EuPA Young Investigator Award 2013

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more recently, lipidomics and glycomics, and (iii) quantitative analyses of proteins using triple ... mass spectrometers for targeted or untargeted approaches.
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EuPA Young Investigator Award 2013夽 Absolute quantification of CSF Tau isoforms by a simple and cost-effective purification and µLC-MS/HRMS I presently work as post doctoral researcher in the Drug Metabolism Research Laboratory (LEMM, Laboratoire d’Etude du Métabolisme des Medicaments). LEMM belongs to the CEA (Commissariat à l’Energie Atomique, French Atomic Commission) and is located at Saclay in the south of Paris. The laboratory, headed by Dr. Christophe Junot, develops analytical and cellular tools to support drug development and biomarker discovery. One of the fields of excellence is the use of mass spectrometry (MS) to investigate biological systems. To this end, the laboratory develops and implements (i) targeted approaches for the elucidation of drug metabolism, intracellular pharmacokinetics, (ii) global approaches for metabolomics, and more recently, lipidomics and glycomics, and (iii) quantitative analyses of proteins using triple quadrupole and high resolution instruments, applied to, protein drugs, biological warfare (toxins, bacteria) and biomarkers of neurodegenerative diseases, area in which I conduct my researches. LEMM is equipped with various mass spectrometers for targeted or untargeted approaches (triple quadrupoles, Orbitrap, Maldi-TOF, Q-TOF, Q-Orbitrap). My research activity is supervised by Dr. François Becher with partnership with the Clinical Proteomics Platform of Montpellier University Hospital headed by Pr. Sylvain Lehmann in the frame of French Hospital Clinical Research Program (PHRC). We are focused on MS strategies development to monitor key biomarkers of Alzheimer disease (AD) in cerebrospinal fluid (CSF) and provide alternative information to supplement immunoassay analyses. We are particularly interested in Tau protein involved in numerous neurological disorders or “tauopathies” including AD. However, detection of Tau in CSF by MS is challenging due to protein heterogeneity (six splicing variants, many

described truncated forms and extensive post-translational modifications) and very low concentration expected in CSF (100 to 1000 pg/ml). To overcome this challenge, we have developed an original extraction at the protein level based on Tau specific physico-chemical properties. Without antibodies requirement, this extraction step allows an effective depletion of major CSF proteins and Tau concentration prior digestion and peptides analysis by MS. To achieve sensitive detection required to quantify CSF Tau peptides, we have also developed a LC-HRMS platform based on Q-Orbitrap mass spectrometer. Thanks to scheduled Parallel Reaction Monitoring (PRM), we are finally able to target 26 Tau peptides whose 22 are successfully detected and quantified in CSF extracts. I have presented these findings at an oral communication during the 61st ASMS conference in Minneapolis. These results have been also the subject of two oral communications by Dr. Christophe Hirtz at the 12th HUPO and 6th CTAD congresses. We have performed absolute quantification of all detected CSF Tau peptides using recombinant labeled Tau and compared our results obtained for t-Tau ELISA on 50 samples from a clinical cohort. Quantification results allow peptides stoichiometry study and inferring major CSF Tau proteoforms. MS data appears more informative and with higher diagnosis utility than t-Tau ELISA to discriminate AD patients from other neurological diseases, especially in the context of shared high CSF tau concentration. Nicolas R. Barthélemy, CEA, iBiTec-S, Service de Pharmacologie et d’Immunoanalyse, Laboratoire d’Etude du Métabolisme des Médicaments, Gif-sur-Yvette, France

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Fig. Superimposed extracted ion chromatograms of 22 Tau peptides obtained from a digest of CSF extract analyzed by scheduled PRM. Red signals correspond to exons inserts specific peptides. Their absolute quantification allows assessment of Tau isoforms in CSF extracts.



Corresponded by Natacha Turck on behalf of the EuPA Conference and Communications Committee (via [email protected])

http://dx.doi.org/10.1016/j.euprot.2013.12.003