BackgroundCurrent quantification methods for mass spectrometry (MS)-based proteomics either do not provide sufficient control of variability or are difficult to implement for routine clinical testing.ResultsWe present here an integrated quantification (InteQuan) method that better controls pre-analytical and analytical variability than the popular quantification method using stable isotope-labeled standard peptides (SISQuan). We quantified 16 lung cancer biomarker candidates in human plasma samples in three assessment studies, using immunoaffinity depletion coupled with multiple reaction monitoring (MRM) MS. InteQuan outperformed SISQuan in precision in all three studies and tolerated a two-fold difference in sample loading. The three studies lasted over six months and encountered major changes in experimental settings. Nevertheless, plasma proteins in low ng/ml to low μg/ml concentrations were measured with a median technical coefficient of variation (CV) of 11.9% using InteQuan. The corresponding median CV using SISQuan was 15.3% after linear fitting. Furthermore, InteQuan surpassed SISQuan in measuring biological difference among clinical samples and in distinguishing benign versus cancer plasma samples.ConclusionsWe demonstrated that InteQuan is a simple yet robust quantification method for MS-based quantitative proteomics, especially for applications in biomarker research and in routine clinical testing.Electronic supplementary materialThe online version of this article (doi:10.1186/1559-0275-12-3) contains supplementary material, which is available to authorized users.
Forest fire smoke influence in urban areas is relatively easy to detect at high concentrations but more challenging to detect at low concentrations. In this study, we present a simplified method that can reliably quantify smoke tracers in an urban environment at relatively low cost and complexity. For this purpose, we used dual-bed thermal desorption tubes with an auto-sampler to collect continuous samples of volatile organic compounds (VOCs). We present the validation and evaluation of this approach using thermal desorption gas chromatography mass spectrometry (TD-GC-MS) to detect VOCs at ppt to ppb concentrations. To evaluate the method, we tested stability during storage, interferences (e.g., water and O3), and reproducibility for reactive and short-lived VOCs such as acetonitrile (a specific chemical tracer for biomass burning), acetone, n-pentane, isopentane, benzene, toluene, furan, acrolein, 2-butanone, 2,3-butanedione, methacrolein, 2,5- dimethylfuran, and furfural. The results demonstrate that these VOCs can be quantified reproducibly with a total uncertainty of ≤30% between the collection and analysis, and with storage times of up to 15 days. Calibration experiments performed over a dynamic range of 10–150 ng loaded on to each thermal desorption tube at different relative humidity showed excellent linearity (r2 ≥ 0.90). We utilized this method during the summer 2019 National Oceanic and Atmospheric Administration (NOAA) Fire Influence on Regional to Global Environments Experiment–Air Quality (FIREX-AQ) intensive experiment at the Boise ground site. The results of this field study demonstrate the method’s applicability for ambient VOC speciation to identify forest fire smoke in urban areas.
With the discovery of hydrogen sulfide as a signaling molecule and a potential therapeutic, measurement of free sulfide in blood – as hydrogen sulfide or hydrosulfide anion – has taken on importance. Here, we demonstrate and validate a method of free sulfide measurement whereby the free sulfide in whole blood is derivatized with excess monobromobimane. The resulting sulfide‐dibimane is subsequently extracted into ethyl acetate, followed by quantitation of sulfide‐dibimane via reverse‐phase HPLC with fluorescence detection. Reaction conditions are validated through 1) characterization of rate of conversion from sulfide to sulfide‐dibimane, 2) analysis of reaction in the presence of potential interferants, and 3) recovery of standard samples from a whole‐blood matrix. We found that reaction conditions of a mixture of acetonitrile and HEPES buffer (50 mM pH 8) gave rapid, clean conversion of sulfide to sulfide‐dibimane in the presence of excess monobromobimane. For whole blood, a 1:1:1 reaction mixture of 200 μl each acetonitrile:HEPES:blood proved optimal. Using this protocol, standard samples were consistently recovered in approximately 76% yield over the range of the assay. Baseline levels of free sulfide in rat blood were found to be about 0.3 – 0.5 μM. Subsequent work has proved the method effective in generating whole‐blood sulfide PK data in multiple species.
Hydrogen sulfide exhibits many characteristics of an endogenous biological mediator and is present at low baseline levels in tissues and blood. A large body of evidence suggests that exogenously applied sulfide may also be suitable as a therapeutic agent. Therefore, a method using the rare 34S sulfur isotope was developed and validated to distinguish exogenously administered sulfide from endogenous sulfide which is predominantly 32S‐sulfide. Sodium 34S‐sulfide was prepared and administered intravenously to Sprague Dawley rats. Concentrations of 32S and 34S‐sulfide were measured in blood and tissues after derivatization with monobromobimane as monobromobimane derivative, separation by reverse phase HPLC and quantification by mass spectroscopy. On intravenous infusion of sodium 34S‐sulfide into rats, blood sulfide concentrations of 34S‐sulfide, but not 32S‐sulfide increased in a dose‐dependent manner indicating that exogenously administered sulfide can be traced and distinguished from endogenous sulfide. The method will be useful in monitoring of the distribution of therapeutic sulfide in tissues and organs.
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