Gaël Cobraiville

Development of a nano-liquid chromatography on chip tandem mass spectrometry method for high-sensitivity hepcidin quantitation

Microfluidic LC systems present undeniable advantages over classical LC in terms of sensitivity. Hepcidin, a peptide marker of clinical disorders linked to iron metabolism, was used as model to demonstrate peptide quantification potentialities of LC-chip coupled to a nanoelectrospray source ion trap mass spectrometer in an aqueous sample. First, stable isotope labelled hepcidin was chosen as internal standard and gradient as well as sample compositions were optimised using design of experiments as development tool. The method was then prevalidated using accuracy profiles in order to select the most appropriate response function and to confirm the ability of the technique to quantify low hepcidin concentration. A reliable and very sensitive quantitation method was finally obtained using this integrated microfluidic technology. Indeed, good results with respect to accuracy, trueness and precision were achieved, as well as a very low limit of quantitation (0.07 ng/ml). Method suitability of nano-LC on chip tandem mass spectrometry for hepcidin quantitation was also demonstrated in complex media such as human plasma.

Discovery and biochemical characterisation of four novel biomarkers for osteoarthritis

Objective Knee osteoarthritis (OA) is a heterogeneous, complex joint pathology of unknown aetiology. Biomarkers have been widely used to investigate OA but currently available biomarkers lack specificity and sensitivity. Therefore, novel biomarkers are needed to better understand the pathophysiological processes of OA initiation and progression. Methods Surface enhanced laser desorption/ionisation-time of flight-mass spectrometry proteomic technique was used to analyse protein expression levels in 284 serum samples from patients with knee OA classified according to Kellgren and Lawrence (K&L) score (0–4). OA serum samples were also compared to serum samples provided by healthy individuals (negative control subjects; NC; n=36) and rheumatoid arthritis (RA) patients (n=25). Proteins that gave similar signal in all K&L groups of OA patients were ignored, whereas proteins with increased or decreased levels of expression were selected for further studies. Results Two proteins were found to be expressed at higher levels in sera of OA patients at all four K&L scores compared to NC and RA, and were identified as V65 vitronectin fragment and C3fpeptide. Of the two remaining proteins, one showed increased expression (unknown protein at m/z of 3762) and the other (identified as connective tissue-activating peptide III protein) was decreased in K&L scores >2 subsets compared to NC, RA and K&L scores 0 or 1 subsets. Conclusion The authors detected four unexpected biomarkers (V65 vitronectin fragment, C3f peptide, CTAP-III and m/z 3762 protein) that could be relevant in the pathophysiological process of OA as having significant correlation with parameters reflecting local inflammation and bone remodelling, as well as decrease in cartilage turnover.

Acute-phase serum amyloid a in osteoarthritis: regulatory mechanism and proinflammatory properties.

Objective To determine if serum amyloid A (A-SAA) could be detected in human osteoarthritic (OA) joints and further clarify if high A-SAA level in joints result from a local production or from a diffusion process from abnormally elevated plasma concentration. Regulatory mechanism of A-SAA expression and its pro-inflammatory properties were also investigated. Methods A-SAA levels in serum and synovial fluid of OA (n = 29) and rheumatoid arthritis (RA) (n = 27) patients were measured and compared to matched-healthy volunteers (HV) (n = 35). In vitro cell cultures were performed on primary joint cells provided from osteoarthritis patients. Regulatory mechanisms were studied using Western-blotting, ELISA and lentiviral transfections. Results A-SAA was statistically increased in OA plasma patients compared to HV. Moreover, A-SAA level in OA plasma and synovial fluid increased with the Kellgren & Lauwrence grade. For all OA and RA patients, A-SAA plasma level was higher and highly correlated with its corresponding level in the synovial fluid, therefore supporting that A-SAA was mainly due to the passive diffusion process from blood into the joint cavity. However, A-SAA expression was also observed in vitro under corticosteroid treatment and/or under IL-1beta stimuli. A-SAA expression was down-regulated by PPAR-γ agonists (genistein and rosiglitazone) and up-regulated by TGF-β1 through Alk1 (Smad1/5) pathway. RhSAA induced proinflammatory cytokines (IL-6, IL-8, GRO-α and MCP-1) and metalloproteinases (MMP-1, MMP-3 and MMP-13) expression in FLS and chondrocytes, which expression was downregulated by TAK242, a specific TLR4 inhibitor. Conclusion Systemic or local A-SAA expression inside OA joint cavity may play a key role in inflammatory process seen in osteoarthritis, which could be counteracted by TLR4 inhibition.

Hepcidin determination in dried blood by microfluidic LC–MS/MS: comparison of DBS and volumetric absorptive microsampling for matrix effect and recovery

BACKGROUND Dried blood analysis experiences a growing interest due to practical, ethical and financial advantages compared with classical wet plasma or serum analysis. Besides classical DBS, new alternatives are commercialized as volumetric absorptive microsampling (VAMS) that are expected to overcome hematocrit influence. RESULTS The feasibility of hepcidin (a peptide hormone) extraction and determination from DBS and VAMS blood sampling was investigated. Experimental design was used to determine the optimal extraction conditions. Matrix effect and extraction recovery were studied and a special attention was paid to phospholipid removal. CONCLUSION The data suggest that the combination of VAMS and phospholipid removal plates provides low matrix effect and high sensitivity, and constitutes an easy and promising protocol for hepcidin analysis.

Whole blood microsampling for the quantitation of estetrol without derivatization by liquid chromatography-tandem mass spectrometry

&NA; Quantitative bioanalysis and especially pharmacokinetic studies are challenging since only low volumes of biological material are available and low concentrations (ng/ml) are often expected. In this context, volumetric absorptive microsampling (VAMS) devices were developed to accurately collect 10 or 20 &mgr;l of whole blood from tested subjects. In this study, we present the development and validation of ultra‐high performance liquid chromatography coupled to tandem mass spectrometry method after VAMS sampling for the quantitation of estetrol (E4), a potentially new medicine for hormone replacement, contraception and osteoporosis therapies. Interestingly, a very simple sample preparation procedure was developed without any derivatization step. Even if lack of sensitivity is a common consideration when using negative ionization mode, we demonstrated in this work that an excellent sensitivity could be reached by carefully optimizing the nature and concentration of the mobile phase additive. After the optimization of every experimental parameter, the stability, selectivity, trueness, precision and accuracy of the final method were successfully demonstrated. In addition, the excellent performances of the method were confirmed by two independent proof‐of‐concept pharmacokinetic studies of E4 after VAMS collection in a murine model. Graphical abstract Figure. No caption available. HighlightsA UHPLC–MS/MS method is proposed for the analysis of estetrol without derivatization.NH4F as LC additive is used to reach needed sensitivity in negative mode.An innovative microsampling technique is used to collect 10 &mgr;l of blood from mice.The method was successfully validated for the quantitation of estetrol.A pharmacokinetic study is performed as proof‐of‐concept.

Apolipoprotein-A1 as a Damage-Associated Molecular Patterns Protein in Osteoarthritis: Ex Vivo and In Vitro Pro-Inflammatory Properties

Osteoarthritis (OA) is associated with a local inflammatory process. Dyslipidemia is known to be an underlying cause for the development of OA. Therefore, lipid and inflammatory levels were quantified ex vivo in blood and synovial fluid of OA patients (n=29) and compared to those of rheumatoid arthritis (RA) patients (n=27) or healthy volunteers (HV) (n=35). The role of apolipoprotein A-I (ApoA1) was investigated in vitro on inflammatory parameters using human joint cells isolated from cartilage and synovial membrane obtained from OA patients after joint replacement. Cells were stimulated with ApoA1 in the presence or not of serum amyloid A (SAA) protein and/or lipoproteins (LDL and HDL) at physiological concentration observed in OA synovial fluid. In our ex vivo study, ApoA1, LDL-C and total cholesterol levels were strongly correlated to each other inside the OA joint cavity whereas same levels were not or weakly correlated to their corresponding serum levels. In OA synovial fluid, ApoA1 was not as strongly correlated to HDL as observed in OA serum or in RA synovial fluid, suggesting a dissociative level between ApoA1 and HDL in OA synovial fluid. In vitro, ApoA1 induced IL-6, MMP-1 and MMP-3 expression by primary chondrocytes and fibroblast-like synoviocytes through TLR4 receptor. HDL and LDL attenuated joint inflammatory response induced by ApoA1 and SAA in a ratio dependent manner. In conclusion, a dysregulated lipidic profile in the synovial fluid of OA patients was observed and was correlated with inflammatory parameters in the OA joint cavity. Pro-inflammatory properties of ApoA1 were confirmed in vitro.

Biomarkers of inflammation and innate immunity in atrophic nonunion fracture

BackgroundNonunion is a failure of healing following a bone fracture. Its physiopathology remains partially unclear and the discovery of new mediators could promote the understanding of bone healing.MethodsThirty-three atrophic nonunion (NU) patients that failed to demonstrate any radiographic improvement for 6 consecutive months were recruited for providing serum samples. Thirty-five healthy volunteers (HV) served as the control group. Proteomics studies were performed using SELDI-TOF–MS and 2D-DIGE approaches, associated or not with Proteominer® preprocessing, to highlight biomarkers specific to atrophic nonunion pathology. Peak intensities were analyzed by two statistical approaches, a nonparametric Mann–Whitney U tests (univariate approach) and a machine-learning algorithm called extra-trees (multivariate approach). Validation of highlighted biomarkers was performed by alternative approaches such as microfluidic LC–MS/MS, nephelometry, western blotting or ELISA assays.ResultsFrom the 35 HV and 33 NU crude serum samples and Proteominer® eluates, 136 spectra were collected by SELDI-TOF–MS using CM10 and IMAC-Cu2+ ProteinChip arrays, and 665 peaks were integrated for extra-trees multivariate analysis. Accordingly, seven biomarkers and several variants were identified as potential NU biomarkers. Their levels of expression were found to be down- or up-regulated in serum of HV vs NU. These biomarkers are inter-α-trypsin inhibitor H4, hepcidin, S100A8, S100A9, glycated hemoglobin β subunit, PACAP related peptide, complement C3 α-chain. 2D-DIGE experiment allowed to detect 14 biomarkers as being down- or up-regulated in serum of HV vs NU including a cleaved fragment of apolipoprotein A-IV, apolipoprotein E, complement C3 and C6. Several biomarkers such as hepcidin, complement C6, S100A9, apolipoprotein E, complement C3 and C4 were confirmed by an alternative approach as being up-regulated in serum of NU patients compared to HV controls.ConclusionTwo proteomics approaches were used to identify new biomarkers up- or down-regulated in the nonunion pathology, which are involved in bone turn-over, inflammation, innate immunity, glycation and lipid metabolisms. High expression of hepcidin or S100A8/S100A9 by myeloid cells and the presence of advanced glycation end products and complement factors could be the result of a longstanding inflammatory process. Blocking macrophage activation and/or TLR4 receptor could accelerate healing of fractured bone in at-risk patients.

Impact of injection solvent composition on protein identification in column-switching chip-liquid chromatography/mass spectrometry

In shotgun proteomics, the gold standard technique is reversed-phase liquid chromatography coupled to mass spectrometry. Many researches have been carried out to study the effects on identification performances of chromatographic parameters such as the stationary phase and column dimensions, mobile phase composition and flow rate, as well as the gradient slope and length. However, little attention is usually paid to the injection solvent composition. In this study, we investigated the effect of the injection solvent on protein identification parameters (number of distinct peptides, amino acid coverage and MS/MS search score) as well as sensitivity. Tryptic peptides from six different proteins, covering a wide range of physicochemical properties, were employed as training set. Design of experiments was employed as a tool to highlight the factors related to the composition of the injection solvent that significantly influenced the obtained results. Optimal results for the training set were applied to analysis of more complex samples. The experiments pointed out optimising the composition of the injection solvent had a strong beneficial effect on all the considered responses. On the basis of these results, an approach to determine optimal conditions was proposed to maximise the protein identification performances and detection sensitivity.

Validation of a new method by nano-liquid chromatography on chip tandem mass spectrometry for combined quantitation of C3f and the V65 vitronectin fragment as biomarkers of diagnosis and severity of osteoarthritis.

Microfluidic liquid chromatography coupled to a nanoelectrospray source ion trap mass spectrometry was used for the absolute and simultaneous quantitation of C3f and the V65 vitronectin fragment in serum. The method was first carefully optimized and then validated in serum biological matrix. Stable isotopes for the two biomarkers of interest were used as stable isotope labeled peptide standards. A weighted 1/x2 quadratic regression for C3f and a weighted 1/x quadratic regression for the V65 vitronectin peptide were selected for calibration curves. Trueness (with a relative bias <10%), precision (repeatability and intermediate precision <15%) and accuracy (risk <15%) of the method were successfully demonstrated. The linearity of results was validated in the concentration range of 2.5-200ng/mL for C3f and 2.5-100ng/mL for the V65 vitronectin fragment. Serum samples (n=147) classified in 7 groups [(healthy volunteers, OA with 5 grades of severity and rheumatoid arthritis (RA) patients] were analyzed with our new quantitative method. Our data confirm that C3f and the V65 vitronectin fragment are biomarkers of OA severity, but also that C3f fragment is further related to OA severity whereas the V65 vitronectin fragment is more related to early OA detection.

Comparison of nanofluidic and ultra-high performance liquid chromatography-tandem mass spectrometry for high sensitive pharmacokinetic studies of estrogens starting from whole blood microsampling

Pharmacokinetic (PK) studies on small animals are challenging as only small volumes of samples are available, in which the analyte is present at low concentration in a complex matrix. In this context, the use of miniaturized analytical techniques may provide undeniable advantages in terms of sensitivity, sample and solvent consumption compared to the reference UHPLC-MS/MS methods In this study, we present the development of a nanofluidic-LC-MS/MS method to analyze two model analytes of therapeutic interest, namely estradiol (E2) and estetrol (E4) after microsampling with volumetric absorptive microsampling (VAMS) devices, an innovative sampling technique to collect small volumes of whole blood. The nanofluidic LC-MS/MS method was developed using an experimental design to find the optimal conditions to analyze both E2 and E4 with the highest sensitivity. Subsequently, the optimized method was validated according to ICH guidelines and compared to a previously developed UHPLC-MS/MS method. A limit of quantitation of 50pg/ml was reached with the LC-chip method, which is 50 times better than UHPLC-MS/MS. Both methods were then critically evaluated from the analytical and operational points of view. Finally, the quantitation of estrogens after whole blood microsampling was compared with the results obtained with the corresponding plasma samples.