Laboratory of Pharmaceutical Biotechnology, Ghent University and 1 Laboratory for Molecular Immunology and Inflammation, Division of Rheumatology, Ghent University Hospital, Ghent, Belgium.
Correspondence to: Dirk Elewaut, Laboratory for Molecular Immunology and Inflammation, Division of Rheumatology, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium, E-mail: Dirk.Elewaut{at}Ugent.be, or Dieter Deforce, Laboratory of Pharmaceutical Biotechnology, Harelbekestraat 72, 9000 Ghent, Belgium, E-mail: Dieter.Deforce{at}Ugent.be
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Abstract |
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KEY WORDS: Proteomics, Two-dimensional gel electrophoresis, Mass spectrometry, Rheumatology, Autoimmunity
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Introduction |
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From genomics to proteomics
The Human Genome Initiative provided us with a blueprint of the human genome [3]. By studying the genome, it became clear that the behaviour of genome products is difficult to predict from the gene sequence alone. This realization has led to a variety of new large-scale disciplines analysing downstream of the genome sequence. The introduction of these new techniques came with a variety of omics terminologies (Fig. 1).
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Transcripts are further translated into proteins, which are considered to be the main carriers of biological activity. One gene can lead to different mRNA molecules, due to alternative splicing. These mRNA species are further translated into proteins. These proteins can become fully active by adding post-translational modifications (PTM) or interaction with other proteins. Due to all these processes, one gene can result in many different protein isoforms. Protein function is highly dynamic and influenced by environmental factors. It not only depends on the amino acid sequence, but also on PTM, degradation and/or compartmentalization of proteins in protein complexes. Proteomics aims at determining protein expression levels, but also protein structure, modifications, localization and interaction with other proteins.
Proteomics is one area among the various omics disciplines, which also include new disciplines, such as metabolomics. Thus, the integration of all these areas, called systems biology, will lead to a comprehensive understanding of cellular biology.
For evident reasons, we will further discuss only proteomics and its applications in the field of rheumatology.
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Types of proteomics |
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Expression proteomics seeks to identify all the protein species present in a proteome of a cell, tissue or organism at a certain time. Structural proteomics, by contrast, aims at identifying the molecular structure, i.e. the amino acid sequence of the protein entities involved in a given process and to relate this information to the database of identified genes [4]. de Hoog et al. defined it as the comprehensive coverage of a single protein or domain structure [2]. Finally, functional proteomics describes the changes in protein abundance and modification during the differentiation, proliferation and signalling of cells, in both qualitative and quantitative terms. It also includes studies of the coordinated expression of proteins, as well as the elucidation of the sequence of regulatory events during all stages which a cell or an organism undergoes during its entire lifespan [4].
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Proteomic approaches |
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2DE is a high-resolution method for separating proteins in two dimensions, according to their isoelectric point (pI) in the first dimension and according to their size (molecular weight) in the second dimension. Firstly, proteins are separated by isoelectric focusing. Therefore, proteins are brought into a small gel strip that contains an immobilized pH gradient (IPG). This gel strip is applied on a plastic backing for easier handling and is further referred to as the IPG strip. When an electric field is applied over this IPG strip, the proteins migrate along the pH gradient in the strip until they reach the pH at which their overall charge is neutral: the isoelectric point (pI) of the protein. The result of this separation is a gel strip containing discrete protein bands at the pH position of their pI. This IPG strip is then applied to a polyacrylamide gel. An electric current drives the focused proteins out of the strip into the gel and separates the proteins according to their size. (For reviews on 2DE and sample preparation see [79].)
One of the greatest strengths of 2DE is its ability to resolve proteins differing in a single charge, and consequently in vivo modifications of proteins can be visualized. Multiple protein isoforms can be analysed by determining factors such as the solubilization conditions and pH range of the IPG strip. Currently, a wide range of pH gradients are available on the market [9, 10].
2D gel visualization
In order to visualize the separated proteins, 2D gels can be stained with a variety of different stains. Detection methods in 2DE can be divided into two large groups: general protein staining and specific detection of PTM. A limited overview of the most commonly used detection methods will be discussed.
Coomassie Blue is probably the most widely used general protein stain. Major advantages are its low cost, ease of use and its compatibility with identification by mass spectrometry. Because of its limited sensitivity (10 ng per protein spot), Coomassie Blue is mostly used for detection of proteins in preparative gels, from which spots are cut to be identified (see section Mass spectrometry and protein identification). Although silver stains are more sensitive (0.51 ng per protein spot), they have a very small linear range and are time-consuming because of the numerous steps involved in the protocol. Moreover, they are less compatible with the mass spectrometry-based identification of gel separated proteins.
Radioactive labelling is a very sensitive method of protein visualization; however, it is hazardous and expensive. It is only applied for very specific analyses, such as protein synthesis (35S) or protein phosphorylation (32P). Fluorescent dyes such as Sypro Ruby and Tangerine dyes are now becoming the standard for general protein staining. They are sensitive (1 ng per protein spot), have a large linear range (over three orders of magnitude), are extremely easy to use and are compatible with mass spectrometry. Fluorescent dyes can also be used to label protein complexes prior to 2DE. This so-called difference gel electrophoresis (DIGE) approach is gaining interest. The method makes it possible to fluorescently label (with cyanine dyes Cy2, Cy3, Cy4) as many as three different complex protein populations prior to mixing them together and running them on the same 2D gel [11].
As the role of post-translational modifications has become more prominent, specific detection methods of protein modifications have been developed in which specifically glycoproteins, phosphoproteins and other modified proteins can be visualized in the gel. For detailed information on detection in proteomic analysis, see reference [12].
2D gel data analysis
Although 2D gel data analysis is complex and laborious, state-of-the art software programs have made this process easier and more straightforward. After acquiring 2D images, protein spots have to be detected. Such a protein spot has three characteristics: the x- and y-axis of the spot, representing the pI of the protein and its molecular mass respectively, and the z-axis, which is a measure for the intensity of the spot and indicates the amount of protein present in the spot. A spot on a 2D gel can therefore correspond to a single protein, an isoform of a protein or a modified protein.
In order to compare spots across several gels, spots have to be matched to each other. This is generally done by highlighting spots that are clearly present in all the gels as marker points. Using these markers, other spots present in the gels are matched to one another. Exploring differences in spot intensity or tracking protein spots that appear or disappear on gels derived from experimental and control conditions is the main goal of functional proteomics.
Spots of interest are further processed and identified by mass spectrometry (MS) (Fig. 2). Briefly, protein spots are cut from the gels and digested by using a sequence-specific protease (e.g. trypsin, chymotrypsin, Arg-C, Asp-N). The resulting mixture of peptides is desalted and analysed by MS.
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Although both proteins and peptides can be analysed, we further discuss only peptide analysis by MS, since this is the way to identify proteins derived from different proteomic approaches.
MALDI
MALDI is a soft ionization technique that uses matrix material in order to get the peptides into the gaseous phase. Protonated peptides are coprecipitated with matrix material on a metal surface and air-dried. The resulting dried spots are irradiated by laser pulses, usually from small nitrogen lasers. The matrix contains small organic molecules with absorbance at the wavelength of the laser (e.g. -cyano-4-hydroxycinnamic acid or dihydrobenzoic acid).
The exact nature of the ionization process in MALDI is largely unknown. During ionization and desorption, matrix molecules pass energy absorbed from the laser light to the charged peptides, which are dispersed into gaseous phase (Fig. 3). Ionized peptides are further analysed in the mass spectrometer.
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Mass analysers
Every mass spectrometer consists of three basic elements: an ionization source, one or more mass analysers and a detector. The names of the mass spectrometers are simply a compilation of their ionization source and the mass analyser. There is a wide range of different types of mass spectrometers, commonly divided into two groups: single MS machines, in which only one mass analyser is present and tandem MS or MSMS machines, in which more mass analysers are present.
The most commonly used single MS analysers are time-of-flight (TOF), quadrupole (Q) and ion-trap. The most generally used MSMS analysers are Q-TOF and TOF-TOF.
The list of mass analysers cited above is far from complete; however, these are the ones that are used most routinely in protein identification. (For extended reading on mass analysers, see specific sections of references [15, 17].)
The great difference between single MS and MSMS analysers is that the latter can not only separate the ions according to their mass, but are also able to select and fragment ions. The benefit of this will become evident in the next section.
Identification of proteins by peptide mass fingerprinting. When ions are passed into a mass spectrometer, they are separated according to their mass. This analysis results in a mass fingerprint of the peptides present in the mixture. These peptides are the result of the cleavage of a particular protein using a sequence-specific protease, such as trypsin. The set of masses obtained by the mass spectrometer is compared with the theoretically expected tryptic peptide masses for each protein present in a certain database (Fig. 5).
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These search engines will subsequently rank the positively identified proteins according to the number of peptide matches. Proteins identified positively by PMF have, on average, a minimum peptide coverage of 20%. Identification of proteins by PMF is often the result of MALDI-TOF analysis.
Identification by tandem mass spectrometry. Mass spectrometers can determine not only the mass of the peptide but also its amino acid sequence, which is typical of MSMS mass analysers. A particular peptide is selected out of a mixture of peptides in the first mass analyser and is subsequently dissociated by collision with an inert gas, such as nitrogen or argon, in a collision cell. During these energetic collisions of the selected peptide and the collision gas, bonds are broken along the peptide backbone. In most applications, this leads to so-called b and y ions, which indicate fragmentation at the amide bond with charge retention on the N or C terminus, respectively [18]. The resulting fragments are analysed by the second mass analyser, producing a tandem mass spectrum, also called the fragmentation spectrum or MSMS spectrum. Each peptide fragment in a series differs from its neighbour by only one amino acid. It is therefore possible to determine the amino acid sequence by considering the mass difference between the neighbouring peaks in a series [18]. The experimental MSMS spectrum is matched against a calculated spectrum for all peptides in the database (e.g. mascot, SEQUEST). A score is calculated which reflects the quality of the match between the experimental spectrum and the theoretical one (Fig. 6).
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Identification by sequence-tag analysis. It is not necessary to obtain a complete amino acid sequence of a peptide in order to be able to identify the protein. A short sequence tag is often sufficient to identify the corresponding protein from which the peptide(s) was (were) derived. A small stretch of amino acids can be combined with the start mass and the end mass of the series, which specifies the exact location of the sequence in the peptide and the known cleavage specificity of the enzyme. Such a peptide sequence will then retrieve from the database (e.g. MS-Tag, mascot, dbEST) one or a few sequences whose theoretical fragmentation pattern is matched against the experimental one.
Tandem mass spectrometry and sequence-tag analysis are routinely obtained by ESI-Q-TOF, MALDI-Q-TOF or MALDI-TOF-TOF mass spectrometers.
Alternative approach: gel-free proteomics
2DE is still widely used in the separation and quantitative analysis of complex protein mixtures. It is by far the best technique to visualize and analyse PTM of proteins; however, the technique has its limitations. These limitations include the low throughput of samples, problems in detecting low-abundance, extremely basic and acidic proteins and proteins with very high or very low molecular weights. Because of these restrictions, several groups have explored alternative separation techniques for proteins using MS [1926]. Many of these alternative separation techniques rely on the ability of tandem mass spectrometers to collect sequence information from a specific peptide even if several other peptides, derived from other proteins, are in the sample at the same time. In order to be able to perform quantitative analysis, stable isotope labelling of the samples is used. This is achieved in different ways: proteins can be labelled metabolically by culturing cells in media that are enriched (e.g. containing 15N salts or 13C-labelled amino acids) or by enzymatic digestion of proteins in 18O-containing water [19, 26]. Proteins can also be labelled at specific sites with an isotopically encoded reagent. One of the best known examples of this technique is the isotope-coded affinity tag (ICAT) method. This method stably labels peptides of two populations of proteins using reactive probes that differ in isotope composition [24]. Proteins from the experimental mixture are labelled with either the heavy reagent, composed of eight deuterium atoms (d8-ICAT), or the light reagent, composed of eight hydrogen atoms (d0-ICAT), and then mixed together. The mixed proteins are digested by a site-specific protease, e.g. trypsin. ICAT-labelled peptides are isolated from the complex mixture using the biotin tag present in the ICAT reagent. Selected peptides are further separated and analysed by liquid chromatography on-line coupled to electrospray tandem mass spectrometry (LC-ESI/MSMS). Mass spectrometry is used to reveal the ratio of isotopic molecular weight peaks that differ by 8 Da, and this gives a measure of the relative amount of each protein present in the original samples [20, 21] (Fig. 7).
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It is important to note that both gel-based and gel-free proteomic approaches have their strengths and drawbacks. It is therefore crucial to find out which method is best suited for achieving the goals that were set out.
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Present status of proteome research in the field of rheumatology |
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Proteomic papers in the field of rheumatology can largely be divided into three groups: protein target identification by differential screening of biological fluids; biomarker searching in rheumatic tissues; and proteomic surveillance of autoantigens.
Protein discovery by differential screening of biological fluids
The comparison of protein patterns in body fluids of diseased and healthy individuals has the potential to identify new diagnostic tools and could subsequently lead to new disease-specific therapies. Analysis of serum and synovial fluids is of great importance in the diagnosis of rheumatic diseases, but also in monitoring a patient's response to a particular medication. Currently available biomarkers are related to the degree of inflammation, but lack straightforward correlation with disease severity or do not change dramatically in response to treatment [29]. Moreover, it is known that joint damage may progress in spite of decreased inflammatory activity and erosions may develop in patients who have few clinical signs of inflammation [30, 31]. In addition, there is a need for serum biomarkers in other rheumatic pathologies, such as spondyloarthropathy (SpA).
Large-scale analysis of synovial fluid and serum provides information on disease-specific differences due to local processes in the inflamed joint, compared with systemic disease symptoms. Several discriminating acute-phase proteins have been identified in serum, plasma and synovial fluid of different rheumatic pathologies using classic proteomic approaches [3236]. Serum amyloid A protein is present in the synovial fluid and plasma of RA patients, but is undetectable in plasma or synovial fluid of osteoarthritis (OA) patients. This acute-phase protein has a crucial role in the very early organization of host defence, but could have a destructive effect in chronic inflammation [33]. Certain fibrinogen isoforms and the calgranulin protein isoforms show disease-associated expression and processing in biological fluid matrices of RA and OA patients [33, 36].
Although the classic proteome approach is able to identify new discriminating targets, it is not applicable for fast screening. Several groups have therefore attempted to directly analyse pathological fluids by mass spectrometry [30, 37, 38]. Liao et al. identified at least 33 possible protein biomarkers in synovial fluid of RA patients by applying a semiquantitative gel-free proteomics approach using internal 13C-labelled peptide standards [30]. Besides members of the S100 protein group, other proteins, such as osteopontin (which plays a role in attachment and invasion of synovial fibroblasts [39]), cyclophilin (a proinflammatory mediator in arthritis [40]), cathepsin B and many others were highly increased in synovial fluid of RA patients with erosions [30]. These newly identified proteins need further validation in larger patient cohorts to evaluate their specificity and sensitivity.
Large-scale proteomic analysis of biological fluids has great potential. However, one must realize that these samples contain highly abundant proteins (e.g. albumin, IgGs). These proteins are detrimental to the detection of other species in the sample. Good sample clean-up or efficient enrichment techniques are therefore pivotal for the investigation of proteins present in smaller concentrations.
Biomarker discovery in rheumatic tissue samples
Potentially interesting samples are not only synovial fluid and serum, but may also include joint components such as synovial tissue and cartilage. The molecular understanding of the process leading to joint destruction is not complete yet. Therefore, large-scale proteome and genome analysis of joint tissue could reveal important information about mechanisms leading to joint damage.
Joint destruction in non-inflammatory arthritis or OA is characterized by degradation of articular cartilage. It is thought that this is due to an imbalance between anabolism and catabolism of the extracellular matrix. Therefore, several groups have been studying the regulation of the protein synthesis and protein secretion by cartilage using proteomic approaches [41, 42].
Proteome analysis of the phenotype of articular cartilage in OA and normal patients could be of great help in understanding the endogenous control mechanisms of matrix turnover in cartilage. Moreover, this information could be of help in tissue engineering during stem-cell differentiation.
Joint destruction in inflammatory arthritis is very different from OA and is initiated by chronic inflammation of the synovial tissue. To unravel the molecular and cellular mechanism of chronic synovitis, synovial proteomes of RA and OA patients have been investigated. The group of Thiessen has used a multi-western blot PowerblotTM (BD Biosciences) for differential protein screening of synovium obtained from OA and RA patients [43]. This commercially available method offers the possibility of analysing over 700 protein species simultaneously. Some differentially expressed proteins were further validated in a larger patient cohort, namely Stat 1, p47phox and MnSOD, which were up-regulated in RA, and cathepsin D, which showed lower expression in RA in comparison to OA. All of these proteins are related to inflammation or cytokine-induced activation, or have been described in tissue damage and joint destruction in arthritic joints [43].
Chronic synovitis is characteristic not only of RA but also of SpA. These two frequent forms of inflammatory arthritis have a different clinical outcome, but have some shared features, such as chronic synovitis. Our group analysed the synovial proteome of RA, SpA and OA patients and came to the conclusion that the synovial proteome of RA patients consists of a unique protein expression pattern in contrast to the synovial proteome of SpA patients [44]. We identified several proteins related to either RA or SpA inflammation, amongst them MRP-8 (calgranulin A), a well-known biomarker for inflammatory arthritis. MRP-8 was highly up-regulated in the inflammatory arthritides in comparison with OA patients, in whom this protein spot was completely absent from the 2D images [44].
Proteome studies of fibroblast-like cells derived from the synovium of patients with inflammatory arthritis can aid in resolving the mechanism(s) behind the synovial hyperplasia and the associated tissue damage [45]. Likewise, tissue derived from inflamed joints of animals can also be helpful in gaining access to the complex pathology of joint inflammation [46].
Proteomic surveillance of autoimmunity
The majority of rheumatic diseases are characterized by autoimmune processes. Identification of autoantigens playing a role in these complex pathologies is extremely important. Also, the presence of autoantibodies in otherwise healthy individuals may perhaps predict the development of autoimmune disease. Furthermore, the appearance of certain autoantibodies might predict the clinical course of a patient with established disease [47].
Using 2DE to separate tissue proteins, autoantibodies against these proteins can be detected in serum or synovial fluid of patients by western blotting. This technique was already being used in rheumatic pathologies in the late 80s [48, 49] and is currently widely applied. A recent example of this technology is described by Xiang et al. [50], who investigated autoantigens present in chondrocytes in both RA and OA patients. Triosephosphate isomerase (TPI) was identified as an OA-specific autoantigen. The autoantibodies recognized multiple epitopes on TPI and the titres were both high in serum and synovial fluid [50].
-Enolase, a well known autoantigen, has also been investigated by proteomics in several autoimmune diseases [51, 52]. Autoantibodies against glycolytic proteins, such as glucose-6-phosphate isomerase [53] and aldolase A [54], have been identified in RA sera. Moreover, dysregulation of glucose metabolism could play a role in the hyperplasia of synoviocytes in RA. Autoantibodies against these proteins might be implicated in the pathology of RA.
Not only the discovery of new autoantigens but also the analysis of specific post-translational modifications of these autoantigens has great potential in proteomic applications in rheumatic pathologies. Immunoreactive spots can clearly be identified using 2DE. These spots can represent a specific isoform of a protein or particular modification of a protein. Some of these PTMs, particularly citrullination of certain proteins, elicit autoimmune reactivity in RA [55, 56]. Other PTMs which may play a key role in the acquisition of autoantigenicity in rheumatic pathologies may be unmasked by proteomics [57].
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A look towards the future |
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There is no doubt that fast screening methods, such as array-based protein chips and quantitative mass spectrometry, will become very important in the future. Using these high-throughput techniques, the potential applications in diagnosis and in monitoring prognostic markers of patients with rheumatic diseases are almost unlimited.
There is, however, still a need for classic proteomic approaches. As the role of post-translational modifications will increase in the future, 2DE could play a part in elucidating specific protein modifications.
Synovial tissue proteomics could help us understand the complex nature of rheumatic pathologies. Techniques like laser-capture microdissection in combination with 2DE and mass spectrometry allow in situ analyses [58]. Specific cell types can be isolated and the disease-associated proteins subsequently determined.
As biomarkers, protein interactions and signalling pathways become apparent during proteome analyses, disease mechanisms may be unravelled that eventually could open new avenues for drug targets in the field of rheumatology.
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Acknowledgments |
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The authors have declared no conflicts of interest.
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References |
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