to be conducted on a scale commensurate with the complexity of its subject matter. 4.8.1 PROTEOME DATABASES The proteome database, consisting of annotated 2DE data obtained under different cellular conditions, is the basic platform from which proteome research addresses specific biological and pharmaceutical questions. It is notable, however, that to date relatively few large-scale databases have emerged into the public domain. The Yeast Protein Database (YPD; http://www.proteome.com/databases/index.html) is one of the best and longest-established large-scale databases and its curators, Proteome Inc. (Beverly, MA), are now constructing WormPD [16], a proteome database for the nematode worm Caenorhabditis elegans, for which the complete genome was recently sequenced. One of the most important database developments during the past year was the construction by Hoffmann-La Roche of a Haemophilus influenzae database, consisting of over 1000 individual 2DE map protein spots from which more than 500 individual proteins have been identified to date [17]. Klose and co-workers [18] have also recently reported their ongoing programme to develop a database for the mouse proteome, with an initial focus on brain proteins. In humans, the databases on bladder cancer constructed by Celis et al. [19] continue to be among the largest and bestannotated currently available, although the next few years are certain to see a major drive to generate major proteome databases for a wide variety of human tissues. The number of smaller proteome databases designed to address specific biological questions in humans and other organisms is now very large and beyond the scope of this review to address. The Expasy website home to the major SWISS-2D PAGE proteomics database and other resources, contains numerous links and remains the best platform from which to explore the world of public proteomics databases.

33

CHAPTER 5 FUTURE SCOPE AND APPLCATION

Companies had succeeded to make similar products and put them in the market. Putting a newly invented, innovative product in the market is not easy. The prices of 2-D digital pens are about 50 dollars to 90 dollars. So the price of smart note taker will be high. But this disadvantage definitely be eliminated in near future. The development of proteomic techniques and technologies has introduced a new dimension into the drug-discovery arena. By elucidating the pathogenesis of human diseases, proteomics couldaid in the discovery of patient-tailored molecular medicine (personalized medicine) of the future. Proteomics has set foot in every stage of the drug-discovery process, from target discovery to clinical trials, often providing new insights and interesting discoveries. Future improvements in this field promise to streamline drug development and to continually meet the quest for safer, more effective and cost-effective therapeutics. We are only at the beginning and it seems as if the sky is the limit. APLICATIONS Convergence between discovery science and hypothesis-driven science

34

Systems biology approaches will detect connections between broad cellular functions and pathways that were neigher apparent nor predictable. Ability to collect data already outstrips our ability to validate, integrate, and interpret it.

Clone desiging Drug discover

CHAPTER 5 ADVANTAGE AND DISADVANTAGE

Advantages 1 Established technology large dynamic range for detection of extralevals. 2 We can detect all types of proteins, 3 Test directly to the chemical components. 4 Economical Disadvantages 1 2 3 4 5 6 Limited/variable sample material Sample degradation (occurs rapidly, even during sample preparation) Vast dynamic range required Post-translational modifications (often skew results) Specificity among tissue, developmental and temporal stages Perturbations by environmental (disease/drugs) conditions

35

Researchers have deemed sequencing the genome easy, as PCR was able to assist in overcoming many of these issues in genomics

CHAPTER 6 CONCLUSION

Performing 2D PAGE separations allows the proteome analysis to be made in a protein form where the size as well as the charge of each respective protein can be determined. It is a great advantage when it comes to the mass spectrometric identification since the database query using publicly available databases (e.g., SWISSPROT, NCBI, or EMBLE) will make the identification more probable. The statistical significance of an identity hit will be higher in cases where the molecular size of the proteins from the 2D gel being identified is accurate. The PI value is additionally a useful physical property to be used, however, this is in many cases altered due to protein modifications such as phosphorylation or sulfatation. The resolution of 2D PAGE-separated proteins is in the order of 15005000 as shown in this work, although the latter high number of protein spots are generally only obtained when metabolic labelling is made. The really big challenge and disadvantage of the 2D PAGE approach is that the gel image annotations made can be performed highly accurately with the high resolving power, but cannot be made ID-approved by mass spectrometry. The main reason for failure of identification is simply due to the fact that the protein spot that is excised and digested has an unfavorable digestion kinetics. The MichaelisMenten kinetics works against you in situations where the substrate levels are low (i.e., the protein spots are faint). It is also a difference in resolution running straightforward 2D PAGE studies and post-gel stainings in comparison to metabolically labeled cells that are separated by 2D PAGE [19]. It should also be stressed that its not really the same experiment that is being performed since the methionine actually was added to the cell culture during the incubation and incorporated the amino acid. Newly synthesized proteins can be analyzed in this way, while the post-gel staining technique does not distinguish in-between newly synthesize and proteins already expressed with a long half-life. The combination of the two techniques is really useful in cell studies where detailed protein mechanisms and processes are being investigated.

36

CHAPTER 7 REFERENCES

www.proteomics.com www.wikipidia.com www.seminarsonly.com www.proteotech.com VanBogelen RA, Schiller EE, Thomas JD, Neidhardt FC: Diagnosis of cellular states of microbial organisms using proteomics. Electrophoresis 1999, 20:2149-2159.

37