Publications by authors named "François Becher"

62 Publications

Mass Spectrometry for Neurobiomarker Discovery: The Relevance of Post-Translational Modifications.

Cells 2022 04 9;11(8). Epub 2022 Apr 9.

CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), SPI, Université Paris-Saclay, 91191 Gif-sur-Yvette, France.

Neurodegenerative diseases are incurable, heterogeneous, and age-dependent disorders that challenge modern medicine. A deeper understanding of the pathogenesis underlying neurodegenerative diseases is necessary to solve the unmet need for new diagnostic biomarkers and disease-modifying therapy and reduce these diseases' burden. Specifically, post-translational modifications (PTMs) play a significant role in neurodegeneration. Due to its proximity to the brain parenchyma, cerebrospinal fluid (CSF) has long been used as an indirect way to measure changes in the brain. Mass spectrometry (MS) analysis in neurodegenerative diseases focusing on PTMs and in the context of biomarker discovery has improved and opened venues for analyzing more complex matrices such as brain tissue and blood. Notably, phosphorylated tau protein, truncated α-synuclein, APP and TDP-43, and many other modifications were extensively characterized by MS. Great potential is underlying specific pathological PTM-signatures for clinical application. This review focuses on PTM-modified proteins involved in neurodegenerative diseases and highlights the most important and recent breakthroughs in MS-based biomarker discovery.
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http://dx.doi.org/10.3390/cells11081279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9031030PMC
April 2022

Multiplex Detection of 24 Staphylococcal Enterotoxins in Culture Supernatant Using Liquid Chromatography Coupled to High-Resolution Mass Spectrometry.

Toxins (Basel) 2022 03 31;14(4). Epub 2022 Mar 31.

Laboratory for Food Safety, ANSES, Université Paris-Est, 91191 Maisons-Alfort, France.

Staphylococcal food poisoning outbreaks are caused by the ingestion of food contaminated with staphylococcal enterotoxins (SEs). Among the 27 SEs described in the literature to date, only a few can be detected using immuno-enzymatic-based methods that are strongly dependent on the availability of antibodies. Liquid chromatography, coupled to high-resolution mass spectrometry (LC-HRMS), has, therefore, been put forward as a relevant complementary method, but only for the detection of a limited number of enterotoxins. In this work, LC-HRMS was developed for the detection and quantification of 24 SEs. A database of 93 specific signature peptides and LC-HRMS parameters was optimized using sequences from 24 SEs, including their 162 variants. A label-free quantification protocol was established to overcome the absence of calibration standards. The LC-HRMS method showed high performance in terms of specificity, sensitivity, and accuracy when applied to 49 enterotoxin-producing strains. SE concentrations measured depended on both SE type and the coagulase-positive staphylococci (CPS) strain. This study indicates that LC-MS is a relevant alternative and complementary tool to ELISA methods. The advantages of LC-MS clearly lie in both the multiplex analysis of a large number of SEs, and the automated analysis of a high number of samples.
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http://dx.doi.org/10.3390/toxins14040249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9031063PMC
March 2022

Top-Down Mass Spectrometry for Trace Level Quantification of Staphylococcal Enterotoxin A Variants.

J Proteome Res 2022 02 30;21(2):547-556. Epub 2021 Dec 30.

Université Paris-Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), SPI, 91191 Gif-sur-Yvette, France.

We addressed here the need for improved sensitivity of top-down mass spectrometry for identification, differentiation, and absolute quantification of sequence variants of SEA, a bacterial toxin produced by and regularly involved in food poisoning outbreaks (FPO). We combined immunoaffinity enrichment, a protein internal standard, and optimized acquisition conditions, either by full-scan high-resolution mass spectrometry (HRMS) or multiplex parallel reaction monitoring (PRM) mode. Deconvolution of full-scan HRMS signal and PRM detection of variant-specific fragment ions allowed confident identification of each SEA variant. Summing the PRM signal of variant-common fragment ions was most efficient for absolute quantification, illustrated by a sensitivity down to 2.5 ng/mL and an assay variability below 15%. Additionally, we showed that relative PRM fragment ion abundances constituted a supplementary specificity criterion in top-down quantification. The top-down method was successfully evaluated on a panel of enterotoxin-producing strains isolated during FPO, in parallel to the conventional whole genome sequencing, ELISA, and bottom-up mass spectrometry methods. Top-down provided at the same time correct identification of the SEA variants produced and precise determination of the toxin level. The raw files generated in this study can be found on PASSEL (Peptide Atlas) under data set identifier PASS01710.
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http://dx.doi.org/10.1021/acs.jproteome.1c00886DOI Listing
February 2022

Differentiation, Quantification and Identification of Abrin and Agglutinin.

Toxins (Basel) 2021 04 18;13(4). Epub 2021 Apr 18.

Biological Toxins, Centre for Biological Threats and Special Pathogens, Robert Koch Institute, Seestr. 10, 13353 Berlin, Germany.

Abrin, the toxic lectin from the rosary pea plant has gained considerable interest in the recent past due to its potential malevolent use. However, reliable and easy-to-use assays for the detection and discrimination of abrin from related plant proteins such as agglutinin or the homologous toxin ricin from are sparse. To address this gap, a panel of highly specific monoclonal antibodies was generated against abrin and the related agglutinin. These antibodies were used to establish two sandwich ELISAs to preferentially detect abrin or agglutinin (limit of detection 22 pg/mL for abrin; 35 pg/mL for agglutinin). Furthermore, an abrin-specific lateral flow assay was developed for rapid on-site detection (limit of detection ~1 ng/mL abrin). Assays were validated for complex food, environmental and clinical matrices illustrating broad applicability in different threat scenarios. Additionally, the antibodies turned out to be suitable for immuno-enrichment strategies in combination with mass spectrometry-based approaches for unambiguous identification. Finally, we were able to demonstrate for the first time how the developed assays can be applied to detect, identify and quantify abrin from a clinical sample derived from an attempted suicide case involving .
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http://dx.doi.org/10.3390/toxins13040284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8073929PMC
April 2021

Optimal anchoring of a foldamer inhibitor of ASF1 histone chaperone through backbone plasticity.

Sci Adv 2021 03 19;7(12). Epub 2021 Mar 19.

Institute Joliot, Commissariat à l'énergie Atomique (CEA), Direction de la Recherche Fondamentale (DRF), CEA Saclay, F91191 Gif-sur-Yvette, France.

Sequence-specific oligomers with predictable folding patterns, i.e., foldamers, provide new opportunities to mimic α-helical peptides and design inhibitors of protein-protein interactions. One major hurdle of this strategy is to retain the correct orientation of key side chains involved in protein surface recognition. Here, we show that the structural plasticity of a foldamer backbone may notably contribute to the required spatial adjustment for optimal interaction with the protein surface. By using oligoureas as α helix mimics, we designed a foldamer/peptide hybrid inhibitor of histone chaperone ASF1, a key regulator of chromatin dynamics. The crystal structure of its complex with ASF1 reveals a notable plasticity of the urea backbone, which adapts to the ASF1 surface to maintain the same binding interface. One additional benefit of generating ASF1 ligands with nonpeptide oligourea segments is the resistance to proteolysis in human plasma, which was highly improved compared to the cognate α-helical peptide.
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http://dx.doi.org/10.1126/sciadv.abd9153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7978421PMC
March 2021

Quantitative Determination of Enterotoxins Types A to I and Variants in Dairy Food Products by Multiplex Immuno-LC-MS/MS.

J Agric Food Chem 2021 Mar 17;69(8):2603-2610. Epub 2021 Feb 17.

Département Médicaments et Technologies pour la Santé (DMTS), SPI, Université Paris-Saclay, CEA, INRAE, 91191 Gif-sur-Yvette, France.

Staphylococcal enterotoxins (SEs) are responsible for frequent food poisoning outbreaks worldwide. Specific identification of SEs is crucial for confirmation of food poisoning, tracking of the incriminated foods or food ingredients, and removal from the food chain. Here, we report on a new food testing protocol addressing the challenge of low abundance of SEs in contaminated food and high sequence heterogeneity. Multiplex ability of targeted high-resolution mass spectrometry was succesfully applied to the simultaneous and quantitative determination of the eight most frequent SEs including sequence variants. In this aim, between three and eight proteotypic peptides of each SE were selected by carefully considering amino acid variations within each type, and sequence homology between types. Quantification of trace levels of SEs directly in food samples was reached by immunoaffinity enrichment and optimized analytical conditions. The assay was validated in dairy food products with a lower limit of quantification down to 0.1 ng/g (in milk), and quantification of SEs was successfully demonstrated in real-life samples collected during staphylococcal food poisoning outbreaks. Importantly, the ability of the method to detect diverse sequence variants was also illustrated. By enabling for the first time the simultaneous quantification of the eight most frequent SEs, the new mass spectrometry-based assay would facilitate the laboratory confirmation of positive samples in situation of food poisoning outbreaks.
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http://dx.doi.org/10.1021/acs.jafc.0c07545DOI Listing
March 2021

Ricin Antibodies' Neutralizing Capacity against Different Ricin Isoforms and Cultivars.

Toxins (Basel) 2021 01 29;13(2). Epub 2021 Jan 29.

Paris-Saclay University, CEA, INRAE, Medicines and Healthcare Technologies Department (DMTS), SPI, 91191 Gif-sur-Yvette, France.

Ricin, a highly toxic protein from , is considered a potential biowarfare agent. Despite the many data available, no specific treatment has yet been approved. Due to their ability to provide immediate protection, antibodies (Abs) are an approach of choice. However, their high specificity might compromise their capacity to protect against the different ricin isoforms (D and E) found in the different cultivars. In previous work, we have shown the neutralizing potential of different Abs (43RCA-G1 (anti ricin A-chain) and RB34 and RB37 (anti ricin B-chain)) against ricin D. In this study, we evaluated their protective capacity against both ricin isoforms. We show that: (i) RB34 and RB37 recognize exclusively ricin D, whereas 43RCA-G1 recognizes both isoforms, (ii) their neutralizing capacity in vitro varies depending on the cultivar, and (iii) there is a synergistic effect when combining RB34 and 43RCA-G1. This effect is also demonstrated in vivo in a mouse model of intranasal intoxication with ricin D/E (1:1), where approximately 60% and 40% of mice treated 0 and 6 h after intoxication, respectively, are protected. Our results highlight the importance of evaluating the effectiveness of the Abs against different ricin isoforms to identify the treatment with the broadest spectrum neutralizing effect.
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http://dx.doi.org/10.3390/toxins13020100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7911099PMC
January 2021

Quantitative Assessment of SARS-CoV-2 Virus in Nasopharyngeal Swabs Stored in Transport Medium by a Straightforward LC-MS/MS Assay Targeting Nucleocapsid, Membrane, and Spike Proteins.

J Proteome Res 2021 02 26;20(2):1434-1443. Epub 2021 Jan 26.

Université Paris-Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), SPI, 91191 Gif sur Yvette, France.

Alternative methods to RT-PCR for SARS-CoV-2 detection are investigated to provide complementary data on viral proteins, increase the number of tests performed, or identify false positive/negative results. Here, we have developed a simple mass spectrometry assay for SARS-CoV-2 in nasopharyngeal swab samples using common laboratory reagents. The method employs high sensitivity and selectivity targeted mass spectrometry detection, monitoring nine constitutive peptides representative of the three main viral proteins and a straightforward pellet digestion protocol for convenient routine applications. Absolute quantification of N, M, and S proteins was achieved by addition of isotope-labeled versions of best peptides. Limit of detection, recovery, precision, and linearity were thoroughly evaluated in four representative viral transport media (VTM) containing distinct total protein content. The protocol was sensitive in all swab media with limit of detection determined at 2 × 10 pfu/mL, corresponding to as low as 30 pfu injected into the LC-MS/MS system. When tested on VTM-stored nasopharyngeal swab samples from positive and control patients, sensitivity was similar to or better than rapid immunoassay dipsticks, revealing a corresponding RT-PCR detection threshold at Ct ∼ 24. The study represents the first thorough evaluation of sensitivity and robustness of targeted mass spectrometry in nasal swabs, constituting a promising SARS-CoV-2 antigen assay for the first-line diagnosis of COVID-19 and compatible with the constraints of clinical settings. The raw files generated in this study can be found on PASSEL (Peptide Atlas) under data set identifier PASS01646.
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http://dx.doi.org/10.1021/acs.jproteome.0c00887DOI Listing
February 2021

Development and Evaluation of an Immuno-MALDI-TOF Mass Spectrometry Approach for Quantification of the Abrin Toxin in Complex Food Matrices.

Toxins (Basel) 2021 01 13;13(1). Epub 2021 Jan 13.

CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), Université Paris Saclay, SPI, 91191 Gif-sur-Yvette, France.

The toxin abrin found in the seeds of has attracted much attention regarding criminal and terroristic misuse over the past decade. Progress in analytical methods for a rapid and unambiguous identification of low abrin concentrations in complex matrices is essential. Here, we report on the development and evaluation of a MALDI-TOF mass spectrometry approach for the fast, sensitive and robust abrin isolectin identification, differentiation and quantification in complex food matrices. The method combines immunoaffinity-enrichment with specific abrin antibodies, accelerated trypsin digestion and the subsequent MALDI-TOF analysis of abrin peptides using labeled peptides for quantification purposes. Following the optimization of the workflow, common and isoform-specific peptides were detected resulting in a ~38% sequence coverage of abrin when testing ng-amounts of the toxin. The lower limit of detection was established at 40 ng/mL in milk and apple juice. Isotope-labeled versions of abundant peptides with high ionization efficiency were added. The quantitative evaluation demonstrated an assay variability at or below 22% with a linear range up to 800 ng/mL. MALDI-TOF mass spectrometry allows for a simple and fast (<5 min) analysis of abrin peptides, without a time-consuming peptide chromatographic separation, thus constituting a relevant alternative to liquid chromatography-tandem mass spectrometry.
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http://dx.doi.org/10.3390/toxins13010052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7828309PMC
January 2021

Cerebrospinal fluid phospho-tau T217 outperforms T181 as a biomarker for the differential diagnosis of Alzheimer's disease and PET amyloid-positive patient identification.

Alzheimers Res Ther 2020 03 17;12(1):26. Epub 2020 Mar 17.

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

Background: Cerebrospinal fluid biomarker profiles characterized by decreased amyloid-beta peptide levels and increased total and phosphorylated tau levels at threonine 181 (pT181) are currently used to discriminate between Alzheimer's disease and other neurodegenerative diseases. However, these changes are not entirely specific to Alzheimer's disease, and it is noteworthy that other phosphorylated isoforms of tau, possibly more specific for the disease process, have been described in the brain parenchyma of patients. The precise detection of these isoforms in biological fluids remains however a challenge.

Methods: In the present study, we used the latest quantitative mass spectrometry approach, which achieves a sensitive detection in cerebrospinal fluid biomarker of two phosphorylated tau isoforms, pT181 and pT217, and first analyzed a cohort of probable Alzheimer's disease patients and patients with other neurological disorders, including tauopathies, and a set of cognitively normal controls. We then checked the validity of our results on a second cohort comprising cognitively normal individuals and patients with mild cognitive impairments and AD stratified in terms of their amyloid status based on PiB-PET imaging methods.

Results: In the first cohort, pT217 but not pT181 differentiated between Alzheimer's disease patients and those with other neurodegenerative diseases and control subjects much more specificity and sensitivity than pT181. T217 phosphorylation was increased by 6.0-fold in patients with Alzheimer's disease whereas T181 phosphorylation was only increased by 1.3-fold, when compared with control subjects. These results were confirmed in the case of a second cohort, in which the pT217 cerebrospinal fluid levels marked out amyloid-positive patients with a sensitivity and a specificity of more than 90% (AUC 0.961; CI 0.874 to 0.995). The pT217 concentrations were also highly correlated with the PiB-PET values (correlation coefficient 0.72; P < 0.001).

Conclusions: Increased cerebrospinal fluid pT217 levels, more than those of pT181, are highly specific biomarkers for detecting both the preclinical and advanced forms of Alzheimer's disease. This finding should greatly improve the diagnosis of Alzheimer's disease, along with the correlations found to exist between pT217 levels and PiB-PET data. It also suggests that pT217 is a promising potential target for therapeutic applications and that a link exists between amyloid and tau pathology.
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http://dx.doi.org/10.1186/s13195-020-00596-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7079453PMC
March 2020

Top-Down and Bottom-Up Proteomics of Circulating S100A8/S100A9 in Plasma of Septic Shock Patients.

J Proteome Res 2020 02 22;19(2):914-925. Epub 2020 Jan 22.

Service de Pharmacologie et Immunoanalyse (SPI), CEA, INRA, Université Paris-Saclay , Gif-sur Yvette F-91191 , France.

Well-characterized prognostic biomarkers and reliable quantitative methods are key in sepsis management. Among damage-associated molecular patterns, S100A8/S100A9 complexes are reported to be markers for injured cells and to improve the prediction of death in septic shock patients. In view of the structural diversity observed for the intracellular forms, insight into circulating complexes and proteoforms is required to establish prognostic biomarkers. Here, we developed top-down and bottom-up proteomics to characterize the association of S100A8 and S100A9 in complexes and major circulating proteoforms. An antibody-free method was developed for absolute quantification of S100A8/S100A9 in a cohort of 49 patients to evaluate the prognostic value on the first day after admission for septic shock. The predominant circulating forms identified by top-down proteomics were S100A8, mono-oxidized S100A8, truncated acetylated S100A9, and S-nitrosylated S100A9. S100A8, truncated acetylated S100A9, and mono-oxidized S100A8 discriminated between survivors and nonsurvivors, along with total S100A8/S100A9 measured by the antibody-free bottom-up method. Overall, new insights into circulating S100A8/S100A9 and confirmation of its prognostic value in septic shock are crucial in qualification of this biomarker. Also, the simple antibody-free assay would support the harmonization of S100A8/S100A9 measurements.
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http://dx.doi.org/10.1021/acs.jproteome.9b00690DOI Listing
February 2020

High plasma level of S100A8/S100A9 and S100A12 at admission indicates a higher risk of death in septic shock patients.

Sci Rep 2019 10 30;9(1):15660. Epub 2019 Oct 30.

Université Paris 7 Cité Sorbonne; UMR INSERM 1160, 110 Avenue de Verdun, Paris, 75010, France.

Biomarkers in sepsis for severity, prediction of outcome or reversibility of organ dysfunction are warranted. Measurements of plasma DAMP levels at admission can reflect the severity of cellular damage in septic shock, which might predict the prognosis and reduce the risk of overtreating patients with costly therapies. We measured plasma levels of two DAMPs, S100A8/S100A9 and S100A12 during the first 24 h of admission of septic shock patients. Forty-nine septic shock patients with a similar SOFA scores were selected from our sepsis database to compare a similar proportion of survivors and non-survivors. Plasma levels of S100A8/S100A9 and S100A12 were compared with healthy volunteers using in-house ELISA. Plasma levels of S100A8/S100A9 and S100A12 (5.71 [2.60-13.63] µg/mL and 0.48 [0.22-1.05] µg/mL) were higher in septic shock patients than in healthy volunteers (1.18 [0.74-1.93] µg/mL and 0.09 [0.02-0.39] µg/mL) (P < 0.0001 and P = 0.0030). Levels of S100A8/S100A9 and S100A12 in non-survivors at day 28 (11.70 [2.85-24.36] µg/mL and 0.62 [0.30-1.64] µg/mL) were significantly higher than in survivors (4.59 [2.16-7.47] µg/mL and 0.30 [0.20-0.49] µg/mL) (P = 0.0420 and P = 0.0248) and correlated well (Spearman r = 0.879, P < 0.0001). The high level of plasma calgranulins at admission in septic shock, were higher in non-survivors compared to survivors. These markers could indicate a higher risk of death when SOFA scores are similar and help the stratification of patients for improved care and therapy selection.
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http://dx.doi.org/10.1038/s41598-019-52184-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821805PMC
October 2019

Very Early Blood Diffusion of the Active Lethal and Edema Factors of Bacillus anthracis After Intranasal Infection.

J Infect Dis 2020 02;221(4):660-667

Unité Biothérapies Anti-Infectieuses et Immunité, Institut de Recherche Biomédicale des Armées, Brétigny-sur-Orge, France.

Background: Lethal and edema toxins are critical virulence factors of Bacillus anthracis. Few data are available on their presence in the early stage of intranasal infection.

Methods: To investigate the diffusion of edema factor (EF) and lethal factor (LF), we use sensitive quantitative methods to measure their enzymatic activities in mice intranasally challenged with a wild-type B anthracis strain or with an isogenic mutant deficient for the protective antigen.

Results: One hour after mouse challenge, although only 7% of mice presented bacteremia, LF and EF were detected in the blood of 100% and 42% of mice, respectively. Protective antigen facilitated the diffusion of LF and EF into the blood compartment. Toxins played a significant role in the systemic dissemination of B anthracis in the blood, spleen, and liver. A mouse model of intoxination further confirmed that LT and ET could diffuse rapidly in the circulation, independently of bacteria.

Conclusions: In this inhalational model, toxins have disseminated rapidly in the blood, playing a significant and novel role in the early systemic diffusion of bacteria, demonstrating that they may represent a very early target for the diagnosis and the treatment of anthrax.
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http://dx.doi.org/10.1093/infdis/jiz497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996859PMC
February 2020

Simultaneous quantification of tau and α-synuclein in cerebrospinal fluid by high-resolution mass spectrometry for differentiation of Lewy Body Dementia from Alzheimer's Disease and controls.

Analyst 2019 Oct;144(21):6342-6351

Service de Pharmacologie et d'Immunoanalyse (SPI), Laboratoire d'Etude du Métabolisme des Médicaments (LEMM), CEA, INRA, Université Paris Saclay, F-91191 Gif-sur-Yvette cedex, France.

Tau and α-synuclein are central in several neurodegenerative diseases, including Alzheimer Disease (AD), Dementia with Lewy Bodies (DLB) and Parkinson Disease (PD). New analytical methods for precise quantification of cerebrospinal fluid (CSF) levels of both tau and α-synuclein are required to differentiate between dementias or monitor therapeutic responses. Notably, levels of total α-synuclein reported by ELISA are inconsistent among studies, impacted by antibody specificity or lack of standardization. Here, we report on the development and validation of a sensitive and robust mass spectrometry-based assay for the simultaneous quantification of tau and α-synuclein in CSF. The optimized workflow avoided any affinity reagents, and involved the combination of two enzymes, Glu-C and trypsin for optimal sequence coverage of α-synuclein acidic C-terminus. Up to 7 α-synuclein peptides were quantified, including the C-terminal peptide (132-140), resulting in a sequence coverage of 54% in CSF. The lower limits of quantification (LLOQ) ranged from 0.1 ng mL-1 to 1 ng mL-1 depending on the peptide. Regarding CSF tau, 4 peptides common to all isoforms were monitored, and LLOQ ranged from 0.5 ng mL-1 to 0.75 ng mL-1. The multiplex method was successfully applied to CSF samples from AD and DLB patients, two clinically overlapping neurodegenerative diseases. CSF α-synuclein levels were significantly lower in DLB patients compared to AD and controls. Moreover, tau and α-synuclein concentrations showed opposite trends in AD and DLB patients, suggesting the benefit of combining the two biomarkers for differentiation of DLB from AD and controls.
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http://dx.doi.org/10.1039/c9an00751bDOI Listing
October 2019

Proposed BoNT/A and /B Peptide Substrates Cannot Detect Multiple Subtypes in the Endopep-MS Assay.

J Anal Toxicol 2020 Mar;44(2):173-179

Division of Laboratory Sciences, Centers for Disease Control and Prevention, National Center for Environmental Health, Buford Hwy, Northeast Atlanta, GA, USA.

Botulinum neurotoxins (BoNTs) are a family of protein toxins consisting of seven known serotypes (BoNT/A-BoNT/G) and multiple subtypes within the serotypes, and all of which cause the disease botulism-a disease of great public health concern. Accurate detection of BoNTs in human clinical samples is therefore an important public health goal. To achieve this goal, our laboratory developed a mass spectrometry-based assay detecting the presence of BoNT via its enzymatic activity on a peptide substrate. Recently, publications reported the use of new peptide substrates to detect BoNT/A and /B with improved results over other peptide substrates. However, the authors did not provide results of their peptide substrate on multiple subtypes of BoNT. In this work, we describe the results of testing the new substrates with multiple BoNT/A and /B subtypes and find that the substrates cannot detect many subtypes of BoNT/A and /B.
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http://dx.doi.org/10.1093/jat/bkz044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6949424PMC
March 2020

Quantification of low abundance Yersinia pestis markers in dried blood spots by immuno-capture and quantitative high-resolution targeted mass spectrometry.

Eur J Mass Spectrom (Chichester) 2019 Jun;25(3):268-277

1 Service de Pharmacologie et d'Immunoanalyse (SPI), CEA, INRA, Université Paris-Saclay, Gif sur Yvette, France.

Plague, caused by the bacterium Yersinia pestis, is still present in several countries worldwide. Besides, Y. pestis has been designated as Tier 1 agent, the highest rank of bioterrorism agents. In this context, reliable diagnostic methods are of great importance. Here, we have developed an original workflow based upon dried blood spot for simplified sampling of clinical specimens, and specific immuno-mass spectrometry monitoring of Y. pestis biomarkers. Targeted proteins were selectively enriched from dried blood spot extracts by multiplex immunocapture using antibody-coated magnetic beads. After accelerated on-beads digestion, proteotypic peptides were monitored by multiplex LC-MS/MS through the parallel reaction monitoring mode. The DBS-IC-MS assay was designed to quantify both F1 and LcrV antigens, although 10-fold lower sensitivity was observed with LcrV. The assay was successfully validated for F1 with a lower limit of quantification at 5 ng·mL in spiked blood, corresponding to only 0.1 ng on spots. In vivo quantification of F1 in blood and organ samples was demonstrated in the mouse model of pneumonic plague. The new assay could help to simplify the laboratory confirmation of positive point of care F1 dipstick.
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http://dx.doi.org/10.1177/1469066718795978DOI Listing
June 2019

Cetuximab pharmacokinetic/pharmacodynamics relationships in advanced head and neck carcinoma patients.

Br J Clin Pharmacol 2019 06 13;85(6):1357-1366. Epub 2019 Apr 13.

Laboratory of Pharmacology, Institut Claudius Regaud, IUCT-Oncopole, Toulouse, France.

Aims: Cetuximab associated with cisplatin and 5-fluorouracil is used to treat patients with inoperable or metastatic head and neck squamous cell carcinomas (HNSCC) up until disease progression or unacceptable toxicities. To date, no biomarkers of efficacy are available to select patients who will benefit from treatment.

Methods: An ancillary pharmacokinetics (PK) exploration was performed in the context of a prospective study investigating circulating-tumour cells vs progression-free survival (PFS). Cetuximab plasma concentrations were analysed according to a population PK model. Individual exposure parameters were confronted with soluble epidermal growth factor receptor (sEGFR) concentrations, tumour response and PFS.

Results: PK data (28 patients, 203 observations) were best described by a two-compartment model with linear elimination. Performance status (PS) significantly correlated to both cetuximab clearance and central volume of distribution with both parameters increasing by 33.3% (95% CI 1-65.6) for each 1-point increase of PS compared to PS = 0. Univariate analysis showed that patients with higher trough cetuximab concentrations at Day 7 (C ) had better tumour response (P = 0.03) and longer PFS (P = 0.035). However, multivariate analysis revealed that only PS and tumour size at baseline remained significantly associated with PFS. Levels of sEGFR increased during cetuximab treatment but were not associated with PFS in the multivariate analysis.

Conclusions: Our study prospectively indicates that PS is likely a confounding factor in the relationship between cetuximab PK and PFS, patients with a poor PS having lower cetuximab plasma exposure and lower PFS.
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http://dx.doi.org/10.1111/bcp.13907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6533440PMC
June 2019

New Antibody-Free Mass Spectrometry-Based Quantification Reveals That C9ORF72 Long Protein Isoform Is Reduced in the Frontal Cortex of Hexanucleotide-Repeat Expansion Carriers.

Front Neurosci 2018 28;12:589. Epub 2018 Aug 28.

Service de Pharmacologie et Immunoanalyse, Laboratoire d'Etude du Métabolisme des Médicaments, Commissariat à l'Énergie Atomique et aux Énergies Alternatives, Institut National de la Recherche Agronomique, Université Paris Saclay, Gif-sur-Yvette, France.

Frontotemporal dementia (FTD) is a fatal neurodegenerative disease characterized by behavioral and language disorders. The main genetic cause of FTD is an intronic hexanucleotide repeat expansion (GC)n in the gene. A loss of function of the C9ORF72 protein associated with the allele-specific reduction of expression is postulated to contribute to the disease pathogenesis. To better understand the contribution of the loss of function to the disease mechanism, we need to determine precisely the level of reduction in C9ORF72 long and short isoforms in brain tissue from patients with mutations. In this study, we developed a sensitive and robust mass spectrometry (MS) method for quantifying C9ORF72 isoform levels in human brain tissue without requiring antibody or affinity reagent. An optimized workflow based on surfactant-aided protein extraction and pellet digestion was established for optimal recovery of the two isoforms in brain samples. Signature peptides, common or specific to the isoforms, were targeted in brain extracts by multiplex MS through the parallel reaction monitoring mode on a Quadrupole-Orbitrap high resolution mass spectrometer. The assay was successfully validated and subsequently applied to frontal cortex brain samples from a cohort of FTD patients with mutations and neurologically normal controls without mutations. We showed that the C9ORF72 short isoform in the frontal cortices is below detection threshold in all tested individuals and the C9ORF72 long isoform is significantly decreased in mutation carriers.
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http://dx.doi.org/10.3389/fnins.2018.00589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6122177PMC
August 2018

Investigating the utility of minimized sample preparation and high-resolution mass spectrometry for quantification of monoclonal antibody drugs.

J Pharm Biomed Anal 2018 Sep 30;159:384-392. Epub 2018 Jul 30.

Department of Pharmacy, University of Copenhagen, Universitetsparken 2, Copenhagen, Denmark. Electronic address:

Determination of the pharmacokinetic (PK) properties of therapeutic monoclonal antibodies (mAbs) is essential for their successful development as drugs. For this purpose, besides the traditional ligand binding assay (LBA), LC-MS/MS method using low resolution mass spectrometers (e.g. triple quadrupole (QqQ)) has become routinely used, however, complicated and lengthy sample pre-treatment (employing immuno-affinity) is often necessary for obtaining sufficient sensitivity and selectivity. In this study, we investigate the capabilities of high-resolution MS instruments for circumventing the complex sample preparation currently needed for sensitive LC-MS/MS-based quantification of mAbs. Employing a simple one-step sample pre-treatment workflow, we compare the ability of three different LC-MS platforms for absolute quantification of a representative monoclonal antibody Rendomab-B1 in serum and plasma. The samples are subjected to protein precipitation with methanol, followed by pellet digestion with trypsin prior to LC-MS analysis. AQUA peptides based on two surrogate mAb peptides selected from an extensive in-silico and experimental screening are used as internal standards. MS/MS acquisitions are developed and systematically examined for 1) a low-resolution QqQ operated in selected reaction monitoring (SRM) acquisition mode, 2) a high-resolution hybrid Quadrupole-Orbitrap (Q-Orbitrap) operated in parallel reaction monitoring (PRM) acquisition mode and 3) a high-resolution hybrid Quadrupole-Time-of-flight (Q-TOF) operated in SRM acquisition mode with enhanced duty cycle (EDC) function. The sensitivity of the high-resolution Q-Orbitrap and Q-TOF methods was significantly higher (LOD of 80 ng/mL) in serum/plasma samples than the low-resolution QqQ method. Finally, the real-world utility of the developed high-resolution MS method with minimized sample handling was demonstrated and validated by determining the PK profile of Rendomab-B1 in mice by a 10-point in vivo study over 15 days.
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http://dx.doi.org/10.1016/j.jpba.2018.07.012DOI Listing
September 2018

On-a-chip tryptic digestion of transthyretin: a step toward an integrated microfluidic system for the follow-up of familial transthyretin amyloidosis.

Analyst 2018 Feb;143(5):1077-1086

Faculté de Pharmacie, Université Paris-Sud, Université Paris-Saclay, Châtenay-Malabry, France.

A microfluidic microreactor for trypsin mediated transthyretin (TTR) digestion has been developed as a step towards the elaboration of a fully integrated microdevice for the detection of a rare and disabling disease, the familial transthyretin amyloidosis (ATTR) which is related to specific TTR mutations. Therefore, an enzymatic microreactor coupled to an analytical step able to monitor the mutation of TTR on specific peptide fragments would allow an accurate monitoring of the treatment efficiency of ATTR. In this study, two types of immobilized trypsin microreactors have been investigated: a new miniaturized, microfluidic fluidized bed packed with trypsin functionalized magnetic particles (MPs), and a thiol-ene (TE) monolith-based chip. Their performances were first demonstrated with N-benzoyl-dl-arginine-4-nitroanilide hydrochloride BApNA, a low molecular weight substrate. High reaction yields (75.2%) have been reached within 0.6 min for the TE-based trypsin microreactor, while a lower yield (12.4%) was obtained for the micro-fluidized bed within a similar residence time. Transposition of the optimized conditions, developed with BApNA, to TTR digestion in the TE-based trypsin microreactor was successfully performed. We demonstrated that the TE-chip can achieve an efficient and reproducible digestion of TTR. This has been assessed by MS detection. In addition, TTR hydrolysis led to the production of a fragment of interest allowing the therapeutic follow-up of more than twenty possible ATTR mutations. High sequence coverage (90%), similar to those obtained with free trypsin, was achieved in a short time (2.4 min). Repeated experiments showed good reproducibility (RSD = 6.8%). These promising results open up the route for an innovative treatment follow-up dedicated to ATTR.
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http://dx.doi.org/10.1039/c7an01737eDOI Listing
February 2018

βAPP Processing Drives Gradual Tau Pathology in an Age-Dependent Amyloid Rat Model of Alzheimer's Disease.

Cereb Cortex 2018 11;28(11):3976-3993

INSERM UMR1169, Université Paris-Sud, Université Paris-Saclay, Orsay, France.

The treatment of Alzheimer's disease (AD) remains challenging and requires a better in depth understanding of AD progression. Particularly, the link between amyloid protein precursor (APP) processing and Tau pathology development remains poorly understood. Growing evidences suggest that APP processing and amyloid-β (Aβ) release are upstream of Tau pathology but the lack of animal models mimicking the slow progression of human AD raised questions around this mechanism. Here, we described that an AD-like βAPP processing in adults wild-type rats, yielding to human APP, βCTF and Aβ levels similar to those observed in AD patients, is sufficient to trigger gradual Tauopathy. The Tau hyperphosphorylation begins several months before the formation of both amyloid plaques and tangle-like aggregates in aged rats and without associated inflammation. Based on a longitudinal characterization over 30 months, we showed that extrasynaptic and emotional impairments appear before long-term potentiation deficits and memory decline and so before Aβ and Tau aggregations. These compelling data allowed us to (1) experimentally confirm the causal relationship between βAPP processing and Tau pathology in vivo and without Tau transgene overexpression, (2) support the amyloidogenic cascade and (3) propose a 4-step hypothesis of prodromal AD progression.
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http://dx.doi.org/10.1093/cercor/bhx260DOI Listing
November 2018

Rapid Detection of Abrin Toxin and Its Isoforms in Complex Matrices by Immuno-Extraction and Quantitative High Resolution Targeted Mass Spectrometry.

Anal Chem 2017 11 20;89(21):11719-11727. Epub 2017 Oct 20.

Service de Pharmacologie et Immunoanalyse (SPI), Laboratoire d'Etude du Métabolisme des Médicaments, CEA, INRA, Université Paris Saclay , F-91191 Gif-sur-Yvette cedex, France.

Abrin expressed by the tropical plant Abrus precatorius is highly dangerous with an estimated human lethal dose of 0.1-1 μg/kg body weight. Due to the potential misuse as a biothreat agent, abrin is in the focus of surveillance. Fast and reliable methods are therefore of great importance for early identification. Here, we have developed an innovative and rapid multiepitope immuno-mass spectrometry workflow which is capable of unambiguously differentiating abrin and its isoforms in complex matrices. Toxin-containing samples were incubated with magnetic beads coated with multiple abrin-specific antibodies, thereby concentrating and extracting all the isoforms. Using an ultrasonic bath for digestion enhancement, on-bead trypsin digestion was optimized to obtain efficient and reproducible peptide recovery in only 30 min. Improvements made to the workflow reduced total analysis time to less than 3 h. A large panel of common and isoform-specific peptides was monitored by multiplex LC-MS/MS through the parallel reaction monitoring mode on a quadrupole-Orbitrap high resolution mass spectrometer. Additionally, absolute quantification was accomplished by isotope dilution with labeled AQUA peptides. The newly established method was demonstrated as being sensitive and reproducible with quantification limits in the low ng/mL range in various food and clinical matrices for the isoforms of abrin and also the closely related, less toxic Abrus precatorius agglutinin. This method allows for the first time the rapid detection, differentiation, and simultaneous quantification of abrin and its isoforms by mass spectrometry.
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http://dx.doi.org/10.1021/acs.analchem.7b03189DOI Listing
November 2017

A simple and rapid LC-MS/MS method for therapeutic drug monitoring of cetuximab: a GPCO-UNICANCER proof of concept study in head-and-neck cancer patients.

Sci Rep 2017 06 2;7(1):2714. Epub 2017 Jun 2.

Medical Oncology Unit, La Timone University Hospital of Marseille, Marseille, France.

Administration of first-in-class anti-EGFR monoclonal antibody cetuximab is contingent upon extensive pharmacogenomic testing. However in addition to tumor genomics, drug exposure levels could play a critical, yet largely underestimated role, because several reports have demonstrated that cetuximab pharmacokinetic parameters, in particular clearance values, were associated with survival in patients. Here, we have developed an original bioanalytical method based upon the use of LC-MS/MS technology and a simplified sample preparation procedure to assay cetuximab in plasma samples from patients, thus meeting the requirements of standard Therapeutic Drug Monitoring in routine clinical practice. When tested prospectively in a pilot study in 25 head-and-neck cancer patients, this method showed that patients with clinical benefit had cetixumab residual concentrations higher than non-responding patients (i.e., 49 ± 16.3 µg/ml VS. 25.8 ± 17 µg/ml, p < 0.01 t test). Further ROC analysis showed that 33.8 µg/ml was the Cmin threshold predictive of response with an acceptable sensitivity (87%) and specificity (78%). Mass spectrometry-based therapeutic drug monitoring of cetuximab in head-and-neck cancer patients could therefore help to rapidly predict cetuximab efficacy and to adapt dosing if required.
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http://dx.doi.org/10.1038/s41598-017-02821-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5457398PMC
June 2017

Detection of Yersinia pestis in Complex Matrices by Intact Cell Immunocapture and Targeted Mass Spectrometry.

Methods Mol Biol 2017 ;1600:69-83

CEA, iBiTec-S, Service de Pharmacologie et d'Immunoanalyse, Bât. 136, 91191, Gif-sur-Yvette, France.

We describe an immunoaffinity-liquid chromatography-tandem mass spectrometry (immuno-LC-MS/MS) protocol for the direct (i.e., without prior culture), sensitive and specific detection of Yersinia pestis in complex matrices. Immunoaffinity enables isolation and concentration of intact bacterial cells from food and environmental samples. After protein extraction and digestion, suitable proteotypic peptides corresponding to three Y. pestis-specific protein markers (murine toxine, plasminogen activator and pesticin) are monitored by targeted LC-MS/MS using the selected reaction monitoring (SRM) mode. This immuno-LC-MS/MS assay has a limit of detection of 2 × 10 CFU/mL in milk or tap water, and 4.5 × 10 CFU in 10 mg of soil.
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http://dx.doi.org/10.1007/978-1-4939-6958-6_7DOI Listing
February 2018

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

In vivo dynamics of active edema and lethal factors during anthrax.

Sci Rep 2016 Mar 21;6:23346. Epub 2016 Mar 21.

Pathogénie des Toxi-Infections Bactériennes, Institut Pasteur, Paris, France.

Lethal and edema toxins are critical virulence factors of Bacillus anthracis. However, little is known about their in vivo dynamics of production during anthrax. In this study, we unraveled for the first time the in vivo kinetics of production of the toxin components EF (edema factor) and LF (lethal factor) during cutaneous infection with a wild-type toxinogenic encapsulated strain in immuno-competent mice. We stratified the asynchronous infection process into defined stages through bioluminescence imaging (BLI), while exploiting sensitive quantitative methods by measuring the enzymatic activity of LF and EF. LF was produced in high amounts, while EF amounts steadily increased during the infectious process. This led to high LF/EF ratios throughout the infection, with variations between 50 to a few thousands. In the bloodstream, the early detection of active LF and EF despite the absence of bacteria suggests that they may exert long distance effects. Infection with a strain deficient in the protective antigen toxin component enabled to address its role in the diffusion of LF and EF within the host. Our data provide a picture of the in vivo complexity of the infectious process.
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http://dx.doi.org/10.1038/srep23346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4800402PMC
March 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

Recommended Mass Spectrometry-Based Strategies to Identify Ricin-Containing Samples.

Toxins (Basel) 2015 Nov 25;7(12):4881-94. Epub 2015 Nov 25.

Centers for Disease Control and Prevention, 4770 Buford Hwy NE, Atlanta, GA 30341, USA.

Ricin is a protein toxin produced by the castor bean plant (Ricinus communis) together with a related protein known as R. communis agglutinin (RCA120). Mass spectrometric (MS) assays have the capacity to unambiguously identify ricin and to detect ricin's activity in samples with complex matrices. These qualitative and quantitative assays enable detection and differentiation of ricin from the less toxic RCA120 through determination of the amino acid sequence of the protein in question, and active ricin can be monitored by MS as the release of adenine from the depurination of a nucleic acid substrate. In this work, we describe the application of MS-based methods to detect, differentiate and quantify ricin and RCA120 in nine blinded samples supplied as part of the EQuATox proficiency test. Overall, MS-based assays successfully identified all samples containing ricin or RCA120 with the exception of the sample spiked with the lowest concentration (0.414 ng/mL). In fact, mass spectrometry was the most successful method for differentiation of ricin and RCA120 based on amino acid determination. Mass spectrometric methods were also successful at ranking the functional activities of the samples, successfully yielding semi-quantitative results. These results indicate that MS-based assays are excellent techniques to detect, differentiate, and quantify ricin and RCA120 in complex matrices.
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http://dx.doi.org/10.3390/toxins7124854DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4690104PMC
November 2015
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