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Sep 1, 2013 (Vol. 33, No. 15)

Purging Protein Profiling Problems

  • Cell-to-Cell Communication

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    The Sweedler laboratory at the University of Illinois at Urbana-Champaign uses a unique approach for collecting neuropeptides from a living brain slice for mass spectrometry characterization.

    Central nervous system neuropeptides are involved in many physiological processes such as circadian rhythms, pain, hunger, feeding, and body-weight regulation. Some neuropeptides undergo functionally important posttranslational modifications.

    Coupled with their small size (3–50 amino acids) and low concentrations in the CNS, these neuropeptides pose an analysis challenge. Measurements based on MS are well suited for neuropeptide research, simultaneously deducing structural and quantitative information about low-quantity compounds.

    “We are interested in knowing how the brain works,” says Jonathan Sweedler, Ph.D., professor at the University of Illinois, Urbana-Champaign.

    “To do that, we develop analytical methods to follow chemical and quantitative changes in neuropeptides alongside with their distribution and dynamics.”

    Neuropeptides impact many signaling pathways and are highly conserved in the animal kingdom. This allows for discovery and validation to be done on well-defined neuronal networks in the simpler nervous systems of marine invertebrates and other laboratory animals.

    Dr. Sweedler’s team utilized creative cell-isolation and sample-preparation methods to inventory the neuropeptides. To determine what neuropeptides affect circadian rhythm, they isolated the small region of the brain responsible for controlling this behavior and performed a comprehensive peptidomic analysis to inventory its neuropeptide content.

    This research found 102 endogenous peptides, including 33 that were previously unidentified, and 12 posttranslational modifications (including amidation, phosphorylation, pyroglutamylation, and acetylation).

    “Next, we looked at circadian neuropeptides in the functional context, by following peptide release from living neurons,” continues Dr. Sweedler.

    They prepared thin slices of the areas of the rodent brain containing its “biological clock.” The neuropeptides released from this area were collected and measured as a function of time of day and electrical stimulation. Both known circadian-rhythm-related neuropeptides and peptides with unknown roles in circadian rhythms were identified.

    “Coupling this information with metabolic profiling generates an amazing informational map of mammalian circadian physiology,” adds Dr. Sweedler. “We develop capabilities to manipulate neurons, use these approaches to learn about the chemistry in individual cells, and determine how this relates to higher order processes such as animal behavior. As our techniques are animal independent, the next step is to apply these discoveries to human health.”

  • Tracking Changes in Protein Isoforms

    Gary Nelsestuen, Ph.D., professor at the University of Minnesota in Minneapolis, maintains that a growing body of evidence points to the absence of a single new clinical test that has resulted from proteomics studies.

    “Most ‘bottom-up’ methods are expensive and suffer from inconsistent protein detection. Perhaps, instead of striving to identify as many proteins as possible, we should focus on approaches that analyze just a few proteins but with excellent predictive value,” he says.

    Dr. Nelsestuen’s work focuses on MALDI-TOF analysis of intact protein isoforms. While seemingly low-tech, this approach allows the screening of a large number of samples in a reproducible and cost-efficient manner.

    Profiling of a 5,000 blood/urine sample set representing a wide variety of disease states revealed important information about biological variations in normal populations.

    “We found that isoform ratios are consistent for each individual,” continues Dr. Nelsestuen. “At the same time, even if the individual ratio falls within the ‘normal’ range, a person may be still very ill. This necessitates comparison of each individual to his/her own baseline to improve the ability to detect change in health status.”

    Change of isoform ratio related to health outcomes is illustrated by the analysis of glyco-isoforms of intact apolipoprotein C3 (ApoC3). A 1.8-fold change in the glyco-isoform ratios correlated with obesity, specifically among subjects eligible for bariatric surgery.

    Bariatric surgery resulted in the rapid change of isoform distribution to that of non-obese individuals, after which the distribution was stable in each individual. Similarly, glyco-isoform ratios were indicative of chronic hepatitis C, liver cirrhosis, and sepsis.

    The information provided by glyco-isoform ratio changes may provide important, novel information for diagnostic, prognostic, and therapy response to metabolic conditions. However, it requires monitoring of each patient over time.

    “Our method was sufficient to provide clinically important information for monitoring kidney transplants,” says Dr. Nelsestuen. “The health of transplanted kidneys is often monitored by frequent biopsies. A non-invasive urine analysis for anomalous protein biomarkers would provide significant advantage over current clinical practice.”

    Remarkably, two ubiquitous isoforms of saposin B, thought to be an activator of lipid degradation, were low or absent in patients with advanced kidney disease, while a number of other components appeared. The team is combining this data with metabolomics profiling to identify early diagnostic biomarkers.

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