Electrophilically reactive compounds have the ability to form adducts with nucleophilic sites in DNA and proteins, constituting a risk for toxic effects. Mass spectrometric detection of adducts to N-terminal valine in hemoglobin (Hb) after detachment by modified Edman degradation procedures is one approach for in vivo monitoring of exposure to electrophilic compounds/metabolites. So far, applications have been limited to one or a few selected reactive species, such as acrylamide and its metabolite glycidamide. This article presents a novel screening strategy for unknown Hb adducts to be used as a basis for an adductomic approach. The method is based on a modified Edman procedure, FIRE, specifically developed for LC-MS/MS analysis of N-terminal valine adducts in Hb detached as fluorescein thiohydantoin (FTH) derivatives. The aim is to detect and identify a priori unknown Hb adducts in human blood samples. Screening of valine adducts was performed by stepwise scanning of precursor ions in small mass increments, monitoring four fragments common for the FTH derivative of valine with different N-substitutions in the multiple-reaction mode, covering a mass range of 135 Da (m/z 503-638). Samples from six smokers and six nonsmokers were analyzed. Control experiments were performed to compare these results with known adducts and to check for artifactual formation of adducts. In all samples of smokers and nonsmokers, seven adducts were identified, of which six have previously been studied. Nineteen unknown adducts were observed, and 14 of those exhibited fragmentation patterns similar to earlier studied FTH derivatives of adducts to valine. Identification of the unknown adducts will be the focus of future work. The presented methodology is a promising screening tool using Hb adducts to indicate exposure to potentially toxic electrophilic compounds and metabolites.

PURPOSE We performed an open-label randomized controlled phase III study comparing treatment outcome and toxicity between radiotherapy (RT) with concomitant cisplatin versus concomitant cetuximab in patients with locoregionally advanced head and neck squamous cell carcinoma (HNSCC; stage III-IV according to the Union for International Cancer Control TNM classification, 7th edition). MATERIALS AND METHODS Eligible patients were randomly assigned 1:1 to receive either intravenous cetuximab 400 mg/m2 1 week before start of RT followed by 250 mg/m2/wk, or weekly intravenous cisplatin 40 mg/m2, during RT. RT was conventionally fractionated. Patients with T3-T4 tumors underwent a second random assignment 1:1 between standard RT dose 68.0 Gy to the primary tumor or dose escalation to 73.1 Gy. Primary end point was overall survival (OS) evaluated using adjusted Cox regression analysis. Secondary end points were locoregional control, local control with dose-escalated RT, pattern of failure, and adverse effects. RESULTS Study inclusion was prematurely closed after an unplanned interim analysis when 298 patients had been randomly assigned. At 3 years, OS was 88% (95% CI, 83% to 94%) and 78% (95% CI, 71% to 85%) in the cisplatin and cetuximab groups, respectively (adjusted hazard ratio, 1.63; 95% CI, 0.93 to 2.86; P = .086). The cumulative incidence of locoregional failures at 3 years was 23% (95% CI, 16% to 31%) compared with 9% (95% CI, 4% to 14%) in the cetuximab versus the cisplatin group (Gray’s test P = .0036). The cumulative incidence of distant failures did not differ between the treatment groups. Dose escalation in T3-T4 tumors did not increase local control. CONCLUSION Cetuximab is inferior to cisplatin regarding locoregional control for concomitant treatment with RT in patients with locoregionally advanced HNSCC. Additional studies are needed to identify possible subgroups that still may benefit from concomitant cetuximab treatment.

Background Untargeted metabolomics datasets contain large proportions of uninformative features that can impede subsequent statistical analysis such as biomarker discovery and metabolic pathway analysis. Thus, there is a need for versatile and data-adaptive methods for filtering data prior to investigating the underlying biological phenomena. Here, we propose a data-adaptive pipeline for filtering metabolomics data that are generated by liquid chromatography-mass spectrometry (LC-MS) platforms. Our data-adaptive pipeline includes novel methods for filtering features based on blank samples, proportions of missing values, and estimated intra-class correlation coefficients. Results Using metabolomics datasets that were generated in our laboratory from samples of human blood, as well as two public LC-MS datasets, we compared our data-adaptive filtering method with traditional methods that rely on non-method specific thresholds. The data-adaptive approach outperformed traditional approaches in terms of removing noisy features and retaining high quality, biologically informative ones. The R code for running the data-adaptive filtering method is provided at https://github.com/courtneyschiffman/Metabolomics-Filtering . Conclusions Our proposed data-adaptive filtering pipeline is intuitive and effectively removes uninformative features from untargeted metabolomics datasets. It is particularly relevant for interrogation of biological phenomena in data derived from complex matrices associated with biospecimens. Electronic supplementary material The online version of this article (10.1186/s12859-019-2871-9) contains supplementary material, which is available to authorized users.

The reaction products of electrophiles in vivo can be measured as adducts to the abundant proteins, hemoglobin (Hb), and human serum albumin (HSA), in human blood samples. During the last decade, methods for untargeted screening of such adducts, called “adductomics”, have used liquid chromatography-mass spectrometry to detect large numbers of previously unknown Hb and HSA adducts. This review presents methodologies that were developed and used in our laboratories for Hb and HSA adductomics, respectively. We discuss critical aspects regarding choice of target protein, sample preparation, mass spectrometry, data evaluation, and strategies for identification of detected unknown adducts. With this review we give an overview of these two methodologies used for protein adductomics and the precursor electrophiles that have been elucidated from the adducts.

Although benzene has long been recognized as a cause of human leukemia, the mechanism by which this simple molecule causes cancer has been problematic. A complicating factor is benzene metabolism, which produces many reactive intermediates, some specific to benzene and others derived from redox processes. Using archived serum from 20 nonsmoking Chinese workers, 10 with and 10 without occupational exposure to benzene (exposed: 3.2-88.9 ppm, controls: 0.002-0.020 ppm), we employed an adductomic pipeline to characterize protein modifications at Cys34 of human serum albumin, a nucleophilic hotspot in extracellular fluids. Of the 47 measured human serum albumin modifications, 39 were present at higher concentrations in benzene-exposed workers than in controls and many of the exposed-control differences were statistically significant. Correlation analysis identified three prominent clusters of adducts, namely putative modifications by benzene oxide and a benzene diolepoxide that grouped with other measures of benzene exposure, adducts of reactive oxygen and carbonyl species, and Cys34 disulfides of small thiols that are formed following oxidation of Cys34. Benzene diolepoxides are potent mutagens and carcinogens that have received little attention as potential causes of human leukemia. Reactive oxygen and carbonyl species-generated by redox processes involving polyphenolic benzene metabolites and by Cyp2E1 regulation following benzene exposure-can modify DNA and proteins in ways that contribute to cancer. The fact that these diverse human serum albumin modifications differed between benzene-exposed and control workers suggests that benzene can increase leukemia risks via multiple pathways involving a constellation of reactive molecules.

Electrophilic compounds have the ability to form adducts with nucleophilic sites in proteins and DNA in tissues, and thereby constitute risks for toxic effects. Adductomic approaches are developed for systematic screening of adducts to DNA and blood proteins, with the aim to detect unknown internal exposures to electrophiles. In a previous adductomic screening of adducts to N-terminals in hemoglobin, using LC-MS/MS, 19 unknown adducts were detected in addition to seven previously identified adducts. The present paper describes the identification of four of these unknown adducts, as well as the strategy used to identify them. Using LC-MS data from the screening, hypotheses about adduct identities were formulated: probable precursor electrophiles with matching molecular weights were suggested based on the molecular weights of the modifications and the retention times of the analytes, in combination with comparisons of theoretical Log P calculations and databases. Reference adducts were generated by incubation of blood samples with the hypothesized precursor electrophiles. The four identified precursor electrophiles, corresponding to the observed unknown adducts, were glyoxal, methylglyoxal, acrylic acid and 1-octen-3-one. Possible origins/exposure sources and toxicological information concerning the electrophilic precursors are discussed. The identified adducts could be explored as possible biomarkers for exposure.

Background Hydroxychloroquine (HCQ) is the standard of care in the treatment of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and other inflammatory rheumatic diseases and potentially for the treatment in COVID-19 patients. Determination of HCQ for therapeutic drug monitoring (TDM) can be performed in whole blood (WB), serum, and plasma. Direct comparisons of WB, serum, and plasma levels of HCQ in patients with SLE have not previously been reported. We describe a method for the determination of HCQ in human blood using liquid chromatography-high-resolution mass spectrometry (LC-HRMS) and compare the suitability of the three sample matrices. Methods A method for the determination of HCQ in human blood using LC-HRMS was developed, validated, and applied for the determination of HCQ levels in WB, serum, and plasma from 26 SLE patients. The reproducibility of the method, in the three matrices, was evaluated using quality control samples and repeated preparations and measurements of patient samples. The performance of the developed method for HCQ measurement in serum was further evaluated by comparison with two previously reported extraction methods. Results The performance of the presented method demonstrated high accuracy and precision. A large range of HCQ concentrations was observed for the SLE patients in all three matrices (WB, serum, and plasma). The mean levels in WB were approximately two-fold the levels in serum and plasma (813 ng/mL compared to 436 ng/mL and 362 ng/mL, respectively). Spiked quality controls showed high reproducibility for all matrices (coefficient of variation, CV, approx. 5%), whereas in patient samples, equally high-precision was only found using WB as the matrix (CV 3%). The CV for serum and plasma was 14% and 39%, respectively. Two alternative methods applied to serum samples did not demonstrate improved precision. Conclusions A LC-HRMS method for the measurement of HCQ in human blood was developed and validated. Whole blood was found to be the superior sample matrix in terms of sample reproducibility. Thus, whole blood samples should be used for HCQ analysis when patients are monitored for HCQ treatment effects. The assay is in clinical use to monitor levels of HCQ in patients.

Electrophiles have the ability to form adducts to nucleophilic sites in proteins and DNA. Internal exposure to such compounds thus constitutes a risk for toxic effects. Screening of adducts using mass spectrometric methods by adductomic approaches offers possibilities to detect unknown electrophiles present in tissues. Previously, we employed untargeted adductomics to detect 19 unknown adducts to N-terminal valine in hemoglobin (Hb) in human blood. This article describes the characterization of one of these adducts, which was identified as the adduct from ethyl vinyl ketone (EVK). The mean adduct level was 40 ± 12 pmol/g Hb in 12 human blood samples; adduct levels from acrylamide (AA) and methyl vinyl ketone (MVK) were quantified for comparison. Using l-valine p-nitroanilide (Val-pNA), introduced as a model of the N-terminal valine, the rate of formation of the EVK adduct was studied, and the rate constant determined to 200 M(-1)h(-1) at 37 °C. In blood, the reaction rate was too fast to be feasibly measured, EVK showing a half-life <1 min. Parallel experiments with AA and MVK showed that the two vinyl ketones react approximately 2 × 10(3) times faster than AA. The EVK-Hb adduct was found to be unstable, with a half-life of 7.6 h. From the mean adduct level measured in human blood, a daily dose (area under the concentration-time-curve, AUC) of 7 nMh EVK was estimated. The AUC of AA from intake via food is about 20 times higher. EVK is naturally present in a wide range of foods and is also used as a food additive. Most probably, naturally formed EVK is a major source to observed adducts. Evaluation of available toxicological data and information on occurrence of EVK indicate that further studies of EVK are motivated. This study illustrates a quantitative strategy in the initial evaluation of the significance of an adduct detected through adduct screening.