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14 | SEPTEMBER 1, 2016 | GENengnews.com | Genetic Engineering & Biotechnology News It is relatively easy to generate raw data with the new experimental approaches. The new approaches, however, require laborato- ries to deal with new data types. "There are no established methods on how to deal with the new data types," warns Dr. Göttgens. "The next couple of years will see a bottle- neck on the computational side." Low-Abundance Proteins in Plasma "Undoubtedly, the biggest challenge in comprehensive or global protein profiling is the dynamic range," says Hamid Mirzaei, Ph.D., assistant professor of biochemistry at the University of Texas Southwest Medi- cal Center. For the cellular proteome, the dynamic range fluctuates from one copy to about ten million copies per cell, and it also varies with the experimental system, with major differences being seen between cell lines and biological fluids such as plasma. Major efforts in Dr. Mirzaei's laboratory are focused on drug discovery, and these ex- periments, mostly conducted in cell lysates as experimental models, are seeking to under- stand the binding sites of specific drugs and their interactions with proteins. Other ef- forts in Dr. Mirzaei's laboratory are focused on biomarkers. The majority of Dr. Mirzaei's biomarker work is performed in plasma. The difference between the dynamic range of plasma and that of cell lysates is estimated to be about five orders of magni- tude. "This is one of the factors that makes it so difficult to profile proteins in the plasma," explains Dr. Mirzaei. For example, a peptide that originates from a highly abundant protein may mask peptides that come from a low-abundance protein, and estimates that the 22 most abundant proteins represent about 99% of the plasma proteins illustrate the challenges posed by the dynamic range. Most proteins that are expressed in dif- ferent tissues reach and reside in the plasma, where their measurement provides prognostic or diagnostic value. "Even though the plasma proteome is the ultimate proteome," notes Dr. Mirzaei, "we are still far from being able to dig really deeply into plasma-type proteomes." Another key factor in protein profiling is ionization efficiency, and this is a variable that to an extent is determined by the mass spectrometry equipment. "In terms of bio- markers, the field of mass spectrometry has seen significant improvements over the past ten years," observes Dr. Mirzaei. "Major companies are providing increasingly accu- rate and sophisticated mass spectrometers, and this is an aspect that can continuously improve." Protein profiling is critically positioned for biomarker identification and develop- ment. "Post-translational modifications of proteins are potential biomarkers, as op- posed to a proteins themselves," asserts Dr. Mirzai. In recent years, there has been increasing interest in profiling post-transla- tional modifications, and efforts are under- way to significantly improve the ability to vi- sualize lower abundance proteins that carry those post-translational modifications. Damage-Related Signaling Investigations led by Stephen J. Elledge, Ph.D., professor of genetics at Harvard Medical School, interrogate phosphorylation events in the budding yeast in response to the DNA damage response. "Phosphorylation," Dr. Elledge points out, "is relatively easier to profile compared to other post-translational modifications." In one recent study, Dr. Elledge and col- leagues used quantitative mass spectrometry to capture the substrates and the signaling pathways involved in the cellular response. The scientists generated an unbiased data- base of phosphorylation targets that con- tained 133 substrates and was enriched for proteins involved in DNA repair, DNA rep- lication, translation, and cell cycle control. This work helped uncover the central po- sition that the DNA damage response occu- pies in the vast cellular signal transduction network and in cellular metabolism. In ad- dition, the work revealed links to TOR sig- naling, inositol phosphate metabolism, and translational regulation. "The limiting factors in capturing phos- phorylation events will always be sensitiv- ity and the quality of the calls," advises Dr. Elledge. "And these factors depend on how much material can be obtained." Obtaining sufficient material for mass spectrometry is particularly critical when profiling post- translational modifications. For certain post-translational modifica- tions, such as phosphorylation, profiling has been facilitated by the availability of affinity reagents. "Affinity reagents allow investiga- tors to profile the proteome at a much deeper level," explains Dr. Elledge. In a study that profiled ubiquitination in response to the DNA damage response using quantitative proteomics, Dr. Elledge and col- leagues reported that the replication protein A (RPA) complex is ubiquitinated at multiple sites. The RPA complex is a protein scaffold that binds single-stranded DNA generated during replication fork stalling and facilitates repair. In mammalian cells, RPA complex ubiquitination is required for homologous recombination at stalled replication forks. The budding yeast and the baker's yeast, two experimental systems extensively used in Dr. Elledge's lab, provide ideal models to understand the functional relevance of the proteins of interest. These models take ad- vantage of the availability of knockout strain collections that help interrogate the presence of a protein in a specific cellular pathway. "The ability to more sensitively detect peptides is critical," says Dr. Elledge. "One thing that is missing in mass spectrometry analyses is that this technique can identify only those peptides that already exist in the database." As a result, a vast amount of experimental data is discarded during mass spectrometry experiments. "This is the dark matter of the proteome," relates Dr. Elledge. "Figuring out what the dark matter contains will make it a lot easier to make new discoveries on the regulation and modification of peptides." One of the key questions is whether there is a way to try to figure out what is being dis- carded from the vast amount of proteomic data. "It is all there, and we are seeing it," Dr. Elledge continues. "But we don't know what we are seeing because we are blind to its significance." Combining Mass Spec and Thermal Shift Profiling Recent studies have reported that brusa- tol, a natural compound, can potently and selectively inhibit Nrf2 activity and sensitize several cancer cell types to chemotherapeutic agents. "We became interested in brusatol after several papers showed that it inhibits the Nrf2 transcription factor," says David Stokoe, Ph.D., senior scientist in discovery oncology at Genentech. Nrf2, a protein critical for the cellular re- sponse to oxidative damage, has dual roles in cancer, inhibiting tumorigenesis in some contexts, and stimulating it in others. These disparate roles are related to mechanisms of activation and dysregulation of the KE- AP1-NRF2 pathway. Dysregulation of the KEAP1-NRF2 pathway has been implicated in inflammation and cardiovascular, neuro- degenerative, and malignant conditions. Studies conducted by Dr. Stokoe and colleagues revealed that at submicromolar concentrations, brusatol can powerfully downregulate Nrf2 expression. "Our goal," recalls Dr. Stokoe, "was to use a new tech- nology, proteomic profiling, to identify the targets of this small molecule." Using a mass spectrometry-based strat- egy, Dr. Stokoe and colleagues interrogated the cellular targets of brusatol in a non-small cell lung cancer cell line. Combining this ap- proach with a cellular thermal shift assay, which exploits changes in the conformation and the thermal stability of proteins when they are bound by small molecules, Dr. Sto- koe, together with his Genentech colleagues Kebing Yu, Ph.D., and Don Kirkpatrick, Ph.D., found that brusatol regulates Nrf2 by an indirect mechanism that involves a global inhibition of protein synthesis. While the expression of most proteins from the proteome decreased, the expression of ribosomal proteins increased, suggesting that brusatol could decrease Nrf2 protein levels by modulating translation. Brusatol decreased the expression of many proteins; notably, proteins with a short half-life were the most powerfully impacted ones. This re- sult validated thermal proteomic profiling by mass spectrometry as a new strategy for target identification and characterization in drug development. Protein Profiling Continued from page 12 DRUG DISCOVERY A key determinant in the outcome of protein profiling experi- ments is the amount of protein that is available for analysis. This determinant depends on multiple variables including the cellular abundance of the protein, the nature of the samples that are to be interrogated, and the capabilities of the experimental approach that is to be used. For example, if mass spectrometry were to be used, ionization efficiency would be a relevant factor. "When it comes to characterizing proteins, a primary constraint that we observe is that there is often very little sample to work with, especially early in the drug development pipeline," says Lisa Newey- Keane, Ph.D., life science sector marketing manager at Malvern Instruments. "During early drug candidate screening, the most desirable tests are the ones that provide relevant and useful data using only microliters or even nanoliters of sample." The ability to extract information from analytical systems while working with very low sample volumes is not the only challenge in protein profiling. Another challenge is the need to reach high enough sensitivity to deliver accurate data. "Also, if possible, a test should be nondestructive, preserving the sample so that it may be reused for further tests." Protein profiling is also challenging because of the dynamic nature of proteins. Proteins have a wide spectrum of half-lives. Also, proteins change in abundance and alter their interactions with binding partners in response to their environment. "Being able to measure proteins under representative conditions, and to know that the tests carried out will produce representative data, is crucial," explains Dr. Newey-Keane. Malvern Instruments recently launched a new platform that combines Taylor dispersion analysis and ultraviolet detection for molecular sizing. This approach provides automated protein and peptide stability measurements, has ultra-low volume sample re- quirements, and is ideally positioned to dynamically characterize proteins, even in complex media. Many of the proteins that are interrogated in biological fluids present promise as biomarkers or for therapeutic interventions. In translational research, the ultimate goal is the ability to transition from the bench to bedside in a manner that is safe and therapeuti- cally effective. The dynamic characterization of proteins is an instru- mental step during this process. "Delivering a protein to the patient in the form that has been identified as clinically therapeutic requires a detailed consideration of a wide range of factors," comments Dr. Newey-Keane. "These fac- tors include stability, storage, the impact of temperature, stress, and other processing conditions, as well as the ease of delivery." n Biophysical Riches from Meager Samples