Evaluation of SP3 for antibody-free quantification of tau in CSF mimic and brain by mass spectrometry.

Tubulin-associated unit (tau) has an important role in the pathogenesis and the diagnosis of Alzheimer's disease (AD) and other tauopathies. In view of the diversity of tau proteoforms, antibody-free methods represent a good approach for unbiased quantification. We adapted and evaluated the single-pot, solid-phase-enhanced sample-preparation (SP3) protocol for antibody-free extraction of the tau protein in cerebro-spinal fluid (CSF) mimic and in human brain. A total of 13 non-modified peptides were quantified by high-resolution mass spectrometry (HRMS) after digestion of tau by trypsin. We significantly improved the basic SP3 protocol by carefully optimizing the organic solvents and incubation time for tau binding, as well as the digestion step for the release directly from the SP3 beads of the 13 tau peptides. These optimizations proved to be primarily beneficial for the most hydrophilic tau peptides, increasing the sequence coverage of recombinant tau. Mean recovery in CSF mimic of the 13 non-modified peptides was of 53%, with LODs ranging from 0.75 to 10 ng/mL. Next, we tested the optimized SP3 protocol on pathological tau extracted from the soluble fraction from an AD brain sample (middle frontal gyrus). We could successfully identify and quantify biologically relevant tau peptides including representative peptides of two isoforms and two phospho-peptides (pTau217 and pTau181).

High-throughput untargeted screening of biotherapeutic macromolecules in equine plasma by UHPLC-HRMS/MS: Application to monoclonal antibodies and Fc-fusion proteins for doping control.

Many innovative biotherapeutics have been marketed in the last decade. Monoclonal antibodies (mAbs) and Fc-fusion proteins (Fc-proteins) have been developed for the treatment of diverse diseases (cancer, autoimmune diseases, and inflammatory disorders) and now represent an important part of targeted therapies. However, the ready availability of such biomolecules, sometimes characterized by their anabolic, anti-inflammatory, or erythropoiesis-stimulating properties, raises concerns about their potential misuse as performance enhancers for human and animal athletes. In equine doping control laboratories, a method has been reported to detect the administration of a specific human biotherapeutic in equine plasma; but no high-throughput method has been described for the screening without any a priori knowledge of human or murine biotherapeutic. In this context, a new broad-spectrum screening method involving UHPLC-HRMS/MS has been developed for the untargeted analysis of murine or human mAbs and related macromolecules in equine plasma. This approach, consisting of a "pellet digestion" strategy performed in a 96-well plate, demonstrates reliable performances at low concentrations (pmol/mL range) with high-throughput capability (≈100 samples/day). Targeting species-specific proteotypic peptides located within the constant parts of mAbs enables the "universal" detection of human biotherapeutics only by monitoring 10 peptides. As proof of principle, this strategy successfully detected different biotherapeutics in spiked plasma samples, and allowed, for the first time, the detection of a human mAb up to 10 days after a 0.12 mg/kg administration to a horse. This development will expand the analytical capabilities of horse doping control laboratories towards protein-based biotherapeutics with adequate sensitivity, throughput, and cost-effectiveness.

A large travel-associated outbreak of iatrogenic botulism in four European countries following intragastric botulinum neurotoxin injections for weight reduction, Türkiye, February to March 2023.

In March 2023, 34 associated cases of iatrogenic botulism were detected in Germany (30 cases), Switzerland (two cases), Austria (one case), and France (one case). An alert was rapidly disseminated via European Union networks and communication platforms (Food- and Waterborne Diseases and Zoonoses Network, EpiPulse, Early Warning and Response System) and the International Health Regulation mechanism; the outbreak was investigated in a European collaboration. We traced sources of the botulism outbreak to treatment of weight loss in Türkiye, involving intragastric injections of botulinum neurotoxin. Cases were traced using a list of patients who had received this treatment. Laboratory investigations of the first 12 German cases confirmed nine cases. The application of innovative and highly sensitive endopeptidase assays was necessary to detect minute traces of botulinum neurotoxin in patient sera. The botulism notification requirement for physicians was essential to detect this outbreak in Germany. The surveillance case definition of botulism should be revisited and inclusion of cases of iatrogenic botulism should be considered as these cases might lack standard laboratory confirmation yet warrant public health action. Any potential risks associated with the use of botulinum neurotoxins in medical procedures need to be carefully balanced with the expected benefits of the procedure.

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

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.

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

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.

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

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 Staphylococcus aureus 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.

Differentiation, Quantification and Identification of Abrin and Abrus precatorius Agglutinin.

Abrin, the toxic lectin from the rosary pea plant Abrus precatorius, 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 Abrus precatorius agglutinin or the homologous toxin ricin from Ricinus communis are sparse. To address this gap, a panel of highly specific monoclonal antibodies was generated against abrin and the related Abrus precatorius agglutinin. These antibodies were used to establish two sandwich ELISAs to preferentially detect abrin or A. precatorius agglutinin (limit of detection 22 pg/mL for abrin; 35 pg/mL for A. precatorius 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 A. precatorius.

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

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.

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

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.

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

Ricin, a highly toxic protein from Ricinus communis, 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.

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.

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 × 103 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.

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

The toxin abrin found in the seeds of Abrus precatorius 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.

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

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.

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

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.

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

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.

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

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.

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

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.

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.

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.

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

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-1 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.

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

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 (Cmin,D7 ) 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.