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cover story cover story Protein Separations Proteomics to Production Fig. 1. Electrophoresis. In 2-D SDS-PAGE, proteins in a mixture are first applied to an IPG strip (panel A). The proteins are focused in the strip to their isoelectric points (B). The strip is then applied to a slab gel containing SDS, in which they are separated by molecular weight (C). MW Investigations of many of the most intriguing biological questions today are dependent on a better understanding of individual proteins. With the human genome now unraveled, the complementary protein products’ roles, most of which remain to be discovered, have become a major focus. Endeavors to map out the proteins associated with their gene counterparts are critical to understanding questions such as the profiles of diseases and how to control them. Many different approaches are directed at these efforts, with protein separation remaining of paramount importance. Protein separation is used to fractionate a complex sample so it can be subjected to further analysis or experimentation. With pure fractions, researchers are able to better understand the function of a particular protein in a biological system. Two distinct but complementary technologies are used to separate most proteins: A + 7.1 7.1 8.4 8.8 8.4 7.1 3.9 8.8 3.9 8.8 8.4 3.7 5.3 3.7 5.3 Before focusing B pH 3 3.9 7.1 3.7 5.3 8.4 7.1 8.4 8.8 8.8 After focusing C 3.7 3.9 5.3 7.1 8.4 SDS-charged proteins resolved according to size in SDS-PAGE gel 8.8 – 10 electrophoresis and chromatography. Both exploit inherent protein characteristics. Electrophoresis — the application of an electric current to a solid-phase support such as a gel in which proteins can migrate — can separate proteins based on their isoelectric point, size (molecular weight), or both (Figure 1). Chromatography — the differential partitioning of proteins between stationary and mobile phases — separates proteins based on their charge, size, hydrophobicity, or affinity for particular compounds (Figure 2). Although both techniques have been in common use for some time, they continue to be used in new scientific fields. Why Separate Proteins Protein separations are invaluable to proteomics, drug discovery, and production. In both areas, steps include preliminary sample preparation, protein identification and characterization, and purification at both laboratory and process scales. Proteomics, or the study of the function of all expressed proteins, is heavily dependent on protein separation. Using the foundations of protein chemistry, proteomics has refined many commonly used techniques such as two-dimensional (2-D) gel electrophoresis. Often called the “workhorse of proteomics”, 2-D gel electrophoresis separates proteins in two dimensions, the first by isoelectric point and the second by size (Figure 1). 2-D separations of complex protein samples reveal less-abundant and often overlooked proteins. Drug discovery is another major driver in the separation of proteins. Pharmaceutical and biotechnology companies search for new proteins and other compounds to cure diseases, utilizing both electrophoresis and chromatography as tools. Screening assays are employed to determine the biological effect of a protein target. In order to perform these assays, researchers must first separate and purify proteins of interest. Chromatographic methods isolate and purify target proteins from other sample material for further screening and identification. These methods allow flexibility in choice of mobile phase and support, in addition to scalability. After proteins of interest are identified, Illustration by Audra Geras by Gabriella Armin, William Gette, and Ursula Snow, Bio-Rad Laboratories, Inc., Hercules, CA USA 12 BioRadiations 112
cover story cover story Fig. 2. Chromatography. A solution containing a mixture of proteins is typically applied to a column containing a stationary support. Proteins in the mixture interact differently with the support, leading to separation. The characteristics of the support and the solutions used to wash and elute the proteins can be modified to optimize separation. large-scale purifications, done exclusively through chromatographic means, are utilized. Analytical assessment of purity is often checked through 1-D gel electrophoresis, a separation technique used as a complement to chromatography. Sample Preparation The first step in any protein separation process is sample preparation. Proteins are labile, so precautions must be taken to minimize protein degradation. Other obstacles in isolating and purifying proteins include limited amount of sample, variable sample composition, the presence of nonprotein contaminants, wide differences in relative protein abundance within a sample, and the existence of isozymes and posttranslational modifications. Optimizing sample preparation can help minimize many of these obstacles. For example, the extraction method and buffer can have a large effect on the yield and quality of the sample and components of interest. Nonprotein contaminants that are extracted along with the proteins may be removed by size exclusion chromatography. Complex protein mixtures can often be fractionated effectively using ion exchange resins that separate proteins based on their charge. Samples that contain a large amount of a contaminating protein, such as albumin in serum samples, can be purified by affinity chromatography to remove the contaminating protein and to enrich less-abundant proteins. Preparative electrophoresis is another approach to sample preparation. The most powerful preparative-scale technologies are continuouselution electrophoresis and liquid-phase isoelectric focusing. Liquid-phase isoelectric focusing has the additional advantage of maintaining the biological activity of proteins. Once samples have been fractionated and partially purified, the next step in the process of both proteomic and drug discovery studies is typically identification and characterization of proteins of interest. Protein Identification and Characterization Rapid, accurate, and reliable methods of identifying a protein of interest are necessary in order to efficiently carry out further studies. Preliminary characterization includes molecular weight and isoelectric point estimation by 2-D gel electrophoresis, specific binding to an antibody or other ligand, and biological activity or other screening assays. More definitive characterization includes peptide mass or sequence determination, X-ray crystallography to determine 3-D structure, evaluation of posttranslational modifications such as glycosylation and phosphorylation, and analyses of the biochemical and cellular functions of the protein. Each of these characterizations helps to positively identify the protein in later studies, and most rely to some extent on the science of protein separation. Molecular weight estimation by SDS-PAGE is routinely used for rapid preliminary identification of proteins, particularly analysis of chromatographic column fractions, based on comparison to migration of suitable protein standards in the same gel. This method may not be reliable for complex samples, which may contain several proteins of the same molecular weight. In this case, 2-D electrophoretic analysis, which separates on the basis of both isoelectric point and size, is often used to identify and quantitate proteins of interest. 2-D gel electrophoresis also increases overall sample resolution, which ultimately leads to an increase in the number of protein identifications possible within a sample. A widely used method for specific protein identification is western blotting. Following 1-D gel electrophoresis, protein bands are transferred to a nitrocellulose or PVDF membrane. The membrane is then incubated with an antibody specific for the protein of interest. An indirect method of detection, such as a secondary antibody-enzyme conjugate, is used to produce a recordable signal at the site of antibody binding. Specific antibodies have other uses, for example in cell localization studies and in inhibition of activity or function studies, which can help in assessing the role of the recognized protein. Separation science is also important for obtaining antibodies. Antibodies are often generated to a protein of interest, to one related to it, or to a synthetic peptide based on sequence information. A partially purified target protein is a requirement for each of these approaches. Obtaining specific antibodies also typically requires separation of these antibodies from contaminating proteins. A partially purified protein can often be identified by sequencing, either by Edman degradation using automated sequencing systems, or by mass spectrometry of peptide digests. Although significant investment in capital equipment and user training is required, even a partial sequence, combined with isoelectric point and molecular weight, can positively identify a protein based on comparison to information in protein and nucleic acid databases. This approach is often combined with 2-D analysis to provide positive identification of protein spots excised from a gel. These methods are extremely accurate and can detect low levels of contamination by other proteins. If the protein is not an exact match to a database entry, knowledge of the sequence and inferred structure of the protein can still provide clues to its function and possible interactions with other proteins, based on its other similarities to known proteins. X-ray crystallography is the definitive method for determining protein structure. Knowledge of a protein’s molecular structure allows a better understanding of how it works and can lead to better drugs and treatments for disease. Effective crystallography experiments require preparation of highly pure protein. Significant effort and expense is required to purify proteins to greater than 99% Proteomics encompasses large-scale, highthroughput, parallel protein profiling. The application of proteomics requires two distinct skill sets: analytical chemistry for protein separation, identification, and characterization, and biochemistry/physiology for interpreting the significance of the analytical measurements. I started my research examining the evolutionary relationship between the kangaroo and platypus by studying their milk whey proteins. Two-dimensional electrophoresis was used for protein purification and Edman degradation provided sequence analysis. My major area of interest and study has been to address the inefficiencies of traditional two-gel separations for analyzing membrane proteins and other hydrophobic proteins. My research has shown that by applying prefractionation and improved solubilization methods, proteins previously considered intractable to 2-D electrophoresis are indeed amenable to the process. During my postdoctoral studies I shifted direction by investigating techniques for profiling phosphorylation states of proteins using mass spectrometry and chemical modification approaches. Nowadays in the pharmaceutical setting, my focus is centered on turning the "data" collected using proteomic approaches into "information" to understand the molecular physiology of our preclinical models and enhance the discovery of new drugs. There are major differences in operating a proteomics lab in an academic as opposed to a pharmaceutical setting. The most striking difference is the requirement to operate as part of a larger team in an industrial setting. The studies are too complex and labor-intensive for any one person to conduct efficiently. Consequently, the process is broken down into functional groups, with total reliance on colleagues to contribute. One of the most rewarding aspects is being involved in tying these pieces together and contributing a sizable volume of novel information to the therapeutic area groups that allow them to make new decisions. A second major difference is the acceptance of proteomics as a powerful discovery-based discipline. We find that there is great value in building protein response databases in a discoveryoriented mode, that then allow hypothesis-driven questions to be addressed. In academia it is more difficult to find support for discovery-driven investments. purity, and chromatography is the most often used technique to achieve this goal. Determination of posttranslational modifications is often required for the growing field of cell regulation studies. Such modifications can be characterized using antibodies, by electrophoresis, often combined with modification-specific stains (for example, to detect phosphorylation), and by mass spectrometric peptide analysis. More complete characterization would include biochemical definition of the role of the protein in the cell. Definitive characterization may require complex assays or other tools. Protein Separation Techniques: A Research Perspective Using Proteomics to Enhance the Discovery of New Drugs Mark Molloy, Senior Scientist, Pfizer Global R&D Pharmaceutical companies use proteomics to gain insight into compound safety, mechanism of action, and for target identification. The concept is to shorten the timeline from idea to drug by applying molecular technologies such as proteomics and expression profiling. An emerging focus is to use proteomics for biomarker discovery, again to shorten the timeline from new drug to market. Improvements in speed, sensitivity, automation, and robustness are key areas for attention. From the viewpoint of pharmaceutical applications, one major goal is an integrated system for allowing rapid, holistic, quantitative protein profiling. Some interesting technology is being developed (e.g., protein microarrays and ICAT), although each new approach comes with its own set of drawbacks. Despite its own limitations, the 2-D gel approach coupled with advances in image analysis is still the most reliable for day-to-day use. Selected Publications Molloy MP et al., Overcoming technical variation and biological variation in quantitative proteomics, Proteomics 3, 1912–1919. (2003) Molloy MP, The challenge of industrializing proteomics, Nat Biotechnol 21, 597 (2003) VanBogelen RA and Molloy MP, Exploring the proteome: reviving emphasis on quantitative protein profiling, Proteomics 3, 1833–1834 (2003) Molloy MP and Witzmann FA, Proteomics: Technologies and applications, Brief Funct Genomics Proteomics 1, 23–39 (2002) Benndorf R et al., HSP22, a new member of the small heat shock protein superfamily, interacts with mimic of phosphorylated HSP27 ((3D)HSP27), J Biol Chem 276, 26753–26761 (2001) Nouwens AS et al., Complementing genomics with proteomics: the membrane subproteome of Pseudomonas aeruginosa PAO1, Electrophoresis 21, 3797–3809 (2000) Santoni V et al., Membrane proteins and proteomics: un amour impossible Electrophoresis 21, 1054–1070 (2000) Wilkins MR et al., High-throughput mass spectrometric discovery of protein post-translational modifications, J Mol Biol 289, 645–657 (1999) Molloy MP et al., Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis, Electrophoresis 19, 837–844 (1998) Walsh BJ et al., The Australian Proteome Analysis Facility (APAF): assembling large scale proteomics through integration and automation, Electrophoresis 19, 1883–1890 (1998) Molloy MP et al., Identification of wallaby milk whey proteins separated by two-dimensional electrophoresis, using amino acid analysis and sequence tagging, Electrophoresis 18, 1073–1078 (1997) 14 BioRadiations 112 BioRadiations 112 15
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