A Randomized, Double-Blind, Placebo-Controlled, Prospective Clinical Trial Evaluating Water-Soluble Chicken Eggshell Membrane for Improvement in Joint Health in Adults with Knee Osteoarthritis.

Osteoarthritis (OA) is a chronic condition that impacts quality of life and functionality for which consumers often seek dietary supplements to provide some relief. The purpose of this double-blind, placebo-controlled clinical trial was to assess the safety and efficacy of a water-soluble chicken eggshell membrane hydrosylate (WSEM) dietary supplement (BiovaFlex®) 450 mg daily on knee function, mobility, and general health and well-being in 88 adults with OA randomized into intervention (n = 44) or placebo (n = 44) groups. Outcomes were assessed periodically over 12 weeks, including the Western Ontario McMaster Osteoarthritis Index (WOMAC), the six-minute walk test (6MWT), knee range of motion (ROM) testing, and safety. Normalized analysis (improvement over baseline) showed that the poorest initial performers benefited the greatest from the WSEM by day 5 in the 6MWT, with the rest of the population showing significant improvement over placebo by week 12. The normalized WOMAC Stiffness score was also significantly improved over placebo by day 5 (P < .05). Without normalization, no statistically significant improvements were seen in WOMAC, 6MWT, and ROM testing. The Product was also found to be safe in this study. In conclusion, daily consumption of WSEM significantly enhanced average individual physical capacity (walking distance and ability), reduced stiffness by the fifth day of supplementation with the greatest benefit seen by the most compromised individuals, and was maintained over 12 weeks. A WSEM dietary supplement may offer a safe option for relief from symptoms and increased mobility for those with OA.

Method for recovery and immunoaffinity enrichment of membrane proteins illustrated with metastatic ovarian cancer tissues.

Integral membrane proteins play key biological roles in cell signaling, transport, and pathogen invasion. However, quantitative clinical assays for this critical class of proteins remain elusive and are generally limited to serum-soluble extracellular fragments. Furthermore, classic proteomic approaches to membrane protein analysis typically involve proteolytic digestion of the soluble pieces, resulting in separation of intra- and extracellular segments and significant informational loss. In this paper, we describe the development of a new method for the quantitative extraction of intact integral membrane proteins (including GPCRs) from solid metastatic ovarian tumors using pressure cycling technology in combination with a new (ProteoSolve-TD) buffer system. This new extraction buffer is compatible with immunoaffinity methods (e.g., ELISA and immunoaffinity chromatography), as well as conventional proteomic techniques (e.g., 2D gels, western blots). We demonstrate near quantitative recovery of membrane proteins EDG2, EDG4, FASLG, KDR, and LAMP-3 by western blots. We have also adapted commercial ELISAs for serum-soluble membrane protein fragments (e.g., sVEGFR2) to measure the tissue titers of their transmembrane progenitors. Finally, we demonstrate the compatibility of the new buffers with immunoaffinity enrichment/mass spectrometric characterization of tissue proteins.

Improved CZE capabilities with new dynamic coatings.

Electroosmotic flow (EOF) is one of the fundamental processes affecting both resolution and separation times in capillary zone electrophoresis (CZE). EOF is a function of pH and buffer composition (zeta potential) in bare-silica capillaries at any pH above 3 and runs in the cathodal direction, decreasing the effective separation length for cationic species. On the other hand, the absence of EOF at low pH can significantly increase separation times, particularly for low pl zwitterionic species. This interdependence of pH and EOF limits the ability to design effective separations by capillary zone electrophoresis. The EOTrol family of dynamic coatings (Target Discovery, Inc., Palo Alto, CA, U.S.A.) allows both the magnitude and direction of EOF to be adjusted independently of the buffer composition and pH. Here we report the use of EOTrol in two challenging CZE separations: (1) inorganic cations with small mobility differences, and (2) the rapid separation of organic zwitterions that are only resolvable at low pH.

Rapid separation of protein isoforms by capillary zone electrophoresis with new dynamic coatings.

Many cellular functions are regulated through protein isoforms. Changes in the expression level or regulatory dysfunctions of isoforms often lead to developmental or pathological disorders. Isoforms are traditionally analyzed using techniques such as gel- or capillary-based isoelectric focusing. However, with proper electro-osmotic flow (EOF) control, isoforms with small pI differences can also be analyzed using capillary zone electrophoresis (CZE). Here we demonstrate the ability to quickly resolve isoforms of three model proteins (bovine serum albumin, transferrin, alpha1-antitrypsin) in capillaries coated with novel dynamic coatings. The coatings allow reproducible EOF modulation in the cathodal direction to a level of 10(-9) m2V(-1)s(-1). They also appear to inhibit protein adsorption to the capillary wall, making the isoform separations highly reproducible both in peak areas and apparent mobility. Isoforms of transferrin and alpha1-antitrypsin have been implicated in several human diseases. By coupling the CZE isoform separation with standard affinity capture assays, it may be possible to develop a cost-effective analytical platform for clinical diagnostics.

Stable isotope methods for high-precision proteomics.

Stable isotope tagging methods provide a useful means of determining the relative expression level of individual proteins between samples in a mass spectrometer with high precision (coefficients of variation less than 10%). Because two or more samples tagged with different numbers of stable isotopes can be mixed before any processing steps, sample-to-sample recovery differences are eliminated. Mass spectrometry also allows post-translational modifications, splice variations and mutations (often unnoticed in immunoassays) to be detected and identified, increasing the clinical relevance of the assay and avoiding the issues of non-specific binding and cross-reactivity observed in immunoassays. Several stable isotope tagging methods are available for use in proteomics research. We discuss the advantages and disadvantages of each technique with respect to biomarker discovery, target validation, efficacy and toxicology screening and clinical diagnostic applications.

Isotope-differentiated binding energy shift tags (IDBEST) for improved targeted biomarker discovery and validation.

Mass spectrometry has proved to be an important tool for protein biomarker discovery, identification and characterization. However, global proteomic profiling strategies often fail to identify known low-abundance biomarkers as a result of the limited dynamic range of mass spectrometry (two to three orders of magnitude) compared with the large dynamic range of protein concentrations in biologic fluids (11 to 12 orders of magnitude for serum). In addition, the number of peptides generated in such methods vastly overwhelms the resolution capacity of mass spectrometers, requiring extensive sample clean-up (e.g., affinity tag, retentate chromatography and/or high-performance liquid chromatography) before mass spectrometry analysis. Baiting and affinity pre-enrichment strategies, which overcome the dynamic range and sample complexity issues of global proteomic strategies, are very difficult to couple to mass spectrometry. This is due to the fact that it is nearly impossible to sort target peptides from those of the bait since there will be many cases of isobaric peptides. IDBEST (Target Discovery, Inc.) is a new tagging strategy that enables such pre-enrichment of specific proteins or protein classes as the resulting tagged peptides are distinguishable from those of the bait by a mass defect shift of approximately 0.1 atomic mass units. The special characteristics of these tags allow: resolution of tagged peptides from untagged peptides through incorporation of a mass defect element; high-precision quantitation of up- and downregulation by using stable isotope versions of the same tag; and potential analysis of protein isoforms through more complete peptide coverage from the proteins of interest.

"Mass defect" tags for biomolecular mass spectrometry.

We present a new class of "mass defect" tags with utility in biomolecular mass spectrometry. These tags, incorporating element(s) with atomic numbers between 17 (Cl) and 77 (Ir), have a substantially different nuclear binding energy (mass defect) from the elements common to biomolecules. This mass defect yields a readily resolvable mass difference between tagged and untagged species in high-resolution mass spectrometers. We present the use of a subset of these tags in a new protein sequencing application. This sequencing technique has advantages over existing mass spectral protein identification methodologies: intact proteins are quickly sequenced and unambiguously identified using only an inexpensive, robust mass spectrometer. We discuss the potential broader utility of these tags for the sequencing of other biomolecules, differential display applications and combinatorial methods.