Editorial Article
Monday March 01, 2010
by Caitlin Smith
Imagine if you could peer into the proteome of your child and see raised warning flags denoting the future development of various diseases. Hopefully there would be something you could do to prevent those maladies. We can’t yet do all of this, but we are making progress on using proteins as biomarkers for various diseases. “The field is rapidly advancing,” says Roberto Campos-Gonzalez, director of the intracellular department at R&D Systems. “On one side we have the fast evolution of the label-free assays such as mass spectrometry, ideal for discovery-based experiments. And on the other side we have more focused approaches and tools designed to monitor and quantify a more defined set of analytes, often within an area or pathway.”
Indeed, it seems that a more focused discovery process is developing. “The trend in recent years in proteomics and biomarker discovery has become a more targeted approach as opposed to a broad exploration of the entire proteome,” says Kate Smith, product manager for expression proteomics at Bio-Rad. “With this realization, sample preparation has become a key area of focus in proteomics. As researchers devote more time to simplifying samples through fractionation and enrichment, the data generated in biomarker discovery experiments [have] become more informative.”
Finding low-abundance biomarkers
One challenge of biomarker discovery is clearing away the abundant proteins in a complex sample to glimpse the low- and medium-abundant proteins that would normally be obscured. One way to do this is by fractionation, where a complex sample is separated into fractions to examine independently. “Researchers have realized that due to sensitivity limitations of their analysis methods and the wide concentration range and vast number of proteins in a sample, they must fractionate to enrich proteins and gain meaningful information,” says Smith. “As biomarker discovery has evolved, researchers have realized that reduction of sample complexity, prior to downstream protein analysis, is critical for producing meaningful, reproducible results.”
ProteoMiner uses a combinatorial library of bead-bound hexapeptides. “Due to the random nature of the hexapeptides, the bead population has such diversity that a binding partner should exist for most, if not all, proteins in a sample,” says Smith. “Since there are a limited number of binding sites per protein, high-abundance proteins quickly saturate the ligands for which they have the highest affinity and excess proteins flow through, while low-abundance proteins continue to bind. Following elution, the low-abundance proteins are enriched while the high-abundance proteins are reduced.”
Besides sample complexity, another challenge in biomarker discovery is sample accessibility. The most notorious are proteins that are difficult to solubilize, such as membrane proteins, very basic or acidic proteins, or proteins with various post-translational modifications. “Other challenges [include] preserving information related to the dynamic structure of the proteome (for example, post-translational modifications) in addition to maintaining quantitative information,” says Smith.
Invitrogen also offers tools to reduce the complexity of samples. Peptide profiling with Invitrogen’s magnetic Dynabeads® gives a quick and consistent way to screen samples. Also, their SILAC™ Protein Identification and Quantitation Kits let you label proteins metabolically, while their ICAT® and iTRAQ™ reagents let you label and track proteins through mass spectrometry using chemical labels. Another tool available from Invitrogen is their ProtoArray® protein microarray, which gives fast protein identification with as little as 10 microliters of sera or plasma.
Big discoveries from tiny tools
A new development in biomarker discovery is the application of nanotechnology—the world of the very small. Shimadzu Scientific Instruments, partnered with Ceres Nanosciences and Nonlinear Dynamics, are set to launch their new Nanotrap Biomarker Discovery Platform in March 2010. “Nanotraps™ are core shell hydrogel particles that provide several unique features over existing bead- or surface-based sample fractionation strategies,” says Scott Kuzdzal, life sciences business leader for biotech products at Shimadzu Scientific Instruments. “Nanotraps™ exclude the more abundant, less interesting carrier proteins and simultaneously harvest the low-level proteins and peptides. They also allow unique amplification and protection capabilities. Now, biomarkers once immeasurable can be harvested directly from complex biofluids, protected from enzymatic degradation, and amplified. These nanotrap properties offer exciting advantages to biomarker researchers, enabling them to tap into and mine the low-level proteome.” The partnership incorporates the Nanotraps™ of Ceres Nanosciences, the Axima MALDI workflow of Shimadzu, and the informatics discovery software Progenesis MALDI of Nonlinear Dynamics.
Kuzdzal says that another new technology is showing promise for analyzing proteins with post-translational modifications. It is “a hybrid mass spectrometer that combines the speed of MALDI, the MSn capabilities of an ion trap and the resolution and mass range of a Time-of-Flight (TOF) analyzer,” he says. “All of these abilities are consolidated in the new Axima Resonance from Shimadzu.”
Multiplexing and high-throughput validation
To accelerate biomarker discovery, R&D Systems offers tools for multiplex analysis. Their bead-based Fluorokine MAP Kits use Luminex xMAP® technology to measure multiple proteins per 50 microliters of sample. They also offer “the Proteome Profiler 96, ideal for small samples and formatted in 96 well plates, and the Proteome Profiler for a rapid and economical analysis of many analytes, simultaneously,” says Campos-Gonzalez. “There are major advantages of our multiplex assays, one being the extensive characterization of the paired antibodies providing exquisite specificity and maximum sensitivity. In addition, our comprehensive product offering covers important proteins in both the extracellular space and the intracellular environment.”
Campos-Gonzalez believes that biomarker discovery is still making forward strides. “For instance, the availability of libraries of siRNAs, and the ability to assay these simultaneously to identify key molecules in disease, has been extremely beneficial. Multiplexing analysis of potential biomarkers will continue to grow in demand. The value of reading multiple analytes, or biomarkers, provides more confidence in the results than those coming from an individual readout.”
Like multiplexing, high-throughput systems provide the opportunity to chug through experiments and gather more data, more quickly. Bio-Rad, together with Bruker Daltonics, offers the Lucid Proteomics System for proteomics-based biomarker discovery. It “provides the ability to do both top-down (i.e., profiling of intact proteins) and bottom-up (i.e., ‘shotgun’ or digest-based) proteomics for biomarker discovery on a single platform,” says Julie Hey, marketing manager for the protein detection business unit at Bio-Rad. “Before the Lucid Proteomics System, researchers had to choose between the two approaches. By using both approaches, you increase the chances of success for discovering a biomarker that has statistical and clinical relevance.”
Hey says that Bio-Rad has spent the last two years ensuring standardization and reproducibility of their products, including a series of downloadable guides for researchers on working through the biomarker discovery process. “The Lucid Proteomics System was developed with particular attention paid to these challenges and gives the user the power to ensure instrument and experimental reproducibility and stability over the course of their discovery project,” says Hey. “I also think that we'll likely see more panels of biomarkers as predictors rather than a single, novel biomarker.” Recent success of the top-down biomarker discovery approach is evidenced by the newly FDA-approved OVA1 assay for ovarian cancer, the first panel of protein biomarkers to gain approval. Discovery of the panel of biomarkers and initial clinical validations were done using Bio-Rad’s SELDI platform. “This shows great promise for the future of using this approach,” says Hey.