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Proteomics Instruments and Methods Angelo DePalma Researcher Lisa Sapp examines a ProXpressions biomarker enrichment plate prior to placing it onto a PerkinElmer automated liquid handling robot. Life scientists haven’t had time to rest on their laurels since the completion of the human genome project. According to Jeff Peterson, CEO of Target Discovery (Palo Alto, CA), the incomplete correlation between protein and gene expression led to the first major realization of the post-genomic era. “That’s when everyone knew that proteomics would be the next big wave,” he told BioScience Technology. Faith in the genome was perhaps wishful thinking. Genes possess simple linear structures that are amplified with polymerase chain reaction (PCR) and analyzed with high-throughput methods. Proteins have unpredictable secondary and tertiary structure, are peppered with glycosylations and other post-translational modifications (PTMs), arise in multiple forms from the same gene, and enjoy no amplification methodology or easy path to high-throughput analysis. Because of protein complexity the tools for unraveling their identities and functions fall short of fulfilling the needs of life scientists. “These methods suffer from fundamental limitations,” says Mr. Peterson. Replacing 2D gels ProteomeLab PF 2D protein fractionation system. Target Discovery’s platform technologies include reagents to improve capillary electrophoresis (CE), a promising but generally under-performing technology. CE’s limitation is its underlying physical principle, electro-osmotic force, which pushes proteins through the capillary too quickly, with unacceptable reproducibility. The company’s zero-EOF dynamic coatings, EOTrol and UltraTrol, allow resolution and comparison of complete, complex cellular proteomes. Another reagent kit, Isotope-Differentiated Binding Energy Shift Tags (IDBEST), offers affinity enrichment of low-abundance biomarkers. IDBEST incorporates Target Discovery’s mass defect tag technology, which, combined with classical isotope-differentiated labeling, enhances sequencing and differential display determinations. Target Discovery offers kits for transferrin and α-antitrypsin, with cancer biomarker products under development. 2D HPLC is making a run at replacing gels but has not been universally adopted. Detlef Schumann, PhD, who directs the Genome Research Institute at the University of Cincinnati, uses 2D gel electrophoresis almost exclusively for large, complex protein mixtures. Schumann shies away from LC methods because, to him, GEP is “by far the most mature, reliable technology out there. I know its limitations, but I also know what we can and cannot see with gels.” Dr. Schumann claims much higher throughput from gels than is possible with 2D LC. “We run up to 40 2D gels a week. I don’t think we could achieve this throughput using multi-dimensional chromatography,” he says. Target Discovery scientists couple technology for mass spec and capillary electrophoresis to enable isoform-specific biomarker discovery and diagnostics. Dr. Schumann realizes, however, that eventually he will switch to LC. Before that happens he would like to see higher sensitivity and more robust, reliable methods. “Methods development time is scarce around here due to the steep learning curve. Since I run a service lab I prefer to let others pave the way. We’ll wait until 2D LC is more mature. It may take another two or three years, but it’s definitely on the horizon.” In any case Dr. Schumann will have many instruments to choose from. Eksigent Technologies’ (Livermore, CA) NanoLC-2D Proteomics System incorporates fully automated 2D HPLC and direct nanoscale pumping without flow splitting. “Precise control of nanoliter per minute flow rates using flow splitting is difficult,” says Karen Hahnenberger, PhD, Senior Product Marketing Manager. Eksigent’s NanoLC systems achieve controllable, precise, nanoliter per minute flows by eliminating standard positive displacement or syringe pumps. Within the NanoLC platform are NanoLC-1D and NanoLC-2D for 1D and 2D chromatography, respectively. “2D LC is an alternative to 2D gels,” explains Dr. Hahnenberger. 2D gels use isoelectric focusing followed by a molecular weight separation. 2D LC offers more flexibility, for example cation exchange followed by reverse phase separations in the second dimension. Simplicity, integration Target Discovery scientist prepares a sample with new isoform-specific biomarker discovery reagents for analysis by mass spec. The flood of scientists entering proteomics begs for simpler, easier-to-use instrumentation, says Agilent’s (Santa Clara, CA) LC/MS Marketing Manager John Michnowicz, PhD. “The large fraction of proteomics instrument sales will be to scientists experimenting with proteomics for the first time,” he says. “To succeed vendors must supply not just a collection of bits and pieces but a complete system.” One could argue that complete systems are expensive, and scientists don’t mind tinkering with “bits and pieces.” Dr. Michnowicz disagrees. “It’s impossible to take the piecemeal approach to proteomics. There are no beginner lessons. You have to dive in. If you want to be truly productive you have to grasp fairly complex equipment and complex experiments from the beginning.” As the manufacturer and primary integrator of MS, chromatography, and consumables, Agilent provides protocols that maximize the potential of their instrument systems. Its primary LC-MS platform, the Protein ID, includes nanoflow LC and ion trap MS, data analysis based on the Spectrum Mill Workbench informatics system, and standard protocols. Body fluids, particularly blood, are the most popular samples for human proteome analysis, which raises the question of how to deal with the blood’s 20,000 to 30,000 different proteins spread over a concentration range of 1010. Most proteins of interest are present at concentrations 106 to 108 lower than human serum albumin, the most common blood protein. “Current technology can’t dig that deeply into the proteome,” notes Dr. Michnowicz. “What most scientists see are top 200-300 proteins.” Agilent offers front-end affinity chromatography tools to allow deeper digging. Its Multiple Affinity Removal System eliminates the top 6 proteins in human serum, while the company’s Macroporous RP column fractionates the remaining proteins into 10-20 pools. Similarly, Beckman Coulter’s mission is to migrate proteome analysis away from individual instruments to “solutions.” “For many years research was a series of activities rather than a process,” comments John Hobbs, Group Product Planning Manager for Proteomics. “Now it’s time to de-focus from technology and instrumentation, and re-focus on results.” click the image to enlarge Differential Display of Partial pI/UV Maps of Colon Cancer Cell Line Before and After Drug Treatment Indicating Differences Between the Proteomes. ( Note - This technique can also be used to detect potential biomarkers when comparing diseased with non-diseased states in bio-fluids or cell lysates ) For example while MS reigns supreme in proteomics, what comes out is only as good as what goes into the instrument. “Our strategy is to simplify the problem as early as possible,” says Mr. Hobbs. Simplification begins with a flow cytometer for sorting cells of interest, enrichment of organelles through centrifugation, followed by automated 2D LC through the company’s ProteomeLab platform systems. For example the ProteomeLab PF 2D protein fractionation system uses isoelectric focusing in the first dimension and nonporous reverse-phase chromatography for the second. Fractions are fed automatically into the mass spectrometer. The brains of ProtomeLab PF 2D is the ProteomeLab Software Suite, which creates a visualization map that resembles a 2D gel and allows comparison of samples side by side. Key to this platform — and Beckman Coulter’s approach in general — are pre-loaded methods. “Professional chromatographers get a bit miffed by these, but end-users love them,” notes Mr. Hobbs. Standardized protocols result in higher reproducibility, and facilitate collaboration between research groups that use them. Beckman Coulter also offers a CE system, the ProteomeLab PA 800, with preloaded methods for isoelectric focusing, glycosylation, peptide separation, and SDS gel, all of which interface with any MS instrument. The company will soon introduce a protein fractionation system that will remove the twelve most abundant proteins in plasma automatically. Using preloaded methods, the system will feed fractions for downstream separations, for example to the PF 2D. Wanted: automation, integration Unlike genomics, which enjoyed a high degree of automation early on, proteomics is still searching for rapid, parallel analytical methods. “Proteomics has been a very manual procedure,” notes Mary Lopez, Business Leader for Analytical Proteomics at PerkinElmer (Boston, MA). PerkinElmer offers liquid handling tools for processing its protein fractionation kits and delivering their output to an MS instrument in a high-throughput manner. The centerpiece of PerkinElmer’s biomarker and classical proteomics platform is PRO-TOF, a MALDI-time-of-flight MS capable of 15,000 samples per day. PRO-TOF is integrated with the MultiPROBE II liquid handling system, which processes samples and spots them for MALDI MS analysis. PE is developing ProXPRESSION biomarker enrichment kits and ProXPRESSION protein enrichment kits. “The combination of those technologies provides a seamless integration for taking a drop of blood all the way through enriching biomarkers, processing samples automatically, and presenting them to a MS,” says Ms. Lopez. Despite the temptation to provide complete proteomic “solutions” some vendors are doing nicely by focusing on niches. “There’s been shift at GE Healthcare, from trying to cover every aspect of proteomic analysis to focusing on value-added features of the proteomics workflow, particularly for protein arrays, 2D GEP-to-MS and LC-to-MS experiments,” explained Joakim Rodin, Head of Product Development, Protein Analysis at GE Healthcare (Chalfont, UK). Sample preparation underpins these activities at the front end, while software supports them at the back end. Two years ago GE Healthcare introduced DeCyder differential analysis software to support its Ettan DIGE (Difference Gel Electrophoresis). Ettan DIGE uses size- and charge-matched resolvable CyDye fluors which, by supplying an internal standard for each protein, permit separation of up to three samples in a single 2D gel. DeCyder addresses protein difference measurements by detecting, matching, and analyzing protein spots in multiplexed fluorescent images. GE Healthcare claims the software can differentiate differences of less than 10% with statistical confidence. DeCyder’s two capabilities are DIA (differential in-gel analysis) for co-detection of image pairs from the same gel, and the BVA (biological variation analysis) image analysis module.