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      Proteomics for biomedicine: a half-completed journey

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      EMBO Molecular Medicine
      WILEY-VCH Verlag

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          Abstract

          Although I studied physics and mathematics until the master's level, I always knew that I wanted to get into biology, whose golden age I was sure was happening in my lifetime. When the opportunity came knocking – in the shape of John Fenn, who was spending a sabbatical at the University of Göttingen – I did not hesitate to take up his offer to come with him to Yale for my PhD. John was working on a far-out idea: that one could ionize biomolecules by applying an electric field to the outlet of a needle through which a liquid was flowing. At first there was little interest in ‘electrospray’, but as soon as it became clear that large, intact proteins survive this process (Mann et al, 1989; Meng et al, 1988), the community was hooked. This included the Nobel Prize committee, who gave John a share of their prize for chemistry in 2002. »I took one of the most ambitious and stressful decisions of my professional life: I bet everything on the success of mass spectrometry-based protein characterization.« Electrospray has been my mainstay ever since. The only interruption was as a post-doc, which I did in Denmark – following my Danish wife, whom I had met at Yale. Peter Roepstorff's group taught me a lot about protein chemistry but they had not acquired electrospray yet, so I missed the initial wave of discoveries done with our new technology! This turned out to be a blessing in disguise, however, because it motivated me to develop software instead. Specifically I attacked the problem of how to identify peptides in sequence databases given a minimal amount of mass spectrometric information. When in 1992 I was offered a group leader position at the European Molecular Biology Laboratory (EMBL), I took one of the most ambitious and stressful decisions of my professional life: I bet everything on the success of mass spectrometry (MS)-based protein characterization. At the time, despite much talk, MS had not become a serious tool for high sensitivity protein detection in biology, which was dominated by chemical methods such as Edman degradation instead. Fortunately, we soon made two crucial breakthroughs: Matthias Wilm devised nanoelectrospray, a miniaturized and highly sensitive form of electrospray, and Andrej Shevchenko developed protocols for liberating proteins from gels so that they could be analysed by MS. Together with our novel bioinformatic algorithms (Mann & Wilm, 1994), and the beginning genome sequencing efforts, this enabled identification of minute amounts of protein – far exceeding the capabilities of Edman sequencing and firmly establishing mass spectrometry in molecular biology research (Wilm et al, 1996). Among the prize catches of these years was the identification of caspase-8 (with Peter Krammer and Vishua Dixit) and the catalytic subunit of telomerase (with Tom Check). I am sure that these successes would not have happened without the high pressure environment at EMBL. Together with complementary work by the Yates and the Aebersold groups, these were the first steps of mass spectrometry in biological research, which eventually led to its establishment as the standard tool for protein identification. After EMBL, my move back to Denmark (and reunion with my family) was made possible by the Danish Natural Research Council. The next 7 years were very fruitful and our group developed many of the technologies that are now in general use – including the method of stable isotope labelling by amino acids in cell culture (SILAC; Ong et al, 2002), which has become a gold standard in quantitative accuracy for proteomics. I continued to be involved in programming too, which gave us quite a competitive edge in devising new strategies to retrieve biologically meaningful data from proteomics. During these years we started to apply proteomics to define the members of protein complexes. First examples included the U1 subunit of the yeast spliceosome (with Reinhard Luehrmann), the entire human spliceosome (with Angus Lamond) and the centrosome (with Erich Nigg). Interaction and organellar proteomics continue to be some of the most promising areas for proteomics and together with Tony Hyman in Dresden we are currently pursuing a large-scale effort to produce a high quality human interactome. We also used proteomics to study mechanisms of growth factor stimulation of SILAC labelled cells. By precipitating and then quantifying tyrosine-phosphorylated proteins at different time points, we could follow the signal as it spread to many and diverse substrates (Blagoev et al, 2004). Some of the proteins that we cloned at the time have become quite important in the growth factor signalling field. Several years later, we extended these experiments to generate a first global picture of ‘early information processing’ by the cell in response to growth stimulus (Olsen et al, 2006). In 2005, I moved to the Max-Planck Institute of biochemistry in Martinsried (Munich) where we diversified into a variety of biological and medical directions while keeping our core emphasis on instrumentation and bioinformatics. The Max-Planck institutes are fantastic environments for science in general and for our work in particular. Here, my group is able to work on the entire chain of proteomics – from technologies of proteomic sample preparation, through chromatographic and mass spectrometric innovations to methods in computational proteomics (Fig 1). Bioinformatics development connected to the analysis of proteomics data sets has always been a major focus in my group but in Martinsried it was kicked into high gear by Jürgen Cox. He developed the MaxQuant suite of proteomics tools that are now widely used by the community to analysed proteomics datasets (Cox & Mann, 2008). We then apply all these technologies to a wide range of biological problems; partly to show that proteomics can be a powerful tool in these different fields. »The most ambitious project we undertook at our new location was to crack a complete proteome.« Figure 1 Workflow of high resolution and quantitative proteomics. The most ambitious project we undertook at our new location was to crack a complete proteome. One of the limitations of proteomics had always been that usually only a small number of proteins were actually identified and quantified. Pre-MS techniques such as 2D gel electrophoresis usually identified a few dozens of proteins, a far cry from the thousands of probes that the microarray community was putting on their chips. To show that the full proteome was amenable to MS analysis, we collaborated with Tobias Walther (now at Yale), to quantify haploid versus diploid yeast (de Godoy et al, 2008). Very recently, we have shrunk the analysis time needed to cover nearly the entire yeast proteome to just a few hours (Nagaraj et al, 2011a). The human proteome is not quite complete yet, but we are getting very close (Nagaraj et al, 2011b). However, these studies are just first steps; in the future the task for proteomics will include detection and quantification of protein isoforms, a nearly complete set of protein modifications and doing all this as a function of time and cellular localization – a full program for at least one more generation of proteomics researchers! »Using a variant of the SILAC technology called ‘spike-in’ or ‘super’ SILAC it is now possible to measure the proteome of tumour biopsies at great depth and with extremely high precision.« Recently, proteomics technologies have become sufficiently evolved that it is now realistic to measure clinical material. Using a variant of the SILAC technology called ‘spike-in’ or ‘super’ SILAC it is now possible to measure the proteome of tumour biopsies at great depth and with extremely high precision (Geiger et al, 2010). This is one of the areas that my group will focus on in the coming years and we already have proof of principle in defining protein expression signatures in difficult to diagnose lymphoma subtypes. It is clear that there is now an opportunity to make a great clinical impact using high resolution and robust proteomics technologies (Fig 2). Figure 2 Proteomic analysis of breast cancer samples (Tamar Geiger, Jacek Wisniewski and Matthias Mann). In conclusion, whenever one really wants to understand biological function, one has to deal with proteins and mass spectrometry is the method of choice to do this. Clearly, the fun is just beginning as we now get to apply the tools developed by the community over the last decades. So, what does the future hold for proteomics and where will it make unique contributions? Areas to watch include the analysis of complete mammalian proteomes, including absolute quantification of cellular proteins, increasingly sophisticated studies of the function of thousands of post-translational modifications as well as protein interaction studies. Proteomics is starting to be used to probe the effects of genome variation between humans at the functional level and I predict that this will be an expanding area. In a slightly longer perspective, proteomics will become an important basis on which systems biological modelling of the cell will be built (Cox & Mann, 2011).

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          Most cited references12

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          Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.

          Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification and quantitation of complex protein mixtures. Here we describe a method, termed SILAC, for stable isotope labeling by amino acids in cell culture, for the in vivo incorporation of specific amino acids into all mammalian proteins. Mammalian cell lines are grown in media lacking a standard essential amino acid but supplemented with a non-radioactive, isotopically labeled form of that amino acid, in this case deuterated leucine (Leu-d3). We find that growth of cells maintained in these media is no different from growth in normal media as evidenced by cell morphology, doubling time, and ability to differentiate. Complete incorporation of Leu-d3 occurred after five doublings in the cell lines and proteins studied. Protein populations from experimental and control samples are mixed directly after harvesting, and mass spectrometric identification is straightforward as every leucine-containing peptide incorporates either all normal leucine or all Leu-d3. We have applied this technique to the relative quantitation of changes in protein expression during the process of muscle cell differentiation. Proteins that were found to be up-regulated during this process include glyceraldehyde-3-phosphate dehydrogenase, fibronectin, and pyruvate kinase M2. SILAC is a simple, inexpensive, and accurate procedure that can be used as a quantitative proteomic approach in any cell culture system.
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            Comprehensive mass-spectrometry-based proteome quantification of haploid versus diploid yeast.

            Mass spectrometry is a powerful technology for the analysis of large numbers of endogenous proteins. However, the analytical challenges associated with comprehensive identification and relative quantification of cellular proteomes have so far appeared to be insurmountable. Here, using advances in computational proteomics, instrument performance and sample preparation strategies, we compare protein levels of essentially all endogenous proteins in haploid yeast cells to their diploid counterparts. Our analysis spans more than four orders of magnitude in protein abundance with no discrimination against membrane or low level regulatory proteins. Stable-isotope labelling by amino acids in cell culture (SILAC) quantification was very accurate across the proteome, as demonstrated by one-to-one ratios of most yeast proteins. Key members of the pheromone pathway were specific to haploid yeast but others were unaltered, suggesting an efficient control mechanism of the mating response. Several retrotransposon-associated proteins were specific to haploid yeast. Gene ontology analysis pinpointed a significant change for cell wall components in agreement with geometrical considerations: diploid cells have twice the volume but not twice the surface area of haploid cells. Transcriptome levels agreed poorly with proteome changes overall. However, after filtering out low confidence microarray measurements, messenger RNA changes and SILAC ratios correlated very well for pheromone pathway components. Systems-wide, precise quantification directly at the protein level opens up new perspectives in post-genomics and systems biology.
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              Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry.

              Molecular analysis of complex biological structures and processes increasingly requires sensitive methods for protein sequencing. Electrospray mass spectrometry has been applied to the high-sensitivity sequencing of short peptides, but technical difficulties have prevented similar success with gel-isolated proteins. Here we report a simple and robust technique for the sequencing of proteins isolated by polyacrylamide gel electrophoresis, using nano-electrospray tandem mass spectrometry. As little as 5 ng protein starting material on Coomassie- or silver-stained gels can be sequenced. Multiple-sequence stretches of up to 16 amino acids are obtained, which identify the protein unambiguously if already present in databases or provide information to clone the corresponding gene. We have applied this method to the sequencing and cloning of a protein which inhibits the proliferation of capillary endothelial cells in vitro and thus may have potential antiangiogenic effects on solid tumours.
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                Author and article information

                Journal
                EMBO Mol Med
                EMBO Mol Med
                emmm
                EMBO Molecular Medicine
                WILEY-VCH Verlag (Weinheim )
                1757-4676
                1757-4684
                February 2012
                : 4
                : 2
                : 75-77
                Affiliations
                simpleDepartment of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Munich, Germany and The NNF Center for Protein Research, Faculty of Health Sciences, University of Copenhagen Copenhagen, Denmark E-mail: mmann@ 123456biochem.mpg.de
                Author notes

                Matthias Mann is awarded the 2012 Louis Jeantet Prize for Medicine for his work on developing and applying proteomics to all areas of basic and medical research. He is the Director of the Department of Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry in Munich, Germany.

                Article
                10.1002/emmm.201100198
                3376839
                22278912
                7dec72dd-e27a-4f87-a525-6eb46c4d6ccc
                Copyright © 2012 EMBO Molecular Medicine
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                Molecular medicine
                Molecular medicine

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