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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

  • Rapid and sensitive determination of protein-nitrotyrosine by ELISA: Application to human plasma. 22276750

    3-Nitrotyrosine (3NT) is known as an important indicator of nitrosative stress and has been linked to various diseases. Our aim was to develop an indirect ELISA (enzyme-linked immunosorbent assay) method suitable for the detection of protein-bound 3NT in clinical plasma and serum samples. Nitrated protein standards and reduced protein standards were prepared. Limit of detection was determined for standards; recovery and reproducibility were determined for human plasma samples. The limit of detection for this method is 1.82±0.56 pmol/mg protein. Mean recovery of standards was 95%. 3NT concentration in plasma samples of obese and normal weight subjects was determined to be between 2 pmol/mg and 19 pmol/mg. No time-consuming sample preparation or expensive laboratory equipment is required, and applied antibodies are commercially available. Sensitivity, rapid analysis time, possibilities of high throughput applications and small sample volumes make this ELISA attractive for use in clinical laboratories.
    Document Type:
    Reference
    Product Catalog Number:
    06-284
    Product Catalog Name:
    Anti-Nitrotyrosine Antibody

  • Large-Scale Filter-Aided Sample Preparation Method for the Analysis of the Ubiquitinome. 28260372

    Protein ubiquitination regulates key cellular functions, including protein homeostasis and signal transduction. The digestion of ubiquitinated proteins with trypsin yields a glycine-glycine remnant bound to the modified lysine residue (K-ε-GG) that can be recognized by specific antibodies for immunoaffinity purification (IAP) and subsequent identification of ubiquitination sites by mass spectrometry. Previous ubiquitinome studies based on this strategy have consistently digested milligram amounts of protein as starting material using in-solution digestion protocols prior to K-ε-GG enrichment. Filter-aided sample preparation (FASP) surpasses in-solution protein digestion in cleavage efficiency, but its performance has thus far been shown for digestion of sample amounts on the order of micrograms. Because cleavage efficiency is pivotal in the generation of the K-ε-GG epitope recognized during IAP, here we developed a large-scale FASP method (LFASP) for digestion of milligram amounts of protein and evaluated its applicability to the study of the ubiquitinome. Our results demonstrate that LFASP-based tryptic digestion is efficient, robust, reproducible, and applicable to the study of the ubiquitinome. We benchmark our results with state-of-the-art ubiquitinome studies and show a ∼3-fold reduction in the proportion of miscleaved peptides with the method presented here. Beyond ubiquitinome analysis, LFASP overcomes the general limitation in sample capacity of standard FASP-based protocols and can therefore be used for a variety of applications that demand a large(r) amount of starting material.
    Document Type:
    Reference
    Product Catalog Number:
    UFC901024
    Product Catalog Name:
    Amicon® Ultra-15 Centrifugal Filter Unit

  • 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)

  • 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

  • Chromatin immunoprecipitation (ChIP): revisiting the efficacy of sample preparation, sonication, quantification of sheared DNA, and analysis via PCR. 22046253

    The "quantitative" ChIP, a tool commonly used to study protein-DNA interactions in cells and tissue, is a difficult assay often plagued with technical error. We present, herein, the process required to merge multiple protocols into a quick, reliable and easy method and an approach to accurately quantify ChIP DNA prior to performing PCR. We demonstrate that high intensity sonication for at least 30 min is required for full cellular disruption and maximum DNA recovery because ChIP lysis buffers fail to lyse formaldehyde-fixed cells. In addition, extracting ChIP DNA with chelex-100 yields samples that are too dilute for evaluation of shearing efficiency or quantification via nanospectrophotometry. However, DNA extracted from the Mock-ChIP supernatant via the phenol-chloroform-isoamyl alcohol (PCIA) method can be used to evaluate DNA shearing efficiency and used as the standard in a fluorescence-based microplate assay. This enabled accurate quantification of DNA in chelex-extracted ChIP samples and normalization to total DNA concentration prior to performing real-time PCR (rtPCR). Thus, a quick ChIP assay that can be completed in nine bench hours over two days has been validated along with a rapid, accurate and repeatable way to quantify ChIP DNA. The resulting rtPCR data more accurately depicts treatment effects on protein-DNA interactions of interest.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
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