Biogerontology 3: 133–136, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands.

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The proteomics of ageing ∗ Gary S. Cobon1,∗∗ , Nicole Verrills1 , Penny Papakostopoulos2, Hayden Eastwood2 & Anthony W. Linnane2 1 Australian

Proteome Analysis Facility, Level 4, F7B, Macquarie University, Sydney, Australia; 2 Centre for Molecular Biology and Medicine, Epworth Medical Centre, Richmond (Melbourne) Australia; ∗∗ Author for correspondence (e-mail: [email protected]; fax: +61-2-98506200)

Key words: ageing, human skeletal muscle, individual variation, proteomics

Abstract Proteomics provides an extremely powerful tool for the study of variations in protein expression between individuals, different disease states and different conditions. One of the major challenges facing the medical profession in the forthcoming decades is to understand the changes that occur in individuals as they become older and to attempt to develop means to improve the quality of life for the otherwise healthy ageing population. The present study describes the first phase of such an investigation in which the protein composition of human skeletal muscle samples from young and aged subjects are compared. These results provide the beginning of a Human Aged Skeletal Muscle Profile reference map which is an essential first step to further investigations.

Introduction Biological processes involve many steps including the transcription of genes into mRNA molecules, translation of those mRNAs into proteins, post-translational modifications of those proteins, the formation of complex protein/protein interactions and subsequent degradation and inactivation of the resulting proteins. These functions are controlled by a complex network of factors and activities that miraculously meld together to form a functional cell. These cells then interact with each other to form a functional organism. All of these activities from DNA replication to cellcell interaction involve proteins. Although preliminary, it has been estimated that the human genome codes for 30–40,000 proteins. The number of genes however does not describe the number of different proteins that comprise a human cell or tissue. Firstly there is the potential for a variety of differential splicing events to occur which would lead to different mRNA molecules being coded by the one open reading frame. Such differential splicing is ∗ Presented at the 1st ASCMG meeting, Melbourne, Australia,

March 19–22, 2001.

known to occur but its extent is not known. By the time the myriad of post translational modifications are considered, such as glycosylation, phosphorylation, lipidation and proteolytic digestion, the number of different proteins that are expressed by a human cells is many of times, more than the coding potential of the organism. None of these differences can be identified by only studying the DNA of the cells or even the transcription of the cells. The study of these proteins, their expression levels, post translational modifications and protein-protein interactions is referred to as proteomics. There is a large number of factors that control the amount of each protein that is expressed by a cell at any particular time. These include the controls on the transcription of the genes, the codon usage, the rates and extent of post translational modification, nature and abundance of proteins with which the gene product interacts, substrate levels and eventually rate of proteolytic degradation. The levels of particular proteins vary with the cell type, the cell growth phase, external stimuli such as hormones, cytokines and growth factors and, of course, age. Even though the DNA within a cell may not alter appreciably, the proteins that are expressed vary considerably. These

134 variations cannot be detected by only investigating the genetic makeup of the cells but they can be identified by investigating the protein profile or the proteome of the cells. The variation in protein expression or posttranslational modification of the proteins leads to variation in the protein-protein interaction within the cells. In turn, these sometimes minor differences in the large number of protein interactions lead to individual-toindividual variations seen among an out-bred population. More substantial differences in protein expression, modification or interaction will lead to disease states such as are observed in many forms of cancer for example. Between these extremes are the more subtle modifications in protein expression, modification and interaction that lead to gradual ageing of cells, tissues and individuals. The rate at which this process occurs differs between individuals for reasons that are not fully understood. If the factors involved in the ageing process could be understood at a detailed biochemical level, it is likely that the process could be modulated to ameliorate the process thereby impacting upon an improved quality of life and life expectancy. Modern developments in biotechnology are providing the tools to enable such investigations to take place. In the first instance, the sequencing of the human and mouse genomes has enabled the identification of some 30,000 open reading frames that can now be individually screened by hybridisation techniques to determine the level of expression of each under a variety of conditions. Such studies will generate enormous amounts of very valuable information.

Proteomics The developments in the protein chemistry area are further contributing to this revolution in biology. In particular the technology referred to as proteomics has enabled detailed analysis of the differences in protein expression between any two cell-types. Proteomics provides a platform technology that is applicable to all biology. The Australian Proteome Analysis Facility (APAF) specialises in proteomics and provides a service to all research and commercial organisations throughout the world. APAF is a Major National Research Facility which has been established at Sydney’s Macquarie University. It has been in full operation for 4 years and specialises in high throughput processing of samples. APAF is

the only such facility in Australia and established collaborations with more than 120 different research organisations during 2000. The technology employed at APAF can be divided into a number of stages. The first is the solubilisation of the proteins within the sample. This may seem trite but it is unfortunately often overlooked by researchers. On several occasions, collaborators have approached us with a proposal to investigate a particular protein of interest to them only to find that the majority of that protein is still in the insoluble membrane phase of the extract. At APAF, we have spent considerable time and effort investigating optimal means to solubilise proteins in a form that it compatible with first dimensional separation by isoelectric focussing. It is not possible to use powerful ionic detergents such as SDS as these coat the proteins with charges that interfere with isoelectric focussing. APAF has developed a series of extraction buffers suitable for different situations. The proteins are then separated by Isoelectric focussing. This technology has improved vastly over the past 5 years which has resulted in the gels being far more reproducible than was the case previously. The major advance has been the development of immobilised pH gradients within a gel matrix attached to plastic strips. Gradients can now be obtained commercially covering a variety of pH ranges which can be broad (e.g. pH 3–7) or narrow covering any of several one-pH units (e.g. 3–4 or 5.5–6.6). This enables the researcher to conduct initial examination of the sample using broad range strips and then to focus in on areas of particular interest with narrow range gels. Alternately, if the pI of a particular protein of interest is known, the most highly resolving fractionation can be selected immediately. In our experience, no matter whether it is a narrow range or broad range resolution, approximately 3000 proteins can be observed on each gel. Thus the narrow range strips enable far greater resolution and enable the low abundance proteins to be observed. The next step involves equilibrating the strip in buffer containing SDS and separation of the proteins in the second dimension based on size by electrophoresis through polyacrylamide gels. Gels can be constructed to resolve proteins of particular interest by altering the polyarylamide concentration. More generally however, gradient gels are selected that resolve proteins over a large size range. This technology has been in use for more than 20 years and is standard in many laboratories throughout the world.

135 The gels are then fixed stained. Traditional stains include Coomassie and silver and are still used quite extensively in many laboratories. However, more recently, fluorescent stains have replaced the traditional stains. SYPRO stains in particular are utilised in many laboratories as they are sensitive, simple to use, safe and, most importantly, the intensity of staining is proportional to the amount of protein in the gel over at least two orders of magnitude. Therefore it is possible to obtain an impression not only of whether a particular protein is present in the extract but also an idea of its abundance in relation to other proteins in the extract. The down side is that these stains are relatively expensive but this is more than compensated by the increased sensitivity. The gels are then scanned and images of the gels are compared using any of a number of different computer programmes that are on the market. Effectively the image of one gel is stained in silico one colour and the other gel another colour. The two images are then superimposed and the differences in colour of the various spots used to provide an estimate of the relative abundance of the proteins on the gel. By this method it is possible to ascertain not only whether a protein is present in one sample but not in another but whether there is a difference of perhaps two to four fold in the abundance of a particular protein. It is these variations in the amount of various protein that makes the difference between the metabolism of one cell type compared with another. It is essential that the gels are of the highest quality in order for this comparison to be meaningful and it is also important to take account of individual-to-individual variations that occur. It is possible to run gels of extracts from several different individuals and to average their stains before the comparison which does reduce this variation. Once differences in the gel profiles have been compared and the proteins that vary between the extracts identified, these proteins are cut from the gels and digested with an endoproteinase, usually trypsin. The digests are then applied to a mass spectrometer grid and the size of the tryptic fragments is determined by Matrix Assisted Laser Desorption Ionisation Time of Flight, Mass Spectrometry (MALDI-TOF Mass Spec). The enormous developments that have been made over the past 2–3 years in sequencing the genomes of a variety of organisms has meant that there are comprehensive databases that can be interrogated with the peptide mass fingerprint information and increasingly this information alone is sufficient to provide an identification of the proteins of interest.

Should more information be required, dual mass spectrometry can be utilised to obtain amino acid sequence information from selected peptides. The advantage of doing so by mass spectrometry is the sensitivity compared with the traditional chemical methodology. As little as a few tens of picomoles of protein is required for more than sufficient sequence to provide protein identification or sufficient sequence to design discriminating PCR primers to clone the genes of interest. The mass spectrometry technology can also be utilised to identify differences in the post-translational modification of various proteins. It is often clear from the gel profiles if there are significant changes in the glyscosylation of the major proteins in the extract. However, the precision of the mass spectrometry is required to identify variations in other modifications such as phosphorylation of the proteins. It is becoming more routine to do so which is adding yet another great deal of power to the analysis of the various disease states. Utilising this technology, it is possible to very rapidly obtain a great deal of information on the differences in the biochemistry of cell types. If the project requires the entire proteome to be determined, several thousand proteins can be studied from one series of gels. Alternately the programme can be designed to concentrate exclusively on one subset of proteins from a particular range of size and/or charge or a subset of proteins that vary in abundance by a predetermined level of abundance. Once the series of variant proteins has been identified, it is often possible to determine the major biochemical pathways that differ between the two cell types. Alternately the major variant proteins can be identified which will provide targets for chemotherapies or immunological intervention. Proteomics analysis, when used together with transcriptional analysis, are extremely powerful tools to study the biochemistry of processes such as ageing. While the transcriptional analysis will lead to the identification of genes that are differentially expressed, the proteome analysis will enable the ultimate identification of the affected pathways. The affected pathways may arise as a direct consequence of modifications in the transcription of the gene coding for the protein, or by such factors as defects in the post translational modification of the protein leading to varied half-life of the protein or defects in the ability of the protein to interact with other proteins in the cell. Thus transcription analysis is valuable to provide an understanding of the molecular basis for the disease or metabolic

136 disorder. However proteome analysis is important to provide potential targets for therapeutics and an understanding of the mechanisms of the disease or disorder. Together they provide the complete picture.

Ageing studies The Centre for Molecular Biology and Medicine (The Centre), in collaboration with The Australian Proteome Analysis Facility (APAF) has initiated an investigation, to create a comparison protein profile ‘a reference map’ of high abundance proteins in skeletal muscle (vastus lateralis) obtained from young and aged subjects. While the work is at a preliminary stage, 7 Vastus Lateralis muscle samples have been run on a variety of 2D gel pH arrays and analysed for common proteins from aged subjects (56–79 years,

[Av.65; Sd.7.4]). Our findings suggest each sample contains approximately 2000 (1737–2234 [Av.2058; Sd.205]) high abundance proteins (detected at a sensitivity of 1 nanogram of protein per spot). Approximately 60% of these proteins (Av. 1112 proteins per sample) were found to be common (defined as occurring in a minimum detection of 6 of the 7 subjects). These high abundance proteins are the beginnings of a Human Aged Skeletal Muscle Protein Profile (reference map). The less extensively matched proteins (Av 946 proteins per sample) we suggest probably reflect the characteristic stochastic variation between ageing individuals. The project is a long term one and the Centre’s work continues towards establishing a protein reference map of skeletal muscle for aged subjects preparatory to the development of a similar map of young muscle samples.