Genetic Engineering & Biotechnology News

MAY15 2018

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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10 | MAY 15, 2018 | | Genetic Engineering & Biotechnology News Mass Spec Is Being Souped Up to Help It Keep Up Continued from page 1 Fortunately, if they receive the right enhancements, gen- eral-purpose instruments can provide reproducible, accurate mass data and formula identification. One such enhance- ment is Cerno Bioscience's novel calibration technology, a software solution that can significantly enhance mass accu- racy (typically 100×), enabling accurate mass measurements for formula identification. In addition to giving accurate mass information, the soft- ware evaluates the isotope fingerprint to show the best match between it and calculated formula profiles to further enhance formula identification through "Spectral Accuracy." This process is incorporated into CLIPS (calibrated line- shape isotope profile search), a key technology incorporated into the software. After the accurate mass confidence level is determined for a mass peak, the candidate formulas are fur- ther analyzed for Spectral Accuracy, reducing the possible can- didates from 30 to 40, for example, to perhaps three or four. The software enhances the discriminating power of routine MS instruments, bringing identification capability downstream to the quality control (QC) technician, reducing costs, acceler- ating the process, and improving the QC process overall. "Our software and algorithms enable formula identifica- tion on the standard QC instrument," asserts Don Kuehl, Ph.D., vice president of marketing and product development at Cerno Bioscience. "[Our technology] provides a much higher level of confidence for identification of impurities and degradants in [chromatographically] well separated samples. "In addition to calibrating for the mass position, our soft- ware also calibrates the line shape. We can then calculate the theoretical isotope pattern to be the same and exactly perfect every time. This is an extremely powerful dimension to fur- ther qualify the identification of an unknown compound." Single-Cell Analysis Another extended capability for quadruple-based tech- nology is single-cell analysis. Traditional metal content mea- surements use nominal mass concentrations, which assume an equal distribution of metals among the cells in a cell popu- lation, overlooking vital information on metal distribution and variation on a unicellular level. Introduced in March 2017, the PerkinElmer Single Cell ICP-MS (SC-ICP-MS) is an automated technology capable of rapidly measuring and quantifying the metal content of live or fixed individual cells down to the attogram-per-cell level, the metal mass distribution, and the number of cells containing the metal. SC-ICP-MS can scan for any metal in the periodic table in cells ranging from 0.2 to 100 µm in size. Accurately quantifying the metal content in an individual cell can offer insight into the uptake and elimination mecha- nisms of metals and/or metal-containing nanoparticles; the mechanisms of interactions between metal-containing drugs and cells; and the distribution of micronutrients, such as zinc, copper, or iron. Hardware includes one of PerkinElmer's NexION family of ICP-MS instruments (capable of acquiring 6 million data points/min at 10- µ sec dwell time), a PerkinElmer Asperon spray chamber (capable of delivering individual, intact cells into the plasma of the ICP-MS), and a microfluidic worksta- tion. Any shape or size well plate can be used, and sample volumes can be as low as 5 µL. The company's Syngistix Single Cell Application Module can be used to perform data collection and processing. The technology has been used to determine the uptake and interaction of toxic metals in bacteria and plant cells, the bioavailability and bioaccumulation of gold particles or ionic gold by algae, the efficiency and effectiveness of drug delivery monitoring cisplatin uptake in ovarian cancer cell, and the usefulness of single-cell approaches in nanomedicine. "Gold nanoparticles (AuNPs) are showing emerging use in biomedicine for cancer diagnosis and therapy," discussed Chady Stephan, Ph.D., senior lead inorganic applications, PerkinElmer. "However, the quantification of AuNPs uptake into cancer cells using elemental analysis has been limited to ensemble measurements, which require digestion of the entire cell population in concentrated acids. "This approach assumes that all cells within a given cell population interact with the same number of AuNPs. To test whether this assumption is correct, new analytical strat- egies were needed that allow quantitative elemental analy- sis at a single-cell level." Isotopic Ratio Outlier Analysis In metabolomics, interlaboratory result comparisons pres- ent an analytic challenge because they must allow for varied methodologies and limited reference standards. Also, they cannot readily adapt an approach that is often used in classi- cal targeted quantification assays, an approach that provides relative quantifications based on the relation of the sample to purchased isotopic standards. In the metabolomics con- text, this process is restrictive and costly because individual analytes require corresponding standards, which may not be available for all metabolites. Isotopic ratio outlier analysis (IROA) allows for the gen- eration of isotopically labeled control standards of an entire metabolome by growing cells in 95% randomly 13 C-labeled glucose. Naturally occurring isotopes are positive; IROA re- verses that and creates a negative effect of n−1 isotopes. The IROA-labeled cells are spiked into the sample. Since the spiked-in amount is known, quantitation for each metab- olite can be performed through a ratiometric approach. This allows pattern comparisons from multiple methods and labo- ratories, and multiple cell lines can be grown with the media. Designed to provide cost savings, IROA makes it easy to find the labeled species and allows untargeted discovery of metabolites that have not yet been curated. A key aspect is that only enzymatic metabolites get labeled, helping to dis- criminate noise and artifacts. "We employed IROA to help other people learn how to use it," states Timothy J. Garrett, Ph.D., associate professor of pathology at the University of Florida and co-director of the high-throughput MS metabolomics core of the Southeast Center for Integrated Metabolomics. "We developed stan- dard operating procedures and improved the software to ensure that it is looking for and picking things correctly. We are also testing to make sure the technique is applicable to human studies." The technique was initially tested in yeast extracts, then it was tested with human-based samples, where it was found to work well. Next steps include evaluating human cell lines, developing the quantitative methods, and scaling up for clini- cal diagnostics. Reference ranges will be developed and test- ed to determine correlation with ranges in the clinical setting as well as approaches defined to identify unknowns. "Metabolism is the closest to phenotype," insists Dr. Gar- rett. "Metabolomics is gearing up to discover new metabolic associations in known and rare diseases to help improve the way we diagnose diseases. "If we can then pull out and understand all the meta- bolic information, it will help us develop better diagnostic paradigms. Years ago, this is what newborn screening did, it transformed the way we diagnosed newborns." Coping with Higher Pressures Samples generated by omics and environmental samples may contain more than 10,000 compounds. Such complex mixtures can overwhelm current liquid chromatography (LC)–MS systems, which lack sufficient resolving power. Although these systems can achieve higher resolving power if they make use of smaller resin particles and longer col- umns, both these changes require that the systems operate at higher pressure. Commercial systems are currently limited to operating between 8,000 and 20,000 psi, depending on column di- mensions. To see if this operational ceiling could be raised, Robert Kennedy, Ph.D., Hobart H. Willard Distinguished University Professor, professor of chemistry, and professor of pharmacology at the University of Michigan, experimented with modifications to a proprietary ultra-high-pressure liq- Drug Discovery Feature Cerno Biosciences provides a technology for formula identification called calibrated line- shape isotope profile search (CLIPS). Unlike approaches to elemental composition that use only mass accuracy for formula identification, CLIPS adds another dimension by matching the full isotope profile of the unknown to the theoretical profile. In the past, this has not been possible due to the uncalibrated line shape in mass spectrometry.

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