Background Biological diagnosis of hemoglobin disorders is a complex process relying on the combination of several analytical techniques to identify Hb variants in a particular sample. Currently, hematology laboratories usually use high-performance liquid chromatography (HPLC), capillary electrophoresis and gel-based methods to characterize Hb variants. Co-elution and co-migration may represent major issues for precise identification of Hb variants, even for the most common ones such as Hb S and C. Methods We adapted a top-down selected reaction monitoring (SRM) electron transfer dissociation (ETD) mass spectrometry (MS) method to fit with a clinical laboratory environment. An automated analytical process with semi-automated data analysis compatible with a clinical practice was developed. A comparative study between a reference HPLC method and the MS assay was performed on 152 patient samples. Results The developed workflow allowed to identify with high specificity and selectivity the most common Hb variants (Hb S and Hb C). Concordance of the MS-based approach with HPLC was 71/71 (100%) for Hb S and 11/11 (100%) for Hb C. Conclusions This top-down SRM ETD method can be used in a clinical environment to detect Hb S and Hb C.

Characterization of protein structure modifications is an important field in mass spectrometry (MS)-based proteomics. Here, we describe a process to quickly and reliably identify a mass change in a targeted protein sequence by top-down mass spectrometry (TD MS) using electron transfer dissociation (ETD). The step-by-step procedure describes how to develop a TD MS method for data acquisition as well as the data analysis process. The described TD MS workflow utilizes diagnostic ions to characterize an unknown sample in a few hours.

Cet article presente trois methodes d’analyse des proteines intactes par spectrometrie de masse appliquees de maniere combinee au diagnostic des maladies de l’hemoglobine ou hemoglobinopathies. La premiere methode est basee sur l’analyse selective de couples ion precurseur – ion produit (« selected reaction monitoring » (SRM)) dans une trappe d’ions et utilise une technique particuliere d’activation des ions precurseurs appelee « electron transfer dissociation » (ETD). Elle permet l’identifi cation specifi que de variants de l’hemoglobine selectionnes pour leur prevalence et leur importance clinique. La seconde methode a ete developpee sur le meme instrument pour la quantifi cation de l’hemoglobine A2. Les ions precurseurs d’interet sont isoles puis detectes sans activation, permettant d’augmenter le rapport signal/bruit et d’atteindre un niveau de precision analytique compatible avec les exigences cliniques. La troisieme methode utilise un spectrometre de masse a haute resolution de type Qq-TOF avec activation ETD pour la caracterisation de variants rares de l’hemoglobine. Cette approche permet la localisation de la sequence mutee dans la chaine d’hemoglobine etudiee. Tous les developpements methodologiques presentes ont ete realises avec comme objectif que l’acquisition, l’analyse et l’interpretation des donnees puissent etre adaptees a un environnement clinique.

Accessing online health content of high quality and reliability presents challenges. Laypersons cannot easily differentiate trustworthy content from misinformed or manipulated content. This article describes complementary approaches for members of the general public and health professionals to find trustworthy content with as little bias as possible. These include the Khresmoi health search engine (K4E), the Health On the Net Code of Conduct (HONcode) and health trust indicator Web browser extensions.

The Health On the Net Foundation (HON) was born in 1996, during the beginning of the World Wide Web, from a collective decision by health specialists, led by the late Jean-Raoul Scherrer, who anticipated the need for online trustworthy health information. Because the Internet is a free space that everyone shares, a search for quality information is like a shot in the dark: neither will reliably hit their target. Thus, HON was created to promote deployment of useful and reliable online health information, and to enable its appropriate and efficient use. Two decades on, HON is the oldest and most valued quality marker for online health information. The organization has maintained its reputation through dynamic measures, innovative endeavors and dedication to upholding key values and goals. This paper provides an overview of the HON Foundation, and its activities, challenges, and achievements over the years.

The ability to accurately quantify proteins in formalin-fixed paraffin-embedded tissues using targeted mass spectrometry opens exciting perspectives for biomarker discovery. We have developed and evaluated a selectedreaction monitoring assay for the human receptor tyrosine-protein kinase erbB-2 (HER2) in formalin-fixed paraffin-embedded breast tumors. Peptide candidates were identified using an untargeted mass spectrometry approach in relevant cell lines. A multiplexed assay was developed for the six best candidate peptides and evaluated for linearity, precision and lower limit of quantification. Results showed a linear response over a calibration range of 0.012 to 100 fmol on column (R(2): 0.99-1.00).The lower limit of quantification was 0.155 fmol on column for all peptides evaluated. The six HER2 peptides were quantified by selected reaction monitoring in a cohort of 40 archival formalin-fixed paraffin-embedded tumor tissues from women with invasive breast carcinomas, which showed different levels of HER2 gene amplification as assessed by standard methods used in clinical pathology. The amounts of the six HER2 peptides were highly and significantly correlated with each other, indicating that peptide levels can be used as surrogates of protein amounts in formalin-fixed paraffin-embedded tissues. After normalization for sample size, selected reaction monitoring peptide measurements were able to correctly predict 90% of cases based on HER2 amplification as defined by the American Society of Clinical Oncology and College of American Pathologists. In conclusion, the developed assay showed good analytical performance and a high agreement with immunohistochemistry and fluorescence in situ hybridization data. This study demonstrated that selected reaction monitoring allows to accurately quantify protein expression in formalin-fixed paraffin-embedded tissues and represents therefore a powerful approach for biomarker discovery studies. The untargeted mass spectrometry data is available via ProteomeXchange whereas the quantification data by selected reaction monitoring is available on the Panorama Public website.

A previous high-resolution metabolomic study pointed out a dysregulation of urinary steroids and bile acids in human cases of acute dioxin exposure. A subset of 24 compounds was highlighted as putative biomarkers. The aim of the current study was (i) to evaluate the 24 biomarkers in an independent human cohort exposed to dioxins released from the incineration fumes of a municipal waste incinerator and; (ii) to identify them by comparison with authentic chemical standards and biosynthesised products obtained with in vitro metabolic reactions. An orthogonal projection to latent structures discriminant analysis built on biomarker profiles measured in the intoxicated cohort and the controls separated both groups with reported values of 93.8%; 100% and 87.5% for global accuracy; sensitivity and specificity; respectively. These results corroborated the 24 compounds as exposure biomarkers; but a definite identification was necessary for a better understanding of dioxin toxicity. Dehydroepiandrosterone 3β-sulfate, androsterone 3α-glucuronide, androsterone 3α-sulfate, pregnanediol 3α-glucuronide and 11-ketoetiocholanolone 3α-glucuronide were identified by authentic standards. Metabolic reactions characterised four biomarkers: glucuronide conjugates of 11β-hydroxyandrosterone; glycochenodeoxycholic acid and glycocholic acid produced in human liver microsomes and glycoursodeoxycholic acid sulfate generated in cytosol fraction. The combination of metabolomics by high-resolution mass spectrometry with in vitro metabolic syntheses confirmed a perturbed profile of steroids and bile acids in human cases of dioxin exposure.

The screening of endocrine disrupting chemicals (EDCs) that may alter steroidogenesis represents a highly important field mainly due to the numerous pathologies, such as cancer, diabetes, obesity, osteoporosis, and infertility that have been related to impaired steroid-mediated regulation. The adrenal H295R cell model has been validated to study steroidogenesis by the Organization for Economic Co-operation and Development (OECD) guideline. However, this guideline focuses solely on testosterone and estradiol monitoring, hormones not typically produced by the adrenals, hence limiting possible in-depth mechanistic investigations. The present work proposes an untargeted steroidomic footprinting workflow based on ultra-high pressure liquid chromatography (UHPLC) coupled to high-resolution MS for the screening and mechanistic investigations of EDCs in H295R cell supernatants. A suspected EDC, triclocarban (TCC), used in detergents, cosmetics, and personal care products, was selected to demonstrate the efficiency of the reported methodology, allowing the simultaneous assessment of a steroidomic footprint and quantification of a selected subset of steroids in a single analysis. The effects of exposure to increasing TCC concentrations were assessed, and the selection of features with database matching followed by multivariate analysis has led to the selection of the most salient affected steroids. Using correlation analysis, 11 steroids were associated with a high, 18 with a medium, and 8 with a relatively low sensitivity behavior to TCC. Among the candidates, 13 identified steroids were simultaneously quantified, leading to the evaluation and localization of the disruption of steroidogenesis caused by TCC upstream of the formation of pregnenolone. The remaining candidates could be associated with a specific steroid class (progestogens and corticosteroids, or androgens) and represent a specific footprint of steroidogenesis disruption by TCC. This strategy was devised to be compatible with medium/high-throughput screening and could be useful for the mechanistic elucidation of EDCs.

Hemoglobin disorder diagnosis is a complex procedure combining several analytical steps. Due to the lack of specificity of the currently used protein analysis methods, the identification of uncommon hemoglobin variants (proteoforms) can become a hard task to accomplish. The aim of this work was to develop a mass spectrometry-based approach to quickly identify mutated protein sequences within globin chain variants. To reach this goal, a top-down electron transfer dissociation mass spectrometry method was developed for hemoglobin β chain analysis. A diagnostic product ion list was established with a color code strategy allowing to quickly and specifically localize a mutation in the hemoglobin β chain sequence. The method was applied to the analysis of rare hemoglobin β chain variants and an (A)γ-β fusion protein. The results showed that the developed data analysis process allows fast and reliable interpretation of top-down electron transfer dissociation mass spectra by nonexpert users in the clinical area.

The use of targeted LC-MS/MS methods for protein quantitation in clinical laboratories implies a careful evaluation of potential sources of analytical interference. In this study, we investigated whether inflammation, which is associated with both the release of proteolytic enzymes and increased expression of acute phase protease inhibitors, is affecting the accuracy of a haptoglobin selected reaction monitoring (SRM) assay.

Differentiating malignant from nonmalignant biliary stenoses is challenging. This could be facilitated by the measurement of cancer biomarkers in bile. We aimed at (i) identifying new cancer biomarkers by comparative proteomic analysis of bile collected from patients with a malignant or benign biliary stenosis (exploratory phase) and (ii) verifying the accuracy of the newly identified potential biomarkers for discriminating malignant versus nonmalignant biliary stenoses in a larger group of patients (confirmation phase). Overall, 66 proteins were found overexpressed (ratio>1.5) in at least one cancer condition using proteomic analysis and 7 proteins were increased in all malignant/nonmalignant disease comparisons. Preliminary screening by immunoblot highlighted carcinoembryonic cell adhesion molecule 6 (CEAM6), a cell surface protein overexpressed in many human cancers, as an interesting candidate biomarker. ELISA subsequently confirmed CEAM6 as a potential bile biomarker for distinguishing malignant from benign biliary stenoses with a receiver operating characteristic (ROC) area under the curve (AUC) of 0.92 (specificity 83%, sensitivity 93%, positive predictive value 93%, and negative predictive value 83%). No significant difference in serum CEAM6 level was found between malignant and nonmalignant samples. Combining bile CEAM6 and serum CA19-9 in a panel further improved diagnostic accuracy for malignant stenoses (AUC 0.96, specificity 83%, sensitivity 97%, positive predictive value 93%, and negative predictive value 91%). CEAM6 measurement in bile could be clinically useful to discriminate between malignant and nonmalignant causes of biliary stenosis. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.

Over the last two decades, numerous genomes of pathogenic bacteria have been fully sequenced and annotated, while others are continuously being sequenced. To date, the sequences of more than 8,500 whole bacterial genomes are publicly available for research purposes. These efforts in high-throughput sequencing simultaneously to progresses in methods allowing to study whole transcriptome and proteome of bacteria provide the basis of comprehensive understanding of metabolism, adaptability to environment, regulation, resistance pathways, or pathogenicity mechanisms of bacterial pathogens. Staphylococcus aureus is a Gram-positive human pathogen causing a wide variety of infections ranging from benign skin infection to life-threatening diseases. Furthermore, the spreading of multidrug-resistant isolates requiring the use of last barrier drugs has resulted in a particular attention of the medical and scientific community to this pathogen. We describe here proteomic methods to prepare, identify, and analyze protein fractions, which allow studying Staphylococcus aureus on the organism level. Besides evaluation of the whole bacterial transcriptome, this approach might contribute to the development of rapid diagnostic tests and to the identification of new drug targets to improve public health.

MS for the evaluation of endocrine disrupting chemicals in H295R cells David Tonoli, Petra Strajhar, Julien Boccard, Denis Hochstrasser, Fabienne Jeanneret, Alex Odermatt, Serge Rudaz 1 School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, Switzerland 2 Department of Human Protein Sciences, Faculty of Medicine, University of Geneva, Geneva, Switzerland Swiss Centre for Applied Human Toxicology, Switzerland Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Basel, Switzerland Department of Genetic and Laboratory Medicine, Geneva University Hospitals, Geneva, Switzerland

Background: Interleukin (IL)-6 is recognised as an important cytokine involved in inflammatory diseases of the central nervous system (CNS).Objective: To perform a large retrospective study designed to test cerebrospinal fluid (CSF) IL-6 levels in the context of neurological diseases, and evaluate its usefulness as a biomarker to help discriminate multiple sclerosis (MS) from other inflammatory neurological diseases (OIND).Patients and Methods: We analyzed 374 CSF samples for IL-6 using a quantitative enzyme-linked immunosorbent assay. Groups tested were composed of demyelinating diseases of the CNS (DD, n = 117), including relapsing-remitting MS (RRMS, n = 65), primary progressive MS (PPMS, n = 11), clinically isolated syndrome (CIS, n = 11), optic neuritis (ON, n = 30); idiopathic transverse myelitis (ITM, n = 10); other inflammatory neurological diseases (OIND, n = 35); and non-inflammatory neurological diseases (NIND, n = 212). Differences between groups were analysed using Kruskal-Wallis test and Mann-Whitney U-test.Results: CSF IL-6 levels exceeded the positivity cut-off of 10 pg/ml in 18 (51.4%) of the 35 OIND samples, but in only three (3.9%) of the 76 MS samples collected. CSF IL-6 was negative for all NIND samples tested (0/212). IL-6 cut-off of 10 pg/ml offers 96% sensitivity to exclude MS.Conclusion: CSF IL-6 may help to differentiate MS from its major differential diagnosis group, OIND.

High throughput protein identification and quantification analysis based on mass spectrometry are fundamental steps in most proteomics projects. Here, we present EasyProt (available at http://easyprot.unige.ch), a new platform for mass spectrometry data processing, protein identification, quantification and unexpected post-translational modification characterization. EasyProt provides a fully integrated graphical experience to perform a large part of the proteomic data analysis workflow. Our goal was to develop a software platform that would fulfill the needs of scientists in the field, while emphasizing ease-of-use for non-bioinformatician users. Protein identification is based on OLAV scoring schemes and protein quantification is implemented for both, isobaric labeling and label-free methods. Additional features are available, such as peak list processing, isotopic correction, spectra filtering, charge-state deconvolution and spectra merging. To illustrate the EasyProt platform, we present two identification and quantification workflows based on isobaric tagging and label-free methods.