Publications by authors named "Jérôme Vialaret"

39 Publications

FTO-mediated cytoplasmic mA demethylation adjusts stem-like properties in colorectal cancer cell.

Nat Commun 2021 03 19;12(1):1716. Epub 2021 Mar 19.

IGF, Univ. Montpellier, CNRS, INSERM, Montpellier, France.

Cancer stem cells (CSCs) are a small but critical cell population for cancer biology since they display inherent resistance to standard therapies and give rise to metastases. Despite accruing evidence establishing a link between deregulation of epitranscriptome-related players and tumorigenic process, the role of messenger RNA (mRNA) modifications in the regulation of CSC properties remains poorly understood. Here, we show that the cytoplasmic pool of fat mass and obesity-associated protein (FTO) impedes CSC abilities in colorectal cancer through its N,2'-O-dimethyladenosine (mA) demethylase activity. While mA is strategically located next to the mG-mRNA cap, its biological function is not well understood and has not been addressed in cancer. Low FTO expression in patient-derived cell lines elevates mA level in mRNA which results in enhanced in vivo tumorigenicity and chemoresistance. Inhibition of the nuclear mA methyltransferase, PCIF1/CAPAM, fully reverses this phenotype, stressing the role of mA modification in stem-like properties acquisition. FTO-mediated regulation of mA marking constitutes a reversible pathway controlling CSC abilities. Altogether, our findings bring to light the first biological function of the mA modification and its potential adverse consequences for colorectal cancer management.
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http://dx.doi.org/10.1038/s41467-021-21758-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7979729PMC
March 2021

Cerebrospinal fluid A beta 1-40 peptides increase in Alzheimer's disease and are highly correlated with phospho-tau in control individuals.

Alzheimers Res Ther 2020 10 2;12(1):123. Epub 2020 Oct 2.

Univ Montpellier, INSERM, CHU Montpellier (CMRR), Montpellier, France.

Background: Amyloid pathology, which is one of the characteristics of Alzheimer's disease (AD), results from altered metabolism of the beta-amyloid (Aβ) peptide in terms of synthesis, clearance, or aggregation. A decrease in cerebrospinal fluid (CSF) level Aβ1-42 is evident in AD, and the CSF ratio Aβ42/Aβ40 has recently been identified as one of the most reliable diagnostic biomarkers of amyloid pathology. Variations in inter-individual levels of Aβ1-40 in the CSF have been observed in the past, but their origins remain unclear. In addition, the variation of Aβ40 in the context of AD studied in several studies has yielded conflicting results.

Methods: Here, we analyzed the levels of Aβ1-40 using multicenter data obtained on 2466 samples from six different cohorts in which CSF was collected under standardized protocols, centrifugation, and storage conditions. Tau and p-tau (181) concentrations were measured using commercially available in vitro diagnostic immunoassays. Concentrations of CSF Aβ1-42 and Aβ1-40 were measured by ELISA, xMAP technology, chemiluminescence immunoassay (CLIA), and mass spectrometry. Statistical analyses were calculated for parametric and non-parametric comparisons, linear regression, correlation, and odds ratios. The statistical tests were adjusted for the effects of covariates (age, in particular).

Results: Regardless of the analysis method used and the cohorts, a slight but significant age-independent increase in the levels of Aβ40 in CSF was observed in AD. We also found a strong positive correlation between the levels of Aβ1-40 and p-tau (181) in CSF, particularly in control patients.

Conclusions: These results indicate that an increase in the baseline level of amyloid peptides, which are associated with an increase in p-tau (181), may be a biological characteristic and possibly a risk factor for AD. Further studies will be needed to establish a causal link between increased baseline levels of Aβ40 and the development of the disease.
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http://dx.doi.org/10.1186/s13195-020-00696-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7532565PMC
October 2020

Hepcidin and ferritin levels in restless legs syndrome: a case-control study.

Sci Rep 2020 07 17;10(1):11914. Epub 2020 Jul 17.

Sleep-Wake Disorders Unit, Department of Neurology, Gui-de-Chauliac Hospital, CHU Montpellier, Montpellier University, Montpellier, France.

The association between restless legs syndrome (RLS) and iron homeostasis remains unclear. We compared serum hepcidin and ferritin levels in patients with RLS and controls, and assessed their relationships with RLS phenotype, drug intake, and history of augmentation syndrome. 102 drug-free RLS patients (age 58.9 [24.5-77.2], 63 females) and 73 controls (age 56.8 [23.46-76.6], 45 females) underwent a polysomnography recording. Hepcidin levels were quantified by ELISA. 34 RLS patients had a second assessment after starting dopaminergic drugs. Ferritin level was low (< 50 µg/l) in 14.7% of patients and 25% of controls, with no between-group differences in the mean values. Hepcidin levels were higher in patients even after adjustment for confounding factors, and excluding participants with low ferritin levels. Ferritin and hepcidin levels were comparable before and after treatment, and between patients with (n = 17) and without history of augmentation. Ferritin and hepcidin levels correlated with age, body mass index, and periodic leg movements. Higher hepcidin levels were associated with older age, older age at RLS onset, less daytime sleepiness and familial RLS. In conclusion, serum hepcidin levels but not ferritin were higher in RLS patients regardless of treatment and history of augmentation. Serum hepcidin may be a more relevant biomarker of RLS than ferritin.
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http://dx.doi.org/10.1038/s41598-020-68851-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7367854PMC
July 2020

In Vivo Large-Scale Mapping of Protein Turnover in Human Cerebrospinal Fluid.

Anal Chem 2019 12 2;91(24):15500-15508. Epub 2019 Dec 2.

Université de Montpellier , 34090 Montpellier , France.

The extraction of accurate physiological parameters from clinical samples provides a unique perspective to understand disease etiology and evolution, including under therapy. We introduce a new methodologic framework to map patient proteome dynamics in vivo, either proteome-wide or in large targeted panels. We applied it to ventricular cerebrospinal fluid (CSF) and could determine the turnover parameters of almost 200 proteins, whereas a handful were known previously. We covered a large number of neuron biology- and immune system-related proteins, including many biomarkers and drug targets. This first large data set unraveled a significant relationship between turnover and protein origin that relates to our ability to investigate organ physiology with protein-labeling strategy specifics. Our data constitute the first draft of CSF proteome dynamics as well as a repertoire of peptides for the community to design new analyses. The disclosed methods apply to other fluids or tissues provided sequential sample collection can be performed. We show that the proposed mathematical modeling applies to other analytical methods in the field.
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http://dx.doi.org/10.1021/acs.analchem.9b03328DOI Listing
December 2019

Detection of amyloid beta peptides in body fluids for the diagnosis of alzheimer's disease: Where do we stand?

Crit Rev Clin Lab Sci 2020 03 29;57(2):99-113. Epub 2019 Oct 29.

INSERM U1183, Laboratoire de Biochimie-Protéomique Clinique, CHU de Montpellier, Université de Montpellier, Montpellier, France.

Alzheimer's disease (AD) is an incurable neurodegenerative disease characterized by progressive decline of cognitive abilities. Amyloid beta peptides (Aβ), Tau proteins and the phosphorylated form of the Tau protein, p-Tau, are the core pathological biomarkers of the disease, and their detection for the diagnosis of patients is progressively being implemented. However, to date, their quantification is mostly performed on cerebrospinal fluid (CSF), the collection of which requires an invasive lumbar puncture. Early diagnosis has been shown to be important for disease-modifying treatment, which is currently in development, to limit the progression of the disease. Nevertheless, the diagnosis is often delayed to the point where the disease has already progressed, and the tools currently available do not allow for a systematic follow-up of patients. Thus, the search for a molecular signature of AD in a body fluid such as blood or saliva that can be collected in a minimally invasive way offers hope. A number of methods have been developed for the quantification of core biomarkers, especially in easily accessible fluids such as the blood, that improve their accuracy, specificity and sensitivity. This review summarizes and compares these approaches, focusing in particular on their use for Aβ detection, the earliest biomarker to be modified in the course of AD. The review also discusses biomarker quantification in CSF, blood and saliva and their clinical applications.
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http://dx.doi.org/10.1080/10408363.2019.1678011DOI Listing
March 2020

Variation of human salivary alpha-amylase proteoforms in three stimulation models.

Clin Oral Investig 2020 Jan 6;24(1):475-486. Epub 2019 Aug 6.

LBPC/PPC - IRMB, CHU de Montpellier, INSERM, Montpellier University, 80 rue Augustin Fliche, Montpellier, France.

Objectives: To evaluate the sAA proteoforms' expression during different stimulation situations.

Materials And Methods: This study evaluated the salivary alpha-amylase (sAA) proteoforms' behavior by western blot (WB) analysis and high-resolution mass spectrometry (LC-MS/MS) in different situations that produce increases in sAA activity. For this purpose, six healthy women with a similar body mass index, age, and fit, underwent different sAA stimulation tests, such as acetic acid stimulation, psychological stress using the standardized Trier social stress test, and physical effort using the Cooper treadmill test.

Results: The three models showed an increase in sAA activity. The WB demonstrated seven common bands observed in the six women (band one at 59 kDa, two at 56 kDa, three at 48 kDa, four at 45 kDa, five at 41 kDa, six at 36 kDa, and seven at 14 kDa), in which sAA protein was identified. The individual WB analysis showed that band two, which corresponded to the native non-glycosylated sAA proteoform, had a higher increase after the three sAA stimulation inducers, and this band was also the only proteoform correlated with sAA activity (r = 0.56, P = 0.001). In addition, when the label-free quantification analysis was performed, the different proteoforms showed different responses depending on the type of stimulation.

Conclusions: This preliminary study showed that the diverse sAA proteoforms' expression depends on the different stimulation models.

Clinical Relevance: This study opens new perspectives and challenges for the use of the different alpha-amylase proteoforms as possible biomarkers in addition to the sAA activity.
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http://dx.doi.org/10.1007/s00784-019-03021-9DOI Listing
January 2020

Intact Protein Analysis by LC-MS for Characterizing Biomarkers in Cerebrospinal Fluid.

Methods Mol Biol 2019 ;1959:163-172

Clinical Proteomics Platform, LBPC, IRMB, CHU Montpellier, Montpellier University, Montpellier, France.

In the field of proteomics, the emerging "top-down" MS-based proteomics approach can be used to obtain a bird's eye view of all intact proteoforms present in a sample. This alternative to the "bottom-up" approach based on tryptic protein digestion processes has some unique advantages for assessing PTMs and sequence variations. However, it requires some dedicated tools for sample preparation and LC-MS analysis, which makes it more complex to handle than the bottom-up approach. In this study, a simple methodology is presented for characterizing intact proteins in biological fluid. This method yields quantitative information using an MS1 profiling approach and makes it possible to identify the proteins regulated under various clinical conditions.
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http://dx.doi.org/10.1007/978-1-4939-9164-8_11DOI Listing
July 2019

Hepcidin: immunoanalytic characteristics.

Ann Biol Clin (Paris) 2018 Dec;76(6):705-715

CHU de Montpellier, Institut de médecine régénératrice et de biothérapies (IRMB), Laboratoire de biochimie-protéomique clinique, CCBHM, Hôpital Saint Eloi, Montpellier, France, Faculté de médecine, Université de Montpellier, France, Inserm U1040, Montpellier, France.

Hepcidin has progressively become essential in clinical practice for the diagnosis and follow-up of a large spectrum of diseases. Anyway, its own biochemical and structural characteristics have complicated and delayed the acquisition of a standardized quantifying tool of the peptide.
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http://dx.doi.org/10.1684/abc.2018.1382DOI Listing
December 2018

Impact of biological matrix on inflammatory protein biomarker quantification based on targeted mass spectrometry.

Bioanalysis 2018 Sep 12;10(17):1383-1399. Epub 2018 Sep 12.

Montpellier University, LBPC/PPC - IRMB, CHU de Montpellier, INSERM, 80 rue Augustin Fliche, Montpellier, France.

Background: Serum and plasma are widely used matrices in biological and clinical studies. To improve reliability and consistency of markers quantification, the influence of these matrices on proteins was evaluated by targeted mass spectrometry.

Results: 65 proteins were quantified in matched blood samples collected in serum, ethylenediaminetetraacetic acid and heparin plasma tubes from 40 healthy and 10 pathological individuals. Only 52% of the proteins were not impacted by any of the biological matrices tested, and the effects on quantification of proteins affected was matrix and protein dependent.

Conclusion: Matrix comparisons using mass spectrometry is therefore recommended to assess the relevance of using surrogate matrix, performing biomarker discovery study or evaluating the clinical use of biomarkers in large clinical cohorts.
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http://dx.doi.org/10.4155/bio-2018-0149DOI Listing
September 2018

Identification of multiple proteoforms biomarkers on clinical samples by routine Top-Down approaches.

Data Brief 2018 Jun 31;18:1013-1021. Epub 2018 Mar 31.

University of Montpellier, LBPC, IRMB, CHU de Montpellier, 34000 Montpellier, France.

Top-Down approaches have an extremely high biological relevance, especially when it comes to biomarker discovery, but the necessary pre-fractionation constraints are not easily compatible with the robustness requirements and the size of clinical sample cohorts. We have demonstrated that intact protein profiling studies could be run on UHR-Q-ToF with limited pre-fractionation (Schmit et al., 2017) [1]. The dataset presented herein is an extension of this research. Proteoforms known to play a role in the pathophysiology process of Alzheimer's disease were identified as candidate biomarkers. In this article, mass spectrometry performance of these candidates are demonstrated.
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http://dx.doi.org/10.1016/j.dib.2018.03.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5996497PMC
June 2018

Regulatory context and validation of assays for clinical mass spectrometry proteomics (cMSP) methods.

Crit Rev Clin Lab Sci 2018 08 23;55(5):346-358. Epub 2018 May 23.

a CHU Montpellier, IRMB, Hôpital Saint Eloi, LBPC et CRB, University of Montpellier , Montpellier , France.

Clinical mass spectrometry proteomics (cMSP) assays are being increasingly used in clinical laboratories for analyzing peptides and proteins. It has therefore become urgent to characterize and validate the methods available for liquid chromatography-tandem mass spectrometry (LC/MS-MS) targeted quantification of peptide and protein biomarkers in biological fluids in the context of in vitro diagnostics. LC-MS/MS for the detection of peptides and proteins is currently the main approach used in the field of cMSP. As a result of their selectivity, low reagent costs and the fact that these methods can be used for absolute quantification and multiplexing, they will likely eventually replace immunoassays. Although LC-MS/MS is known to be the main reference method involved in reference measurement procedures (RMPs), it needs to meet the requirements of in vitro diagnostic (IVD) regulations and standards. This review shows that cMSP is fully compatible with the regulatory IVD requirements and provides an overview of the characterization and validation of the use of LC-MS/MS targeted quantification of clinical protein biomarkers in biological fluids.
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http://dx.doi.org/10.1080/10408363.2018.1470159DOI Listing
August 2018

What sample preparation should be chosen for targeted MS monoclonal antibody quantification in human serum?

Bioanalysis 2018 May 17;10(10):723-735. Epub 2018 May 17.

University of Montpellier, LBPC- IRMB, CHU Montpellier, 80 rue Augustin Fliche, Montpellier, France.

Aim: Monoclonal antibody-based treatment of cancer has been established as one of the most successful therapeutic strategies.

Materials & Methods: In this work, we developed a workflow based on an automated protein-A capture and LC-MS/MS analysis to quantify bevacizumab on patient serum during treatment. This analytical approach was fully validated and compared with a commercially available Monoclonal antibody-based treatment preparation (nanosurface and molecular-orientation limited kit).

Results: The analytical comparison of the two preparative workflows based on protein-A capture gave similar results with a better lower limit of quantification for the nanosurface and molecular-orientation limited kit (0.26986 vs 1.9565 μg/ml).

Conclusion: LC-MS/MS has clear advantages compared with ELISA when considering method development time, multiplexing capacities and absolute quantification with internal standardization.
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http://dx.doi.org/10.4155/bio-2018-0027DOI Listing
May 2018

Nano-flow vs standard-flow: Which is the more suitable LC/MS method for quantifying hepcidin-25 in human serum in routine clinical settings?

J Chromatogr B Analyt Technol Biomed Life Sci 2018 Jun 10;1086:110-117. Epub 2018 Apr 10.

University of Montpellier, CHU Montpellier, IRMB, LBPC, Montpellier F-34000, France. Electronic address:

Hepcidin-25 peptide is a biomarker which is known to have considerable clinical potential for diagnosing iron-related diseases. Developing analytical methods for the absolute quantification of hepcidin is still a real challenge, however, due to the sensitivity, specificity and reproducibility issues involved. In this study, we compare and discuss two MS-based assays for quantifying hepcidin, which differ only in terms of the type of liquid chromatography (nano LC/MS versus standard LC/MS) involved. The same sample preparation, the same internal standards and the same MS analyzer were used with both approaches. In the field of proteomics, nano LC chromatography is generally known to be more sensitive and less robust than standard LC methods. In this study, we established that the performances of the standard LC method are equivalent to those of our previously developed nano LC method. Although the analytical performances were very similar in both cases. The standard-flow platform therefore provides the more suitable alternative for accurately determining hepcidin in clinical settings.
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http://dx.doi.org/10.1016/j.jchromb.2018.04.003DOI Listing
June 2018

Association between serum hepcidin level and restless legs syndrome.

Mov Disord 2018 04 8;33(4):618-627. Epub 2018 Feb 8.

CHU Montpellier, Institut de Recherche en Biothérapie, hôpital St Eloi, Laboratoire de Biochimie Protéomique Clinique et CCBHM, INSERM U1040, Montpellier, France.

Background: To better understand the role of iron homeostasis dysregulation in restless legs syndrome, we compared serum hepcidin and ferritin levels in drug-free patients with primary restless legs syndrome and healthy controls and studied the relationship between hepcidin level and restless legs syndrome severity.

Methods: One hundred and eight drug-free patients with primary restless legs syndrome (65 women; median age, 61.5 years) and 45 controls (28 women; median age, 53.9 years) were enrolled. Inclusion criteria were: normal ferritin level (>50 ng/mL) and absence of iron disorders, chronic renal or liver failure, and inflammatory or neurological diseases. Each subject underwent a thorough clinical examination and a polysomnography assessment. Serum hepcidin-25 was quantified using a validated mass spectrometry method. Restless legs syndrome severity was evaluated according to the International Restless Legs Syndrome Study Group.

Results: Despite no group difference between normal ferritin levels and demographic features, serum hepcidin level and hepcidin/ferritin ratio were higher in patients than in controls. Hepcidin level and hepcidin/ferritin ratio, but not ferritin level, were positively correlated with periodic leg movements during sleep and wakefulness in the whole sample. Hepcidin level seem to be associated with restless legs syndrome severity in a complex U-shaped relationship, without relationship with age at restless legs syndrome onset, positive family history, sleep and depressive symptoms, genetic background, and polysomnographic measurements. No relationship was found between ferritin level and restless legs syndrome severity.

Conclusion: In drug-free patients with primary restless legs syndrome, hepcidin level is higher than in controls and may be associated with restless legs syndrome clinical severity. This result emphasizes the complex peripheral iron metabolism deregulation in restless legs syndrome, opening potential perspectives for a personalized approach with a hepcidin antagonist. © 2018 International Parkinson and Movement Disorder Society.
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http://dx.doi.org/10.1002/mds.27287DOI Listing
April 2018

Assessing a multiplex-targeted proteomics approach for the clinical diagnosis of periodontitis using saliva samples.

Bioanalysis 2018 01 15;10(1):35-45. Epub 2017 Dec 15.

University of Montpellier, LBPC - IRMB, CHU de Montpellier, 80 rue Augustin Fliche, 34 295 Montpellier, France.

Aim: The present study focused on the research of new biomarkers based on the liquid chromatography-multiple-reaction monitoring (MRM) proteomic profile in whole saliva of patients with periodontitis compared with periodontal healthy patients.

Methods: A 30-min multiplexed liquid chromatography-MRM method was used for absolute quantification of 35 plasma biomarkers in saliva from control patients and patients with periodontitis.

Results: Three proteins namely hemopexin, plasminogen and α-fibrinogen were shown to be clearly related to the presence of periodontitis compared with healthy patients. Apolipoprotein H was found to discriminate for the first time chronic and aggressive periodontitis.

Conclusion: Our results indicate that this innovative MRM method could be used to screen for periodontitis in clinical environment. Furthermore, apolipoprotein H was found to be a discriminant biomarker of aggressive periodontitis.
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http://dx.doi.org/10.4155/bio-2017-0218DOI Listing
January 2018

Towards a routine application of Top-Down approaches for label-free discovery workflows.

J Proteomics 2018 03 30;175:12-26. Epub 2017 Aug 30.

Laboratoire de Biochimie et Protéomique Clinique, Institut de Médecine Régénératrice et de Biothérapie, CHU de Montpellier - Hôpital St. Eloi, 34000 Montpellier, France.

Thanks to proteomics investigations, our vision of the role of different protein isoforms in the pathophysiology of diseases has largely evolved. The idea that protein biomarkers like tau, amyloid peptides, ApoE, cystatin, or neurogranin are represented in body fluids as single species is obviously over-simplified, as most proteins are present in different isoforms and subjected to numerous processing and post-translational modifications. Measuring the intact mass of proteins by MS has the advantage to provide information on the presence and relative amount of the different proteoforms. Such Top-Down approaches typically require a high degree of sample pre-fractionation to allow the MS system to deliver optimal performance in terms of dynamic range, mass accuracy and resolution. In clinical studies, however, the requirements for pre-analytical robustness and sample size large enough for statistical power restrict the routine use of a high degree of sample pre-fractionation. In this study, we have investigated the capacities of current-generation Ultra-High Resolution Q-Tof systems to deal with high complexity intact protein samples and have evaluated the approach on a cohort of patients suffering from neurodegenerative disease. Statistical analysis has shown that several proteoforms can be used to distinguish Alzheimer disease patients from patients suffering from other neurodegenerative disease.

Significance: Top-down approaches have an extremely high biological relevance, especially when it comes to biomarker discovery, but the necessary pre-fractionation constraints are not easily compatible with the robustness requirements and the size of clinical sample cohorts. We have demonstrated that intact protein profiling studies could be run on UHR-Q-ToF with limited pre-fractionation. The proteoforms that have been identified as candidate biomarkers in the-proof-of concept study are derived from proteins known to play a role in the pathophysiology process of Alzheimer disease.
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http://dx.doi.org/10.1016/j.jprot.2017.08.003DOI Listing
March 2018

Clinical perspectives of dried blood spot protein quantification using mass spectrometry methods.

Crit Rev Clin Lab Sci 2017 05 10;54(3):173-184. Epub 2017 Apr 10.

a Laboratory of Biochemistry and Clinical Proteomics, CHU Montpellier , Institute of Regenerative Medicine & Biotherapy , Montpellier , France.

Although dried blood spot (DBS) sampling methods have been used since the 1960s, they have recently attracted renewed interest because of the development of new clinical applications. In addition to their other advantages, DBS methods can now be used to quantify many blood proteins using the latest highly sensitive and robust, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) approaches such as multiple reaction monitoring. The DBS blood sampling approach could provide a useful alternative means of conducting blood sampling for routine clinical purposes and patients' follow-up. In this review, we examine the current use of DBS for LC-MS/MS protein quantification in clinical settings and discuss potential clinical applications.
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http://dx.doi.org/10.1080/10408363.2017.1297358DOI Listing
May 2017

Cerebrospinal fluid levels of orexin-A and histamine, and sleep profile within the Alzheimer process.

Neurobiol Aging 2017 05 18;53:59-66. Epub 2017 Jan 18.

University of Montpellier, Montpellier, France; INSERM U1061, Montpellier, France; Sleep Unit, Narcolepsy Reference Center, Department of Neurology, Gui de Chauliac Hospital, Montpellier, France.

To better understand how sleep/wake dysregulation affects Alzheimer's disease (AD), we compared the cerebrospinal fluid (CSF) orexin and histamine/tele-methylhistamine (HA/t-MHA) levels of 82 patients (41 probable-AD-high level of evidence, 41 mild cognitive impairment MCI-due-to-AD), 24 other neurologic disorders (OND) and 24 controls. We determined the relationships between these biomarkers, the CSF AD biomarkers concentrations, and the clinical sleep profile. CSF orexin-A but not HA/t-MHA levels were higher in MCI and AD than OND and controls. CSF orexin-A is correlated to CSF amyloid-βin MCI and AD, independently of age, gender, MMSE, total-tau/phosphorylated-tau, HA or sleep parameters. Nighttime sleep duration was longer in MCI and AD patients than controls. In MCI, nighttime sleep duration negatively correlated with CSF amyloid-β and MMSE. To conclude, CSF orexin-A but not HA/t-HMA was upregulated in AD and correlated with amyloid-β level. Our data suggested a change in the sleep-wake pattern at an early stage of the disease that needs further investigation to deeply explain the mechanistic interplay between sleep and Alzheimer.
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http://dx.doi.org/10.1016/j.neurobiolaging.2017.01.011DOI Listing
May 2017

Quantification of hepcidin-25 in human cerebrospinal fluid using LC-MS/MS.

Bioanalysis 2017 Feb 20;9(4):337-347. Epub 2017 Jan 20.

Laboratoire de Biochimie et de Protéomique Clinique - Institut de Médecine Régénérative et Biothérapies (LBPC-IRMB), CHU de Montpellier, 34250 Montpellier, France.

Aim: Hepcidin, the main iron metabolism regulator, can be detected in various biological fluids. Here, we describe a quantitative method of LC-MS/MS to quantify the 25 amino acid isoform of hepcidin (hepcidin-25) in human cerebrospinal fluid (CSF). Results & methodology: Samples were prepared through protein precipitation followed by solid phase extraction (SPE) and injected into a triple-quadrupole mass spectrometer. Validation of our method included determination of LOQ (0.1 ng/ml), repeatability, intermediate precision, recovery and linearity (up to 25 ng/ml). Hepcidin-25 was subsequently quantified in 36 human CSF samples and its concentration ranged from 0.21 to 3.54 ng/ml.

Conclusion: This is the first time that hepcidin-25 can be reliably quantified in human CSF. This may open interesting perspectives for the management of iron-related neurological disorders.
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http://dx.doi.org/10.4155/bio-2016-0240DOI Listing
February 2017

Clinical mass spectrometry proteomics (cMSP) for medical laboratory: What does the future hold?

Clin Chim Acta 2017 Apr 2;467:51-58. Epub 2016 Jun 2.

CHU de Montpellier, hôpital St Eloi Université de Montpellier and INSERM U1183, IRMB, Laboratoire de Biochimie Protéomique Clinique, Montpellier, France.

Background: Mass spectrometry (MS) methods are being widely used these days in medical laboratories for quantifying many small molecular analytes as well as for microbiological purposes.

Methods: Little use has been made so far, however, of MS for analyzing peptides and proteins in clinical laboratory (an approach known as clinical MS proteomics (cMSP)). The explanation for this situation may be that cMSP assays are more complex to implement than conventional assays, require large investments in terms of equipment and training, and have not yet been sufficiently validated for clinical applications. In addition, the protein analysis assays currently used in medical laboratories mostly meet both laboratory and clinical requirements in terms of analytical performances, ease of use, and turn-around-time.

Results: With the spread of MS methods in laboratories, increasing interest seems to be focusing on the development of MS for quantifying new analytes. MALDI-TOF MS methods have already been replacing classical methods of bacterial classification in clinical laboratories, for example, and this can be said to be an important step in this direction.

Conclusions: In this paper, the literature available on the topic of clinical MS proteomics is reviewed and the pre-analytical, analytical, and post-analytical challenges which will have to be met in connection with this approach are discussed.
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http://dx.doi.org/10.1016/j.cca.2016.06.001DOI Listing
April 2017

Proteomic profile of cerebrospinal fluid in patients with multiple sclerosis using two dimensional gel electrophoresis.

Br J Biomed Sci 2016 Jul 2;73(3):143-146. Epub 2016 Jun 2.

d Faculty of Medicine, Department of Anesthesia and Surgical Care , Alexandria University , Alexandria , Egypt.

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http://dx.doi.org/10.1080/09674845.2016.1186310DOI Listing
July 2016

From radioimmunoassay to mass spectrometry: a new method to quantify orexin-A (hypocretin-1) in cerebrospinal fluid.

Sci Rep 2016 05 11;6:25162. Epub 2016 May 11.

CHU Montpellier, Institut de Recherche en Biothérapie, hôpital St Eloi, Laboratoire de Biochimie Protéomique Clinique et CRB, Montpellier, F-34000 France.

I(125) radioimmunoassay (RIA) is currently the standard technique for quantifying cerebrospinal fluid (CSF) orexin-A/hypocretin-1, a biomarker used to diagnose narcolepsy type 1. However, orexin-A RIA is liable to undergo cross-reactions with matrix constituents generating interference, high variability between batches, low precision and accuracy, and requires special radioactivity precautions. Here we developed the first quantitative mass spectrometry assay of orexin-A based on a multiple reaction monitoring (MRM) approach. This method was tested in keeping with the Clinical and Laboratory Standards Institute (CLSI) guidelines and its clinical relevance was confirmed by comparing patients with narcolepsy type 1 versus patients with other neurological conditions. The results obtained using MRM and RIA methods were highly correlated, and Bland-Altman analysis established their interchangeability. However, the MRM values had a wider distribution and were 2.5 time lower than the RIA findings. In conclusion, this method of assay provides a useful alternative to RIA to quantify orexin-A, and may well replace it not only in narcolepsy type 1, but also in the increasing number of pathologies in which the quantification of this analyte is relevant.
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http://dx.doi.org/10.1038/srep25162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4863245PMC
May 2016

Differential Mass Spectrometry Profiles of Tau Protein in the Cerebrospinal Fluid of Patients with Alzheimer's Disease, Progressive Supranuclear Palsy, and Dementia with Lewy Bodies.

J Alzheimers Dis 2016 ;51(4):1033-43

CHU Montpellier, IRMB, hôpital St Eloi, Laboratoire de Biochimie Protéomique Clinique et CRB, INSERM-UM U1183, Montpellier, France.

Microtubule-associated Tau proteins are major actors in neurological disorders, the so-called tauopathies. In some of them, and specifically in Alzheimer's disease (AD), hyperphosphorylated forms of Tau aggregate into neurofibrillary tangles. Following and understanding the complexity of Tau's molecular profile with its multiple isoforms and post-translational modifications represent an important issue, and a major analytical challenge. Immunodetection methods are, in fact, limited by the number, specificity, sensitivity, and capturing property of the available antibodies. Mass spectrometry (MS) has recently allowed protein quantification in complex biological fluids using isotope-labeled recombinant standard for absolute quantification (PSAQ). To study Tau proteins, which are found at very low concentrations within the cerebrospinal fluid (CSF), we relied on an innovative two-step pre-fractionation strategy, which was not dependent on immuno-enrichment. We then developed a sensitive multiplex peptide detection capability using targeted high-resolution MS to quantify Tau-specific peptides covering its entire sequence. This approach was used on a clinical cohort of patients with AD, progressive supranuclear palsy (PSP), and dementia with Lewy body (DLB) and with control non-neurodegenerative disorders. We uncovered a common CSF Tau molecular profile characterized by a predominance of central core expression and 1N/3R isoform detection. While PSP and DLB tau profiles showed minimal changes, AD was characterized by a unique pattern with specific modifications of peptide distribution. Taken together these results provide important information on Tau biology for future therapeutic interventions, and improved molecular diagnosis of tauopathies.
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http://dx.doi.org/10.3233/JAD-150962DOI Listing
December 2016

Tau Protein Quantification in Human Cerebrospinal Fluid by Targeted Mass Spectrometry at High Sequence Coverage Provides Insights into Its Primary Structure Heterogeneity.

J Proteome Res 2016 Feb 19;15(2):667-76. Epub 2016 Jan 19.

CEA, iBiTec-S , Service de Pharmacologie et d'Immunoanalyse, Laboratoire d'Etude du Métabolisme des Médicaments, Gif-sur-Yvette 91191, France.

Tau protein plays a major role in neurodegenerative disorders, appears to be a central biomarker of neuronal injury in cerebrospinal fluid (CSF), and is a promising target for Alzheimer's disease immunotherapies. To quantify tau at high sensitivity and gain insights into its naturally occurring structural variations in human CSF, we coupled absolute quantification using protein standard with the multiplex detection capability of targeted high-resolution mass spectrometry (MS) on a Quadrupole-Orbitrap instrument. Using recombinant tau we developed a step-by-step workflow optimization including an extraction protocol that avoided affinity reagents and achieved the monitoring of 22 tau peptides uniformly distributed along the tau sequence. The lower limits of quantification ranged (LLOQ) from 150 to 1500 pg/mL depending on the peptide. Applied to endogenous CSF tau, up to 19 peptides were detected. Interestingly, there were significant differences in the abundance of peptides depending on their position in the sequence, with peptides from the tau mid-domain appearing significantly more abundant than peptides from the N- and C-terminus domains. This MS-based strategy provided results complementary to those of previous ELISA or Western Blot studies of CSF tau and could be applied to tau monitoring in human CSF cohorts.
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http://dx.doi.org/10.1021/acs.jproteome.5b01001DOI Listing
February 2016

Development of new quantitative mass spectrometry and semi-automatic isofocusing methods for the determination of Apolipoprotein E typing.

Clin Chim Acta 2016 Feb 19;454:33-8. Epub 2015 Dec 19.

CHRU de Montpellier, Hôpital St Eloi Université de Montpellier, INSERM U1183, IRMB, Laboratoire de Biochimie Protéomique Clinique, Montpellier, France. Electronic address:

Background: Apolipoprotein E (Apo E) is a 36 Kda glycoprotein involved in lipid transport. It exists in 3 major isoforms: E2, E3 and E4. ApoE status is known to be a major risk factor for late-onset Alzheimer's and cardiovascular diseases. Genotyping is commonly used to obtain ApoE status but can show technical issues with ambiguous determinations. Phenotyping can be an alternative, not requiring genetic material. We evaluated the ability to accurately type ApoE isoforms by 2 phenotyping tests in comparison with genotyping.

Methods: Two phenotyping techniques were used: (1) LC-MS/MS detection of 4 ApoE specific peptides (6490 Agilent triple quadripole): After its denaturation, serum was either reduced and alkylated, or only diluted, and then trypsin digested. Before analysis, desalting, evaporation and resuspension were performed. (2) Isoelectric focusing and immunoprecipitation: serum samples were neuraminidase digested, delipidated and electrophoresed on Hydragel ApoE (Sebia agarose gel) using Hydrasys 2 Scan instrument (Sebia, Lisses, France). ApoE isoforms bands were directly immunofixed in the gel using a polyclonal anti human ApoE antibody. Then, incubation of the gel with HRP secondary antibody followed by TTF1/TTF2 substrate allowed the visualization of ApoE bands. The results of the two techniques were compared to genotyping.

Results: Sera from 35 patients previously genotyped were analyzed with the 2 phenotyping techniques. 100% concordance between both phenotyping assays was obtained for the tested phenotypes (E2/E2, E2/E3, E2/E4, E3/E3, E3/E4, E4/E4). When compared to genotyping, 3 samples were discordant. After reanalyzing them by both phenotyping tests and DNA sequencing, 2/3 discrepancies were confirmed. Those can be explained by variants or rare ApoE alleles or by unidentified technical issues. 102 additional samples were then tested on LC-MS/MS only and compared to genotyping. The data showed 100% concordance.

Conclusion: Our 2 phenotyping methods represent a valuable alternative to genotyping. LC-MS/MS has the advantage of being fully specific, with identification of the different isoforms and can be considered as a reference method. Sebia isofocusing technique was concordant with LC-MS/MS. Plus, it is a rapid, semi-automated assay that can be easily implemented in clinical laboratories.
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http://dx.doi.org/10.1016/j.cca.2015.12.020DOI Listing
February 2016

Absolute quantification of 35 plasma biomarkers in human saliva using targeted MS.

Bioanalysis 2016 9;8(1):43-53. Epub 2015 Dec 9.

Laboratoire de Biochimie et de Protéomique Clinique- Institut de Médecine Régénérative et Biothérapies (LBPC-IRMB), CHU de Montpellier, 80 rue Augustin Fliche, Montpellier, France.

Background: Although the use of human saliva for diagnosing disease has been known to be of great clinical potential, few attempts have been made so far to develop its use. In this work, we developed an MRM-MS approach for 35 plasma biomarkers using human saliva in a clinical environment.

Methods & Results: A 30-min micro LC-MS/MS run in MRM mode was conducted in order to quantify the 35 plasma proteins in human saliva. Sample preparation procedures were performed in quadruplicate and analyzed in duplicate. Results show that 32 of the 35 plasma proteins were quantified in human saliva using calibration curves in the 2- log10 dynamic ranges with excellent linearity.

Discussion/conclusion: Our MRM method is compatible with routine measurements in daily clinical practice.
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http://dx.doi.org/10.4155/bio.15.228DOI Listing
September 2016

Stable Isotope Labeling by Amino acid in Vivo (SILAV): a new method to explore protein metabolism.

Rapid Commun Mass Spectrom 2015 Oct;29(20):1917-25

CHRU de Montpellier, Université de Montpellier and INSERM U1183, IRMB, Laboratoire de Biochimie Protéomique Clinique, Montpellier, France.

Rationale: Intravenous administration of stable isotope labeled amino acid ((13)C6-leucine) to humans recently made it possible to study the metabolism of specific biomarkers in cerebrospinal fluid (CSF) using targeted mass spectrometry (MS). This labeling approach could be of great interest for monitoring many leucine-containing peptides in parallel, using high-resolution MS. This will make it possible to quantify the rates of synthesis and clearance of a large range of proteins in humans with a view to obtaining new insights into protein metabolism processes and the pathophysiology of diseases such as Alzheimer's disease.

Methods: Proteins from human lumbar and ventricular CSF samples collected at different times after intravenous (13)C6-leucine infusion were digested enzymatically with LysC/trypsin after being denatured, reduced and alkylated. Desalted tryptic peptides were fractionated using Strong Cation eXchange chromatography (SCX) and analyzed using nanoflow liquid chromatography (nano-LC) coupled to a QTOF Impact II (Bruker Daltonics) mass spectrometer. Data-dependent acquisition (DDA) mode was used to identify and quantify light and heavy (13)C6-leucine peptides. The ratios of (13)C6-leucine incorporation were calculated using the Skyline software program in order to determine the rates of appearance and clearance of proteins in the CSF.

Results: After SCX fractionation and quadrupole time-of-flight (QTOF) analysis, 4528 peptides containing leucine were identified in five fractions prepared from 40 μL of CSF. Upon analyzing one of these fractions, 66 peptides (2.7%) corresponding to 61 individual proteins had significant and reproducible rate of (13)C6-leucine incorporation at various time points. The plots of the light-to-heavy peptide ratios showed the existence of proteins with different patterns of appearance and clearance in the CSF.

Conclusions: The Stable Isotope Labeling Amino acid in Vivo (SILAV) method presented here, which yields unprecedented information about protein metabolism in humans, constitutes a promising new approach which certainly holds great potential in the field of clinical proteomics.
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http://dx.doi.org/10.1002/rcm.7289DOI Listing
October 2015

Antibody-free quantification of seven tau peptides in human CSF using targeted mass spectrometry.

Front Neurosci 2015 1;9:302. Epub 2015 Sep 1.

Laboratoire de Biochimie et de Protéomique Clinique - Institut de Médecine Régénérative et Biothérapies, Centre Hospitalier Universitaire de Montpellier Montpellier, France.

Tau protein concentration in cerebrospinal fluid (CSF) is currently used as a sensitive and specific biomarker for Alzheimer's disease. Its detection currently relies on ELISA but the perspective of using mass spectrometry (MS) to detect its different proteoforms represents an interesting alternative. This is however an analytical challenge because of its low concentration in the CSF, a biological fluid collected in small volume by lumbar puncture, and with a high structural heterogeneity. To overcome these issues, instead of using immunocapture as previously done, we rather relied on an original two steps pre-fractionation technique of CSF: perchloric acid (PCA) followed by micro solid phase extraction (μSPE). We could then measure seven tau trypsic peptides by Multiple Reaction Monitoring (MRM) on a triple quadrupole mass spectrometer. Quantification was performed using isotopically labeled (15)N- recombinant tau protein as internal standard and validated using CSF pools with low, medium, or high tau concentrations (HTCs). Repeatability, intermediate precision, linearity, limit of quantification (LOQ), and recovery were calculated for the different peptides. This new MRM assay, which allowed for the first time CSF tau protein quantification without immunocapture, has important potential application to follow tau metabolism in both diagnostic and therapeutic research.
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http://dx.doi.org/10.3389/fnins.2015.00302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4555028PMC
September 2015

Quantitative detection of amyloid-β peptides by mass spectrometry: state of the art and clinical applications.

Clin Chem Lab Med 2015 Sep;53(10):1483-93

Alzheimer's disease (AD) is the most common form of dementia in humans, and a major public health concern with 35 million of patients worldwide. Cerebrospinal fluid (CSF) biomarkers being early diagnostic indicators of AD, it is essential to use the most efficient analytical methods to detect and quantify them accurately. These biomarkers, and more specifically amyloid-β (Aβ) peptides, are measured in routine clinical practice using immunoassays. However, there are several limits to this immunodetection in terms of specificity and multiplexing of the multiple isoforms of the Aβ peptides. To overcome these issues, the quantification of these analytes by mass spectrometry (MS) represents an interesting alternative, and several assays have been described over the past years. This article reviews the different Aβ peptides quantitative MS-based approaches published so far, compares their pre-analytical phase, and the different quantitative strategies implemented that might be suitable for clinical applications.
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http://dx.doi.org/10.1515/cclm-2014-1048DOI Listing
September 2015

The calcium-dependent protein kinase CPK7 acts on root hydraulic conductivity.

Plant Cell Environ 2015 Jul 15;38(7):1312-20. Epub 2014 Dec 15.

Biochimie et Physiologie Moléculaire des Plantes, INRA/CNRS/SupAgro/UM2, UMR 5004, 2 Place Viala, Montpellier Cedex 1, 34060, France.

The hydraulic conductivity of plant roots (Lp(r)) is determined in large part by the activity of aquaporins. Mechanisms occurring at the post-translational level, in particular phosphorylation of aquaporins of the plasma membrane intrinsic protein 2 (PIP2) subfamily, are thought to be of critical importance for regulating root water transport. However, knowledge of protein kinases and phosphatases acting on aquaporin function is still scarce. In the present work, we investigated the Lp(r) of knockout Arabidopsis plants for four Ca(2+)-dependent protein kinases. cpk7 plants showed a 30% increase in Lp(r) because of a higher aquaporin activity. A quantitative proteomic analysis of wild-type and cpk7 plants revealed that PIP gene expression and PIP protein quantity were not correlated and that CPK7 has no effect on PIP2 phosphorylation. In contrast, CPK7 exerts a negative control on the cellular abundance of PIP1s, which likely accounts for the higher Lp(r) of cpk7. In addition, this study revealed that the cellular amount of a few additional proteins including membrane transporters is controlled by CPK7. The overall work provides evidence for CPK7-dependent stability of specific membrane proteins.
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http://dx.doi.org/10.1111/pce.12478DOI Listing
July 2015