Publications by authors named "Virginie Brun"

39 Publications

Well Plate Maker: A user-friendly randomized block design application to limit batch effects in largescale biomedical studies.

Bioinformatics 2021 Feb 4. Epub 2021 Feb 4.

Univ. Grenoble Alpes, CEA, Inserm, BGE U1038, Grenoble, 38000, France.

Summary: Many factors can influence results in clinical research, in particular bias in the distribution of samples prior to biochemical preparation. Well Plate Maker is a user-friendly application to design single- or multiple-well plate assays. It allows multiple group experiments to be randomized and therefore helps to reduce possible batch effects. Although primarily fathered to optimize the design of clinical sample analysis by high throughput mass spectrometry (e.g. proteomics or metabolomics), it includes multiple options to limit edge-of-plate effects, to incorporate control samples, or to limit cross-contamination. It thus fits the constraints of many experimental fields.

Availability And Implementation: Well Plate Maker is implemented in R and available at Bioconductor repository (https://bioconductor.org/packages/wpm) under the open source Artistic 2.0 license. In addition to classical scripting, it can be used through a graphical user interface, developed with Shiny technology.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btab065DOI Listing
February 2021

PepS: An Innovative Microfluidic Device for Bedside Whole Blood Processing before Plasma Proteomics Analyses.

Anal Chem 2021 01 15;93(2):683-690. Epub 2020 Dec 15.

Univ. Grenoble Alpes, CEA, Inserm, IRIG, BGE, EDyP, F-38000 Grenoble, FRANCE.

Immunoassays have been used for decades in clinical laboratories to quantify proteins in serum and plasma samples. However, their limitations make them inappropriate in some cases. Recently, mass spectrometry (MS) based proteomics analysis has emerged as a promising alternative method when seeking to assess panels of protein biomarkers with a view to providing protein profiles to monitor health status. Up to now, however, translation of MS-based proteomics to the clinic has been hampered by its complexity and the substantial time and human resources necessary for sample preparation. Plasma matrix is particularly tricky to process as it contains more than 3000 proteins with concentrations spanning an extreme dynamic range (10). To address this preanalytical challenge, we designed a microfluidic device (PepS) automating and accelerating blood sample preparation for bottom-up MS-based proteomics analysis. The microfluidic cartridge is operated through a dedicated compact instrument providing fully automated fluid processing and thermal control. In less than 2 h, the PepS device allows bedside plasma separation from whole blood, volume metering, depletion of albumin, protein digestion with trypsin, and stabilization of tryptic peptides on solid-phase extraction sorbent. For this first presentation, the performance of the PepS device was assessed using discovery proteomics and targeted proteomics, detecting a panel of three protein biomarkers routinely assayed in clinical laboratories (alanine aminotransferase 1, C-reactive protein, and myoglobin). This innovative microfluidic device and its associated instrumentation should help to streamline and simplify clinical proteomics studies.
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http://dx.doi.org/10.1021/acs.analchem.0c02270DOI Listing
January 2021

The blood copper isotopic composition is a prognostic indicator of the hepatic injury in Wilson disease.

Metallomics 2020 11 15;12(11):1781-1790. Epub 2020 Oct 15.

Univ Lyon, ENSL, Univ Lyon 1, CNRS UMR 5276, LGL-TPE, F-69007, Lyon, France.

Wilson disease (WD) is an autosomal recessive disorder of copper (Cu) metabolism. The gene responsible for WD, ATP7B, is involved in the cellular transport of Cu, and mutations in the ATP7B gene induce accumulation of Cu in the liver and ultimately in the brain. In a pilot study, the natural variations of copper stable isotope ratios (Cu/Cu) in the serum of WD patients have been shown to differ from that of healthy controls. In the present study, we challenged these first results by measuring the Cu/Cu ratios in the blood of treated (n = 25), naïve patients (n = 11) and age matched healthy controls (n = 75). The results show that naïve patients and healthy controls exhibit undistinguishable Cu/Cu ratios, implying that the Cu isotopic ratio cannot serve as a reliable diagnostic biomarker. The type of treatment (d-penicillamine vs. triethylenetetramine) does not affect the Cu/Cu ratios in WD patients, which remain constant regardless of the type and duration of the treatment. In addition, the Cu/Cu ratios do not vary in naïve patients after the onset of the treatment. However, the Cu/Cu ratios decrease with the degree of liver fibrosis and the gradient of the phenotypic presentation, i.e. presymptomatic, hepatic and neurologic. To get insights into the mechanisms at work, we study the effects of the progress of the WD on the organism by measuring the Cu concentrations and the Cu/Cu ratios in the liver, feces and plasma of 12 and 45 week old Atp7b mice. The evolution of the Cu/Cu ratios is marked by a decrease in all tissues. The results show that Cu accumulates in the liver preferentially to Cu due to the preferential cellular entry of Cu and the impairment of the Cu exit by ceruloplasmin. The hepatic accumulation of monovalent Cu is likely to fuel the production of free radicals, which is potentially an explanation of the pathogenicity of WD. Altogether, the results suggest that the blood Cu/Cu ratio recapitulates WD progression and is a potential prognostic biomarker of WD.
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http://dx.doi.org/10.1039/d0mt00167hDOI Listing
November 2020

Analytical techniques for multiplex analysis of protein biomarkers.

Expert Rev Proteomics 2020 04 19;17(4):257-273. Epub 2020 May 19.

Ephraim Katzir Department of Biotechnology Engineering, ORT Braude College , Karmiel, Israel.

Introduction: The importance of biomarkers for pharmaceutical drug development and clinical diagnostics is more significant than ever in the current shift toward personalized medicine. Biomarkers have taken a central position either as companion markers to support drug development and patient selection, or as indicators aiming to detect the earliest perturbations indicative of disease, minimizing therapeutic intervention or even enabling disease reversal. Protein biomarkers are of particular interest given their central role in biochemical pathways. Hence, capabilities to analyze multiple protein biomarkers in one assay are highly interesting for biomedical research.

Areas Covered: We here review multiple methods that are suitable for robust, high throughput, standardized, and affordable analysis of protein biomarkers in a multiplex format. We describe innovative developments in immunoassays, the vanguard of methods in clinical laboratories, and mass spectrometry, increasingly implemented for protein biomarker analysis. Moreover, emerging techniques are discussed with potentially improved protein capture, separation, and detection that will further boost multiplex analyses.

Expert Commentary: The development of clinically applied multiplex protein biomarker assays is essential as multi-protein signatures provide more comprehensive information about biological systems than single biomarkers, leading to improved insights in mechanisms of disease, diagnostics, and the effect of personalized medicine.
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http://dx.doi.org/10.1080/14789450.2020.1763174DOI Listing
April 2020

A liver-targeting Cu(i) chelator relocates Cu in hepatocytes and promotes Cu excretion in a murine model of Wilson's disease.

Metallomics 2020 06;12(6):1000-1008

Univ. Grenoble Alpes, CEA, CNRS, IRIG, LCBM, F-38000 Grenoble, France.

Copper chelation is the most commonly used therapeutic strategy nowadays to treat Wilson's disease, a genetic disorder primarily inducing a pathological accumulation of Cu in the liver. The mechanism of action of Chel2, a liver-targeting Cu(i) chelator known to promote intracellular Cu chelation, was studied in hepatic cells that reconstitute polarized epithelia with functional bile canaliculi, reminiscent of the excretion pathway in the liver. The interplay between Chel2 and Cu localization in these cells was demonstrated through confocal microscopy using a fluorescent derivative and nano X-ray fluorescence. The Cu(i) bound chelator was found in vesicles potentially excreted in the canaliculi. Moreover, injection of Chel2 either intravenously or subcutaneously to a murine model of Wilson's disease increased excretion of Cu in the faeces, confirming in vivo biliary excretion. Therefore, Chel2 turns out to be a possible means to collect and excrete hepatic Cu in the faeces, hence restoring the physiological pathway.
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http://dx.doi.org/10.1039/d0mt00069hDOI Listing
June 2020

Fetuin-A and thyroxin binding globulin predict rituximab response in rheumatoid arthritis patients with insufficient response to anti-TNFα.

Clin Rheumatol 2020 Sep 24;39(9):2553-2562. Epub 2020 Mar 24.

GREPI EA 7408, Université Grenoble Alpes, 38000, Grenoble, France.

Objectives: Rheumatoid arthritis (RA) is a debilitating disease, but patient management and treatment have been revolutionized since the advent of bDMARDs. However, about one third of RA patients do not respond to specific bDMARD treatment without clear identified reasons. Different bDMARDs must be tried until the right drug is found. Here, we sought to identify a predictive protein signature to stratify patient responsiveness to rituximab (RTX) among patients with an insufficient response to a first anti-TNFα treatment.

Methods: Serum samples were collected at baseline before RTX initiation. A proteomics study comparing responders and nonresponders was conducted to identify and select potential predictive biomarkers whose concentration was measured by quantitative assays. Logistic regression was performed to determine the best biomarker combination to predict good or nonresponse to RTX (EULAR criteria after 6 months' treatment).

Results: Eleven biomarkers potentially discriminating between responders and nonresponders were selected following discovery proteomics. Quantitative immunoassays and univariate statistical analysis showed that fetuin-A and thyroxine binding globulin (TBG) presented a good capacity to discriminate between patient groups. A logistic regression analysis revealed that the combination of fetuin-A plus TBG could accurately predict a patient's responsiveness to RTX with an AUC of 0.86, sensitivity of 80%, and a specificity of 79%.

Conclusion: In RA patients for whom a first anti-TNFα treatment has failed, the serum abundance of fetuin-A and TBG before initiating RTX treatment is an indicator for their response status at 6 months. ClinicalTrials.gov identifier: NCT01000441. Key Points • Proteomic analysis revealed 11 putative predictive biomarkers to discriminate rituximab responder vs. nonresponder RA patients. • Fetuin-A and TBG are significantly differentially expressed at baseline in rituximab responder vs. nonresponder RA patients. • Algorithm combining fetuin-A and TBG accurately predicts response to rituximab in RA patients with insufficient response to TNFi.
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http://dx.doi.org/10.1007/s10067-020-05030-6DOI Listing
September 2020

Comprehensive and comparative exploration of the Atp7b mouse plasma proteome.

Metallomics 2020 02 9;12(2):249-258. Epub 2019 Dec 9.

Univ. Grenoble Alpes, CEA, Inserm, IRIG, BGE, 38000 Grenoble, France.

Wilson's disease (WD), a rare genetic disease caused by mutations in the ATP7B gene, is associated with altered expression and/or function of the copper-transporting ATP7B protein, leading to massive toxic accumulation of copper in the liver and brain. The Atp7b mouse, a genetic and phenotypic model of WD, was developed to provide new insights into the pathogenic mechanisms of WD. Many plasma proteins are secreted by the liver, and impairment of liver function can trigger changes to the plasma proteome. High standard proteomics workflows can identify such changes. Here, we explored the plasma proteome of the Atp7b mouse using a mass spectrometry (MS)-based proteomics workflow combining unbiased discovery analysis followed by targeted quantification. Among the 367 unique plasma proteins identified, 7 proteins were confirmed as differentially abundant between Atp7b mice and wild-type littermates, and were directly linked to WD pathophysiology (regeneration of liver parenchyma, plasma iron depletion, etc.). We then adapted our targeted proteomics assay to quantify human orthologues of these proteins in plasma from copper-chelator-treated WD patients. The plasma proteome changes observed in the Atp7b mouse were not confirmed in these samples, except for alpha-1 antichymotrypsin, levels of which were decreased in WD patients compared to healthy individuals. Plasma ceruloplasmin was investigated in both the Atp7b mouse model and human patients; it was significantly decreased in the human form of WD only. In conclusion, MS-based proteomics is a method of choice to identify proteome changes in murine models of disrupted metal homeostasis, and allows their validation in human cohorts.
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http://dx.doi.org/10.1039/c9mt00225aDOI Listing
February 2020

Bioinformatics Tools and Workflow to Select Blood Biomarkers for Early Cancer Diagnosis: An Application to Pancreatic Cancer.

Proteomics 2019 11 10;19(21-22):e1800489. Epub 2019 Oct 10.

University of Grenoble Alpes, INSERM, CEA, IRIG-BGE, U1038, Grenoble, 38000, France.

Secretome proteomics for the discovery of cancer biomarkers holds great potential to improve early cancer diagnosis. A knowledge-based approach relying on mechanistic criteria related to the type of cancer should help to identify candidates from available "omics" information. With the aim of accelerating the discovery process for novel biomarkers, a set of tools is developed and made available via a Galaxy-based instance to assist end-users biologists. These implemented tools proceed by a step-by-step strategy to mine transcriptomics and proteomics databases for information relating to tissue specificity, allow the selection of proteins that are part of the secretome, and combine this information with proteomics datasets to rank the most promising candidate biomarkers for early cancer diagnosis. Using pancreatic cancer as a case study, this strategy produces a list of 24 candidate biomarkers suitable for experimental assessment by MS-based proteomics. Among these proteins, three (SYCN, REG1B, and PRSS2) were previously reported as circulating candidate biomarkers of pancreatic cancer. Here, further refinement of this list allows to prioritize 14 candidate biomarkers along with their associated proteotypic peptides for further investigation, using targeted MS-based proteomics. The bioinformatics tools and the workflow implementing this strategy for the selection of candidate biomarkers are freely accessible at http://www.proteore.org.
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http://dx.doi.org/10.1002/pmic.201800489DOI Listing
November 2019

Ultrasensitive Quantification of Recombinant Proteins Using AAA-MS.

Methods Mol Biol 2019 ;2030:1-10

Univ. Grenoble Alpes, CEA, Inserm, U1038 BIG-BGE, Grenoble, France.

Recombinant proteins are essential components of therapeutic, biotechnological, food, and household products. In some cases, recombinant proteins must be purified and their quantity and/or concentration precisely determined. In this chapter, we describe a protocol for the quantification of purified recombinant proteins. The protocol is based on a microwave-assisted acidic hydrolysis of the target protein followed by high-resolution mass spectrometry (HRMS) analysis of the hydrolytic products. Absolute quantification is obtained by adding controlled amounts of labeled amino acids that serve as standards.
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http://dx.doi.org/10.1007/978-1-4939-9639-1_1DOI Listing
April 2020

Designing an In Silico Strategy to Select Tissue-Leakage Biomarkers Using the Galaxy Framework.

Methods Mol Biol 2019 ;1959:275-289

Université Grenoble Alpes, CEA, Inserm, BGE U1038, Grenoble, France.

Knowledge-based approaches using large-scale biological ("omics") data are a powerful way to identify mechanistic biomarkers, provided that scientists have access to computational solutions even when they have little programming experience or bioinformatics support. To achieve this goal, we designed a set of tools under the Galaxy framework to allow biologists to define their own strategy for reproducible biomarker selection. These tools rely on retrieving experimental data from public databases, and applying successive filters derived from information relating to disease pathophysiology. A step-by-step protocol linking these tools was implemented to select tissue-leakage biomarker candidates of myocardial infarction. A list of 24 candidates suitable for experimental assessment by MS-based proteomics is proposed. These tools have been made publicly available at http://www.proteore.org , allowing researchers to reuse them in their quest for biomarker discovery.
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http://dx.doi.org/10.1007/978-1-4939-9164-8_18DOI Listing
July 2019

Protein Biomarker Discovery in Non-depleted Serum by Spectral Library-Based Data-Independent Acquisition Mass Spectrometry.

Methods Mol Biol 2019 ;1959:129-150

Université Grenoble Alpes, CEA, Inserm, BGE U1038, Grenoble, France.

In discovery proteomics experiments, tandem mass spectrometry and data-dependent acquisition (DDA) are classically used to identify and quantify peptides and proteins through database searching. This strategy suffers from known limitations such as under-sampling and lack of reproducibility of precursor ion selection in complex proteomics samples, leading to somewhat inconsistent analytical results across large datasets. Data-independent acquisition (DIA) based on fragmentation of all the precursors detected in predetermined isolation windows can potentially overcome this limitation. DIA promises reproducible peptide and protein quantification with deeper proteome coverage and fewer missing values than DDA strategies. This approach is particularly attractive in the field of clinical biomarker discovery, where large numbers of samples must be analyzed. Here, we describe a DIA workflow for non-depleted serum analysis including a straightforward approach through which to construct a dedicated spectral library, and indications on how to optimize chromatographic and mass spectrometry analytical methods to produce high-quality DIA data and results.
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http://dx.doi.org/10.1007/978-1-4939-9164-8_9DOI Listing
July 2019

Liver cancer-associated changes to the proteome: what deserves clinical focus?

Expert Rev Proteomics 2018 09 19;15(9):749-756. Epub 2018 Sep 19.

b National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd), Carlos III National Institute of Health , Madrid , Spain.

Introduction: Hepatocellular carcinoma (HCC) is recognized as the fifth most common neoplasm and currently represents the second leading form of cancer-related death worldwide. Despite great progress has been done in the understanding of its pathogenesis, HCC represents a heavy societal and economic burden as most patients are still diagnosed at advanced stages and the 5-year survival rate remain below 20%. Early detection and revolutionary therapies that rely on the discovery of new molecular biomarkers and therapeutic targets are therefore urgently needed to develop precision medicine strategies for a more efficient management of patients. Areas covered: This review intends to comprehensively analyse the proteomics-based research conducted in the last few years to address some of the principal still open riddles in HCC biology, based on the identification of molecular drivers of tumor progression and metastasis. Expert commentary: The technical advances in mass spectrometry experienced in the last decade have significantly improved the analytical capacity of proteome wide studies. Large-scale protein and protein variant (post-translational modifications) identification and quantification have allowed detailed dissections of molecular mechanisms underlying HCC progression and are already paving the way for the identification of clinically relevant proteins and the development of their use on patient care.
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http://dx.doi.org/10.1080/14789450.2018.1521277DOI Listing
September 2018

Systematic quantitative analysis of H2A and H2B variants by targeted proteomics.

Epigenetics Chromatin 2018 01 12;11(1). Epub 2018 Jan 12.

INSERM U1038, CEA, BIG-BGE, Univ. Grenoble Alpes, Grenoble, France.

Background: Histones organize DNA into chromatin through a variety of processes. Among them, a vast diversity of histone variants can be incorporated into chromatin and finely modulate its organization and functionality. Classically, the study of histone variants has largely relied on antibody-based assays. However, antibodies have a limited efficiency to discriminate between highly similar histone variants.

Results: In this study, we established a mass spectrometry-based analysis to address this challenge. We developed a targeted proteomics method, using selected reaction monitoring or parallel reaction monitoring, to quantify a maximum number of histone variants in a single multiplexed assay, even when histones are present in a crude extract. This strategy was developed on H2A and H2B variants, using 55 peptides corresponding to 25 different histone sequences, among which a few differ by a single amino acid. The methodology was then applied to mouse testis extracts in which almost all histone variants are expressed. It confirmed the abundance profiles of several testis-specific histones during successive stages of spermatogenesis and the existence of predicted H2A.L.1 isoforms. This methodology was also used to explore the over-expression pattern of H2A.L.1 isoforms in a mouse model of male infertility.

Conclusions: Our results demonstrate that targeted proteomics is a powerful method to quantify highly similar histone variants and isoforms. The developed method can be easily transposed to the study of human histone variants, whose abundance can be deregulated in various diseases.
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http://dx.doi.org/10.1186/s13072-017-0172-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5767011PMC
January 2018

Proteomic characterization of human exhaled breath condensate.

J Breath Res 2018 02 20;12(2):021001. Epub 2018 Feb 20.

Université Grenoble-Alpes, F-38000, Grenoble, France. CEA, BIG, Biologie à Grande Echelle, F-38054, Grenoble, France. Inserm, Unité 1038, F-38054, Grenoble, France.

To improve biomedical knowledge and to support biomarker discovery studies, it is essential to establish comprehensive proteome maps for human tissues and biofluids, and to make them publicly accessible. In this study, we performed an in-depth proteomics characterization of exhaled breath condensate (EBC), a sample obtained non-invasively by condensation of exhaled air that contains submicron droplets of airway lining fluid. Two pooled samples of EBC, each obtained from 10 healthy donors, were processed using a straightforward protocol based on sample lyophilization, in-gel digestion and liquid chromatography tandem-mass spectrometry analysis. Two 'technical' control samples were processed in parallel to the pooled samples to correct for exogenous protein contamination. A total of 229 unique proteins were identified in EBC among which 153 proteins were detected in both EBC pooled samples. A detailed bioinformatics analysis of these 153 proteins showed that most of the proteins identified corresponded to proteins secreted in the respiratory tract (lung, bronchi). Eight proteins were salivary proteins. Our dataset is described and has been made accessible through the ProteomeXchange database (dataset identifier: PXD007591) and is expected to be useful for future MS-based biomarker studies using EBC as the diagnostic specimen.
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http://dx.doi.org/10.1088/1752-7163/aa9e71DOI Listing
February 2018

Introducing plasma/serum glycodepletion for the targeted proteomics analysis of cytolysis biomarkers.

Talanta 2017 Aug 19;170:473-480. Epub 2017 Apr 19.

Université Grenoble-Alpes, F-38000 Grenoble, France; CEA, BIG, Biologie à Grande Echelle, F-38054 Grenoble, France; INSERM, U1038, F-38054 Grenoble, France. Electronic address:

A major class of clinical biomarkers is constituted of intracellular proteins which are leaking into the blood following ischemia, exposure to toxic xenobiotics or mechanical aggression. Their ectopic presence in plasma/serum is an indicator of tissue damage and raises a warning signal. These proteins, referred to as cytolysis biomarkers, are generally of cytoplasmic origin and as such, are devoid of glycosylation. In contrast, most plasma/serum proteins originate from the hepatic secretory pathway and are heavily glycosylated (at the exception of albumin). Recent advances in targeted proteomics have supported the parallelized evaluation of new blood biomarkers. However, these analytical methods must be combined with prefractionation strategies that reduce the complexity of plasma/serum matrix. In this article, we present the glycodepletion method, which reverses the hydrazide-based glycocapture concept to remove plasma/serum glycoproteins from plasma/serum matrix and facilitates the detection of cytolysis biomarkers. Glycodepletion was integrated to a targeted proteomics pipeline to evaluate 4 liver cytolysis biomarker candidates in the context of acetaminophen-induced acute hepatitis.
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http://dx.doi.org/10.1016/j.talanta.2017.04.042DOI Listing
August 2017

Multiplex and accurate quantification of acute kidney injury biomarker candidates in urine using Protein Standard Absolute Quantification (PSAQ) and targeted proteomics.

Talanta 2017 Mar 13;164:77-84. Epub 2016 Nov 13.

Université Grenoble-Alpes, F-38000 Grenoble, France; CEA, BIG, Biologie à Grande Echelle, F-38054 Grenoble, France; INSERM, U1038, F-38054 Grenoble, France. Electronic address:

There is a need for multiplex, specific and quantitative methods to speed-up the development of acute kidney injury biomarkers and allow a more specific diagnosis. Targeted proteomic analysis combined with stable isotope dilution has recently emerged as a powerful option for the parallelized evaluation of candidate biomarkers. This article presents the development of a targeted proteomic assay to quantify 4 acute kidney injury biomarker candidates in urine samples. The proteins included in the assessed panel consisted of myo-inositol oxygenase (MIOX), phosphoenolpyruvate carboxykinase 1 (PCK1), neutrophil gelatinase-associated lipocalin (NGAL) and liver fatty acid-binding protein (L-FABP). The proteomic assay combined an antibody-free sample preparation and a liquid chromatography-selected reaction monitoring (LC-SRM) analysis pipeline. For accurate quantification of the selected candidates, we used PSAQ (Protein Standard Absolute Quantification) standards which are isotopically labeled versions of the target proteins. When added directly to the biological samples, these standards improve detection specificity and quantification accuracy. The multiplexed assay developed for the 4 biomarker candidates showed excellent analytical performance, in line with the recommendations of health authorities. Tests on urine from two small patient cohorts and a group of healthy donors confirmed the relevance of NGAL and L-FABP as biomarkers for AKI diagnosis. The assay is readily adaptable to other biomarker candidates and should be very useful for the simultaneous and accurate quantification of multiple biomarkers.
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http://dx.doi.org/10.1016/j.talanta.2016.11.023DOI Listing
March 2017

A proteomics assay to detect eight CBRN-relevant toxins in food.

Proteomics 2017 01 23;17(1-2). Epub 2016 Dec 23.

Université Grenoble-Alpes, Grenoble, France.

A proteomics assay was set up to analyze food substrates for eight toxins of the CBRN (chemical, biological, radiological and nuclear) threat, namely ricin, Clostridium perfringens epsilon toxin (ETX), Staphylococcus aureus enterotoxins (SEA, SEB and SED), shigatoxins from Shigella dysenteriae and entero-hemorragic Escherichia coli strains (STX1 and STX2) and Campylobacter jejuni cytolethal distending toxin (CDT). The assay developed was based on an antibody-free sample preparation followed by bottom-up LC-MS/MS analysis operated in targeted mode. Highly specific detection and absolute quantification were obtained using isotopically labeled proteins (PSAQ standards) spiked into the food matrix. The sensitivity of the assay for the eight toxins was lower than the oral LD50 which would likely be used in a criminal contamination of food supply. This assay should be useful in monitoring biological threats. In the public-health domain, it opens the way for multiplex investigation of food-borne toxins using targeted LC-MS/MS.
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http://dx.doi.org/10.1002/pmic.201600357DOI Listing
January 2017

Staphylococcal Enterotoxin O Exhibits Cell Cycle Modulating Activity.

Front Microbiol 2016 15;7:441. Epub 2016 Apr 15.

International Center for Infectiology ResearchLyon, France; CNRS UMR5308, Inserm U1111, Ecole Normale Supérieure de Lyon - Université Lyon 1Lyon, France; Institut des Agents Infectieux, Hospices Civils de LyonLyon, France.

Maintenance of an intact epithelial barrier constitutes a pivotal defense mechanism against infections. Staphylococcus aureus is a versatile pathogen that produces multiple factors including exotoxins that promote tissue alterations. The aim of the present study is to investigate the cytopathic effect of staphylococcal exotoxins SEA, SEG, SEI, SElM, SElN and SElO on the cell cycle of various human cell lines. Among all tested exotoxins only SEIO inhibited the proliferation of a broad panel of human tumor cell lines in vitro. Evaluation of a LDH release and a DNA fragmentation of host cells exposed to SEIO revealed that the toxin does not induce necrosis or apoptosis. Analysis of the DNA content of tumor cells synchronized by serum starvation after exposure to SEIO showed G0/G1 cell cycle delay. The cell cycle modulating feature of SEIO was confirmed by the flow cytometry analysis of synchronized cells exposed to supernatants of isogenic S. aureus strains wherein only supernatant of the SElO producing strain induced G0/G1 phase delay. The results of yeast-two-hybrid analysis indicated that SEIO's potential partner is cullin-3, involved in the transition from G1 to S phase. In conclusion, we provide evidence that SEIO inhibits cell proliferation without inducing cell death, by delaying host cell entry into the G0/G1 phase of the cell cycle. We speculate that this unique cell cycle modulating feature allows SEIO producing bacteria to gain advantage by arresting the cell cycle of target cells as part of a broader invasive strategy.
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http://dx.doi.org/10.3389/fmicb.2016.00441DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4832122PMC
May 2016

Multiplex quantification of protein toxins in human biofluids and food matrices using immunoextraction and high-resolution targeted mass spectrometry.

Anal Chem 2015 Aug 29;87(16):8473-80. Epub 2015 Jul 29.

†CEA, DSV, iBiTec-S, Laboratoire d'études du métabolisme des médicaments, 91191 Gif-sur-Yvette, France.

The development of rapid methods for unambiguous identification and precise quantification of protein toxins in various matrices is essential for public health surveillance. Nowadays, analytical strategies classically rely on sensitive immunological assays, but mass spectrometry constitutes an attractive complementary approach thanks to direct measurement and protein characterization ability. We developed here an innovative multiplex immuno-LC-MS/MS method for the simultaneous and specific quantification of the three potential biological warfare agents, ricin, staphylococcal enterotoxin B, and epsilon toxin, in complex human biofluids and food matrices. At least 7 peptides were targeted for each toxin (43 peptides in total) with a quadrupole-Orbitrap high-resolution instrument for exquisite detection specificity. Quantification was performed using stable isotope-labeled toxin standards spiked early in the sample. Lower limits of quantification were determined at or close to 1 ng·mL(-1). The whole process was successfully applied to the quantitative analysis of toxins in complex samples such as milk, human urine, and plasma. Finally, we report new data on toxin stability with no evidence of toxin degradation in milk in a 48 h time frame, allowing relevant quantitative toxin analysis for samples collected in this time range.
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http://dx.doi.org/10.1021/acs.analchem.5b01900DOI Listing
August 2015

Absolute and multiplex quantification of antibodies in serum using PSAQ™ standards and LC-MS/MS.

Bioanalysis 2015 21;7(10):1237-51. Epub 2015 Apr 21.

2Centre d'Immunologie Pierre Fabre (CIPF), 5 Av. Napoléon III, BP 60497, 74164 Saint-Julien-en-Genevois, France.

Background: In preclinical studies, monoclonal antibodies (mAbs) are traditionally assayed by ligand-binding-assays. Recently, quantitative liquid chromatography mass spectrometry (MS)-based assays have emerged which circumvent a number of challenges. These assays may also be multiplex, making them potentially compatible with pharmacokinetic assays for combined antibody therapies.

Materials & Methods: We combined a quantitative MS-based approach with the protein standard for absolute quantification (PSAQ™) strategy to simultaneously quantify three mAb variants presenting minor sequence differences. Stable isotopically labeled mAbs were produced and used as quantification standards. Titration curves were performed to assess the analytical performances of the method. LC-MS/MS and ELISA data were cross-compared.

Results: The approach presented provides similar accuracy and precision than ELISA, while being multiplex and faster to develop. It has applications at all stages of the pharmaceutical development.
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http://dx.doi.org/10.4155/bio.15.56DOI Listing
February 2016

DIGESTIF: a universal quality standard for the control of bottom-up proteomics experiments.

J Proteome Res 2015 Feb 30;14(2):787-803. Epub 2014 Dec 30.

Promise Advanced Proteomics , F-38040 Grenoble, France.

In bottom-up mass spectrometry-based proteomics analyses, variability at any step of the process, particularly during sample proteolysis, directly affects the sensitivity, accuracy, and precision of peptide detection and quantification. Currently, no generic internal standards are available to control the quality of sample processing steps. This makes it difficult to assess the comparability of MS proteomic data obtained under different experimental conditions. Here, we describe the design, synthesis, and validation of a universal protein standard, called DIGESTIF, that can be added to any biological sample. The DIGESTIF standard consists of a soluble recombinant protein scaffold to which a set of 11 artificial peptides (iRT peptides) with good ionization properties has been incorporated. In the protein scaffold, the amino acids flanking iRT peptide cleavage sites were selected either to favor or hinder protease cleavage. After sample processing, the retention time and relative intensity pattern of the released iRT peptides can be used to assess the quality of sample workup, the extent of digestion, and the performance of the LC-MS system. Thus, DIGESTIF can be used to standardize a broad spectrum of applications, ranging from simple replicate measurements to large-scale biomarker screening in biomedical applications.
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http://dx.doi.org/10.1021/pr500834zDOI Listing
February 2015

Mass spectrometry-based workflow for accurate quantification of Escherichia coli enzymes: how proteomics can play a key role in metabolic engineering.

Mol Cell Proteomics 2014 Apr 29;13(4):954-68. Epub 2014 Jan 29.

Commisariat à l'Energie Atomique et aux Energies Alternatives (CEA), Institut de Recherches en Technologie et Sciences pour le Vivant (iRTSV), Biologie à Grande Echelle, F-38054 Grenoble, France;

Metabolic engineering aims to design high performance microbial strains producing compounds of interest. This requires systems-level understanding; genome-scale models have therefore been developed to predict metabolic fluxes. However, multi-omics data including genomics, transcriptomics, fluxomics, and proteomics may be required to model the metabolism of potential cell factories. Recent technological advances to quantitative proteomics have made mass spectrometry-based quantitative assays an interesting alternative to more traditional immuno-affinity based approaches. This has improved specificity and multiplexing capabilities. In this study, we developed a quantification workflow to analyze enzymes involved in central metabolism in Escherichia coli (E. coli). This workflow combined full-length isotopically labeled standards with selected reaction monitoring analysis. First, full-length (15)N labeled standards were produced and calibrated to ensure accurate measurements. Liquid chromatography conditions were then optimized for reproducibility and multiplexing capabilities over a single 30-min liquid chromatography-MS analysis. This workflow was used to accurately quantify 22 enzymes involved in E. coli central metabolism in a wild-type reference strain and two derived strains, optimized for higher NADPH production. In combination with measurements of metabolic fluxes, proteomics data can be used to assess different levels of regulation, in particular enzyme abundance and catalytic rate. This provides information that can be used to design specific strains used in biotechnology. In addition, accurate measurement of absolute enzyme concentrations is key to the development of predictive kinetic models in the context of metabolic engineering.
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http://dx.doi.org/10.1074/mcp.M113.032672DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3977194PMC
April 2014

PSAQ™ standards for accurate MS-based quantification of proteins: from the concept to biomedical applications.

J Mass Spectrom 2012 Oct;47(10):1353-63

CEA, IRTSV, Biologie à Grande Echelle, F-38054, Grenoble, France.

Absolute protein quantification, i.e. determining protein concentrations in biological samples, is essential to our understanding of biological and physiopathological phenomena. Protein quantification methods based on the use of antibodies are very effective and widely used. However, over the last ten years, absolute protein quantification by mass spectrometry has attracted considerable interest, particularly for the study of systems biology and as part of biomarker development. This interest is mainly linked to the high multiplexing capacity of MS analysis, and to the availability of stable-isotope-labelled standards for quantification. This article describes the details of how to produce, control the quality and use a specific type of standard: Protein Standard Absolute Quantification (PSAQ™) standards. These standards are whole isotopically labelled proteins, analogues of the proteins to be assayed. PSAQ standards can be added early during sample treatment, thus they can correct for protein losses during sample prefractionation and for incomplete sample digestion. Because of this, quantification of target proteins is very accurate and precise using these standards. To illustrate the advantages of the PSAQ method, and to contribute to the increase in its use, selected applications in the biomedical field are detailed here.
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http://dx.doi.org/10.1002/jms.3106DOI Listing
October 2012

Introducing AAA-MS, a rapid and sensitive method for amino acid analysis using isotope dilution and high-resolution mass spectrometry.

J Proteome Res 2012 Jul 21;11(7):3929-36. Epub 2012 Jun 21.

CEA, IRTSV, Biologie à Grande Echelle, F-38054 Grenoble, France.

Accurate quantification of pure peptides and proteins is essential for biotechnology, clinical chemistry, proteomics, and systems biology. The reference method to quantify peptides and proteins is amino acid analysis (AAA). This consists of an acidic hydrolysis followed by chromatographic separation and spectrophotometric detection of amino acids. Although widely used, this method displays some limitations, in particular the need for large amounts of starting material. Driven by the need to quantify isotope-dilution standards used for absolute quantitative proteomics, particularly stable isotope-labeled (SIL) peptides and PSAQ proteins, we developed a new AAA assay (AAA-MS). This method requires neither derivatization nor chromatographic separation of amino acids. It is based on rapid microwave-assisted acidic hydrolysis followed by high-resolution mass spectrometry analysis of amino acids. Quantification is performed by comparing MS signals from labeled amino acids (SIL peptide- and PSAQ-derived) with those of unlabeled amino acids originating from co-hydrolyzed NIST standard reference materials. For both SIL peptides and PSAQ standards, AAA-MS quantification results were consistent with classical AAA measurements. Compared to AAA assay, AAA-MS was much faster and was 100-fold more sensitive for peptide and protein quantification. Finally, thanks to the development of a labeled protein standard, we also extended AAA-MS analysis to the quantification of unlabeled proteins.
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http://dx.doi.org/10.1021/pr3003326DOI Listing
July 2012

Mass spectrometry-based absolute protein quantification: PSAQ™ strategy makes use of "noncanonical" proteotypic peptides.

Proteomics 2012 Apr;12(8):1217-21

CEA, IRTSV, Biologie à Grande Echelle, Grenoble, France.

Absolute quantification of proteins using isotope dilution mass spectrometry requires the selection of proteotypic peptides. When choosing these peptides, a certain number of rules must be respected. Several of these were established to safeguard against quantification errors resulting from the isotopically labeled standard peptides not behaving in the same way as the peptides to be quantified. Of all absolute quantification methods using isotope dilution, Protein Standard for Absolute Quantification (PSAQ(TM) ) offers the maximal protein sequence coverage. In the present study, we show that the PSAQ method presents a previously unreported advantage for protein quantification as it makes use of Met/Cys-containing peptides and peptides-containing miscleavages in addition to proteotypic peptides. By increasing the total number of peptides that can be considered, robustness of quantification is improved, paving the way for a facilitated quantification of low abundant and/or low-molecular-weight proteins.
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http://dx.doi.org/10.1002/pmic.201100538DOI Listing
April 2012

Development of a Protein Standard Absolute Quantification (PSAQ™) assay for the quantification of Staphylococcus aureus enterotoxin A in serum.

J Proteomics 2012 Jun 6;75(10):3041-9. Epub 2011 Dec 6.

CEA, IRTSV, Biologie à Grande Echelle, F-38054 Grenoble, France.

Enterotoxin A (SEA) is a staphylococcal virulence factor which is suspected to worsen septic shock prognosis. However, the presence of SEA in the blood of sepsis patients has never been demonstrated. We have developed a mass spectrometry-based assay for the targeted and absolute quantification of SEA in serum. To enhance sensitivity and specificity, we combined an immunoaffinity-based sample preparation with mass spectrometry analysis in the selected reaction monitoring (SRM) mode. Absolute quantification of SEA was performed using the PSAQ™ method (Protein Standard Absolute Quantification), which uses a full-length isotope-labeled SEA as internal standard. The lower limit of detection (LLOD) and lower limit of quantification (LLOQ) were estimated at 352pg/mL and 1057pg/mL, respectively. SEA recovery after immunocapture was determined to be 7.8±1.4%. Therefore, we assumed that less than 1femtomole of each SEA proteotypic peptide was injected on the liquid chromatography column before SRM analysis. From a 6-point titration experiment, quantification accuracy was determined to be 77% and precision at LLOQ was lower than 5%. With this sensitive PSAQ-SRM assay, we expect to contribute to decipher the pathophysiological role of SEA in severe sepsis. This article is part of a Special Issue entitled: Proteomics: The clinical link.
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http://dx.doi.org/10.1016/j.jprot.2011.11.031DOI Listing
June 2012

Accurate quantification of cardiovascular biomarkers in serum using Protein Standard Absolute Quantification (PSAQ™) and selected reaction monitoring.

Mol Cell Proteomics 2012 Feb 11;11(2):M111.008235. Epub 2011 Nov 11.

CEA, IRTSV, Biologie à Grande Echelle, F-38054 Grenoble, France.

Development of new biomarkers needs to be significantly accelerated to improve diagnostic, prognostic, and toxicity monitoring as well as therapeutic follow-up. Biomarker evaluation is the main bottleneck in this development process. Selected Reaction Monitoring (SRM) combined with stable isotope dilution has emerged as a promising option to speed this step, particularly because of its multiplexing capacities. However, analytical variabilities because of upstream sample handling or incomplete trypsin digestion still need to be resolved. In 2007, we developed the PSAQ™ method (Protein Standard Absolute Quantification), which uses full-length isotope-labeled protein standards to quantify target proteins. In the present study we used clinically validated cardiovascular biomarkers (LDH-B, CKMB, myoglobin, and troponin I) to demonstrate that the combination of PSAQ and SRM (PSAQ-SRM) allows highly accurate biomarker quantification in serum samples. A multiplex PSAQ-SRM assay was used to quantify these biomarkers in clinical samples from myocardial infarction patients. Good correlation between PSAQ-SRM and ELISA assay results was found and demonstrated the consistency between these analytical approaches. Thus, PSAQ-SRM has the capacity to improve both accuracy and reproducibility in protein analysis. This will be a major contribution to efficient biomarker development strategies.
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http://dx.doi.org/10.1074/mcp.M111.008235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3277748PMC
February 2012

Production and use of stable isotope-labeled proteins for absolute quantitative proteomics.

Methods Mol Biol 2011 ;753:93-115

CEA, DSV, iRTSV, Laboratoire d'Etude de la Dynamique des Protéomes, Grenoble, France.

In the field of analytical chemistry, stable isotope dilution assays are extensively used in combination with liquid chromatography-mass spectrometry (LC-MS) to provide confident quantification results. Over the last decade, the principle of isotope dilution has been adopted by the proteomic community in order to accurately quantify proteins in biological samples. In these experiments, a protein's concentration is deduced from the ratio between the MS signal of a tryptic peptide and that of a stable isotope-labeled analog, which serves as an internal standard. The first isotope dilution standards introduced in proteomics were chemically synthesized peptides incorporating a stable isotope-tagged amino acid. These isotopically labeled peptide standards, which are currently widely used, are generally added to samples after protein isolation and digestion. Thus, if protein enrichment is necessary, they do not allow correction for protein losses that may occur during sample pre-fractionation, nor do they allow the tryptic digestion yield to be taken into account. To reduce these limitations we have developed the PSAQ (Protein Standard Absolute Quantification) strategy using full-length stable isotope-labeled proteins as quantification standards. These standards and the target proteins share identical biochemical properties. This allows standards to be spiked into samples at an early stage of the analytical process. Thanks to this possibility, the PSAQ method provides highly accurate quantification results, including for samples requiring extensive biochemical pre-fractionation. In this chapter, we describe the production of full-length stable isotope-labeled proteins (PSAQ standards) using cell-free expression devices. The purification and quality control of protein standards, crucial for good-quality and accurate measurements, are also detailed. Finally, application of the PSAQ method to a typical protein quantification assay is presented.
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http://dx.doi.org/10.1007/978-1-61779-148-2_7DOI Listing
September 2011

A critical evaluation of self-interaction chromatography as a predictive tool for the assessment of protein-protein interactions in protein formulation development: a case study of a therapeutic monoclonal antibody.

Eur J Pharm Biopharm 2010 May 25;75(1):16-25. Epub 2010 Jan 25.

Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians-Universität München, Germany.

The aim of this study was to establish and evaluate a screening method for the physical characterization of protein-protein interactions of therapeutic proteins based on the determination of the osmotic second virial coefficient (B(22)). B(22) of an IgG1 was measured by self-interaction chromatography (SIC) and was compared to data obtained from static light scattering (SLS). As assessed by Fourier transform infrared spectroscopy (FTIR), the protein coupling to chromatography particles had no relevant influence on the three-dimensional native structure of the IgG1. B(22) variations could be measured for physiological relevant excipient concentrations. Significant positive B(22) values were observed for the following solution conditions of the investigated antibody: (i) acidic pH conditions, (ii) low buffer concentrations, (iii) low salt concentrations and (iv) high amino acid concentrations. B(22) was compared to IgG1 stability data derived from a study conducted for 12weeks at 40 degrees C. A concentration of 5mM histidine, which was the most promising buffer candidate according to B(22), showed a slightly better physical stability (as assessed by turbidity and size exclusion chromatography) compared to the other tested formulations. This is confirmed in a stress study investigating the colloidal stability. Thus, measuring protein-protein interactions with SIC appeared as a promising screening tool for physical characterization of protein formulations for cases in which the protein stability is governed by interparticle interactions.
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http://dx.doi.org/10.1016/j.ejpb.2010.01.009DOI Listing
May 2010

Correlation of protein-protein interactions as assessed by affinity chromatography with colloidal protein stability: a case study with lysozyme.

Pharm Dev Technol 2010 Jul-Aug;15(4):421-30

Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians-University Munich, Munich, Germany.

Lysozyme-lysozyme interactions were assessed in the native state at 25 degrees C as well in the denatured state at 80 degrees C by affinity chromatography in order to measure the osmotic second virial coefficient (B). This parameter allows us to better understand protein aggregation pathways and colloidal protein stability. Repulsive interactions (B > 0) were weakened for both protein states by increasing salt concentration and by increasing the pH value toward lysozyme pI. This decrease was more pronounced in the denatured state, most likely caused by changes in electrostatic interactions and the formation of hydrophobic clusters. The lysozyme formulations presenting the more repulsive conditions (B > 0), as derived from the osmotic second virial coefficient, showed better colloidal stability under mechanical and thermal stresses. As expected, B values are much more negative for the interactions in the denatured state compared to the data obtained for the native state, reflecting a strong tendency of denatured lysozyme to aggregate. Thus, measurement of protein interactions by affinity chromatography allows us to gain information on protein interactions in both native and denatured states as well as to predict solution conditions prone for improving protein colloidal stability.
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http://dx.doi.org/10.3109/10837450903262074DOI Listing
October 2010