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  • Measurement of human surfactant protein-B turnover in vivo from tracheal aspirates using targeted proteomics. 20178338

    We describe a method to measure protein synthesis and catabolism in humans without prior purification and use the method to measure the turnover of surfactant protein-B (SP-B). SP-B, a lung-specific, hydrophobic protein essential for fetal-neonatal respiratory transition, is present in only picomolar quantities in tracheal aspirate samples and difficult to isolate for dynamic turnover studies using traditional in vivo tracer techniques. Using infusion of [5,5,5-(2)H(3)] leucine and a targeted proteomics method, we measured both the quantity and kinetics of SP-B tryptic peptides in tracheal aspirate samples of symptomatic newborn infants. The fractional synthetic rate (FSR) of SP-B measured using the most abundant proteolytic fragment, a 10 amino acid peptide from the carboxy-terminus of proSP-B (SPTGEWLPR), from the circulating leucine pool was 0.035 +/- 0.005 h(-1), and the fractional catabolic rate was 0.044 +/- 0.003 h(-1). This technique permits high-throughput and sensitive measurement of turnover of low abundance proteins with minimal sample preparation.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Paraoxonase-3, a Putative Circulating Antioxidant, Is Systemically Up-Regulated in Late Gestation in the Fetal Rat, Sheep, and Human. 20463093

    Context: Surfactant is a successful therapeutic based on supplementing preterm infants with a substance that would normally have been up-regulated in late gestation. Although prematurity is associated with oxidative stress, no effective antioxidant therapy has yet been identified. Objective: Our objective was to identify endogenous antioxidants involved in fetal preparation for birth. Design: We performed transcript profiling of fetal rat lung and intestine at 16 d gestational age (dGA) and 20 dGA with out-of-sample validation. Gene expression was then measured in fetal sheep tissues, comparing 1) advancing GA, 2) exogenous maternal dexamethasone (compared with saline, at 130 dGA), and 3) fetal adrenalectomy at 115-118 d on levels at term. Protein levels were compared in human umbilical cord serum using Western blot. Results: Four transcripts were up-regulated more than 20-fold on the array in both rat lung and intestine. One of these, paraoxonase-3 (Pon3), had been identified as a putative circulating antioxidant. Up-regulation of Pon3 mRNA in rat lung, intestine, and liver was confirmed in siblings (all P < 0.001). Pon3 mRNA levels in fetal sheep lung and intestine increased 5.1- and 5.3-fold, respectively (both P < 0.001) between 100 and 145 dGA and were strongly correlated with plasma cortisol (both P < 0.001). Fetal sheep pulmonary Pon3 transcript level was increased 55% (P = 0.01) by dexamethasone and reduced 74% (P < 0.001) by adrenalectomy. Term human infants had more than 6-fold higher umbilical cord serum levels of Pon3 than preterm (24-28 wk GA) infants (P < 0.001). Conclusions: Pon3, a putative circulating antioxidant, was systemically up-regulated in late-gestation rat, sheep, and human fetuses and is a candidate therapeutic in preterm human infants.
    Document Type:
    Reference
    Product Catalog Number:
    2100
    Product Catalog Name:
    Protein-Concentrate Kit (Micro)

  • Cross-sample validation provides enhanced proteome coverage in rat vocal fold mucosa. 21423617

    The vocal fold mucosa is a biomechanically unique tissue comprised of a densely cellular epithelium, superficial to an extracellular matrix (ECM)-rich lamina propria. Such ECM-rich tissues are challenging to analyze using proteomic assays, primarily due to extensive crosslinking and glycosylation of the majority of high M(r) ECM proteins. In this study, we implemented an LC-MS/MS-based strategy to characterize the rat vocal fold mucosa proteome. Our sample preparation protocol successfully solubilized both proteins and certain high M(r) glycoconjugates and resulted in the identification of hundreds of mucosal proteins. A straightforward approach to the treatment of protein identifications attributed to single peptide hits allowed the retention of potentially important low abundance identifications (validated by a cross-sample match and de novo interpretation of relevant spectra) while still eliminating potentially spurious identifications (global single peptide hits with no cross-sample match). The resulting vocal fold mucosa proteome was characterized by a wide range of cellular and extracellular proteins spanning 12 functional categories.
    Document Type:
    Reference
    Product Catalog Number:
    AB1954
    Product Catalog Name:
    Anti-Fibronectin Antibody

  • Considerations when measuring urinary albumin: precision, substances that may interfere, and conditions for sample storage. 1764788

    The measurement of small but abnormal amounts of albumin in urine is important in evaluating kidney disease in people with diabetes mellitus, hypertension, or possible adverse health effects from exposure to nephrotoxins. Routine laboratory methods for measuring albumin are not sensitive enough to measure the amounts that are significant in urine (less than 30 mg/L). In our laboratory we used three immunoassays for measuring urinary albumin: enzyme-linked immunosorbent assay (EIA), radioimmunoassay (RIA), and immunoturbidimetric assay (IT). We calculated the CVs of the three methods, investigated potential interfering substances at three times their normal concentrations, and stored urine under different conditions to find the best way to protect the sample until assay. The potential interferents we checked were transferrin, urea, beta 2-microglobulin, retinol-binding protein, creatinine, kappa and lambda light chains, IgG, hemoglobin, ketone, and glucose. The stability study involved two study temperatures (-20 and -70 degrees C) and four treatments (centrifuging or filtering, before or after storage). We found the following: the RIA had the lowest CV; the results from the interference study showed no interference from normal physiological concentrations of the substances investigated; storage at -70 degrees C regardless of the treatment should be adequate to prevent loss of albumin immunoreactivity.
    Document Type:
    Reference
    Product Catalog Number:
    20-176
    Product Catalog Name:
    100X GTPγS, 10mM

  • Spinning-disk confocal microscopy of yeast. 20946826

    Spinning-disk confocal microscopy is an imaging technique that combines the out-of-focus light rejection of confocal microscopy with the high sensitivity of wide-field microscopy. Because of its unique features, it is well suited to high-resolution imaging of yeast and other small cells. Elimination of out-of-focus light significantly improves the image contrast and signal-to-noise ratio, making it easier to resolve and quantitate small, dim structures in the cell. These features make spinning-disk confocal microscopy an excellent technique for studying protein localization and dynamics in yeast. In this review, I describe the rationale behind using spinning-disk confocal imaging for yeast, hardware considerations when assembling a spinning-disk confocal scope, and methods for strain preparation and imaging. In particular, I discuss choices of objective lens and camera, choice of fluorescent proteins for tagging yeast genes, and methods for sample preparation.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
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