Tristan Martineau (Presenter)
Université de Sherbrooke
Bio: Tristan Martineau obtained his bachelors’s degree in biochemistry in 2015. He is currently doing a master’s degree at the Université de Sherbrooke, in the Nuclear Radiation Sciences and Biomedical Imaging Program. Personally, he is also involved as a trainer in a “Ocean Rescue” endeavour, for which he has won numerous awards. It shows his dedication to help people. He is presently working on his project to develop a tandem mass spectrometry method for the simultaneous analysis of proteins related to podocyturia from urine samples collected on filter paper. He has given lectures at the Inter-Faculty Symposium at the Université de Sherbrooke. He will participate at the “Réseau de médecine génétique appliquée meeting in June, and the Faculty of Medicine and Health Sciences Symposium later on. He wants to investigate podocyturia in patients with Fabry disease, preeclampsia patients, and those with chronic kidney diseases. His goal is to provide better patient monitoring, establish normal values for podocyturia using mass spectrometry, and perform a technological transfer of the method to the clinical field. He is proactive and not afraid to try new paths in science to reach his goal. He constantly seeks solutions to the various issues arising while doing research. He is always aiming at robust results. He is dynamic and he works well with other students and research assistants. He loves to share his findings with colleagues during a research symposium, or other scientific and laboratory meetings. His goal is to develop an innovative method by the end of his master’s degree to investigate podocyturia in patients using a mass spectrometry approach, and thus facilitate the diagnosis, and offer a new powerful tool to monitor patients with renal diseases.
Authorship: Tristan Martineau (1), Michel Boutin (1), Christiane Auray-Blais (1)
(1) Université de Sherbrooke, Sherbrooke, QC
| Short Abstract Urinary podocytes are potential biomarkers for several kidney diseases. Current analytical techniques for podocyte evaluation are complex, tedious, and the interpretation of results is operator-dependent. The main objective of the project is to develop and validate a simple and robust tandem mass spectrometry method for the simultaneous analysis of specific proteins related to podocyturia using urine samples dried on filter paper in Fabry disease patients, women with preeclampsia, and patients with chronic kidney diseases. The method is based on the elution of urine from the filter paper and the production of specific tryptic peptides. The goals are to facilitate the diagnosis, provide better management, monitoring and long-term follow up of patients by transferring this method to the clinical field. |
Long Abstract
Introduction
The presence of urinary podocytes (podocyturia) is one of the signs of kidney abnormalities (1). Being essential in the glomeruli capillary sheets for the development of the primitive urine, podocytes ensure the retention of molecules of high molecular masses (> 60 kDa) in plasma by their intercellular-junctions (2). Currently, the measurement of proteinuria (defined by a concentration of > 30 mg protein/mmol creatinine in the urine) and the evaluation of glomerular filtration rate (< 60 mL/min/1.73 m2) are tools used to assess kidney damages (3). However, these tools are often late signs of a disease, which can eventually delay the administration of treatment due to a late diagnosis confirmation (4). The use of tandem mass spectrometry for the quantitation of proteins related to podocyturia represents a potential alternative for early diagnosis of patients affected with various genetic or non-genetic kidney diseases.
Objectives
Current techniques for the analysis of podocyturia are complex, tedious, time-consuming, and lead to marked variability in the interpretation of results. Therefore, the main objective of the project was to develop and validate a simple and robust tandem mass spectrometry method for the simultaneous analysis of specific proteins related to podocyturia, such as podocin, podocalyxin, synaptopodin, and nephrin from urine samples dried on a filter paper. The secondary objectives were to study podocyturia in three specific groups of patients: 1) women with preeclampsia; 2) patients with Fabry disease; and 3) patients with chronic kidney diseases. Control samples will be analyzed to establish normal values. The objectives of the study aim to facilitate the diagnosis, and better management of these patients.
Method
Urine samples are collected on Whatman GE-903 filter paper and dried at room temperature. A 5 cm filter paper disc is punched out for the analysis. A heavy labelled internal standard for each peptide analyzed is then added to the filter paper disc. The proteins adsorbed on the filter paper are eluted with water:acetonitrile by high speed orbital shaking for 1 hour and precipitated overnight with trichloroacetic acid (TCA). The resulting pellet is solubilized in 50 mM ammonium bicarbonate. The cysteine disulfide bridges are reduced with dithiothreitol (DTT) (30 min at 37oC), followed by the cysteine carbamidomethylation with iodoacetamide (20 min at room temperature in the dark). Thereafter, the sample is enzymatically digested with a trypsin:protein ratio between 1:25 to 1:100 to cleave proteins after the lysine and arginine residues, except if they are followed by a proline residue. This step is crucial to generate specific peptides from podocyte proteins which can afterwards be analyzed by tandem mass spectrometry. Tryptic peptides are purified by a solid-phase extraction procedure, using a mixed-mode strong cation-exchange (MCX) cartridge to remove salts and to reduce the matrix effect. After evaporation, samples are resuspended and injected into an Acquity I-class Ultra-Performance Liquid Chromatography system coupled to a Xevo TQ-S tandem mass spectrometer (UPLC-MS/MS from Waters Corps.). The analyses are performed by positive electrospray and characteristic peptide signals are acquired during a multiple reaction monitoring (MRM) experiment.
Results
The optimization of parameters was first performed with bovine serum albumin (BSA) due to its low cost. After several assays, our results show that the optimal condition for protein elution from the filter paper is obtained by using a water:acetonitrile (80:20) solution. The precipitation by TCA was done to concentrate all proteins from a high volume of elution. Primary results show that 0.1 µg/µL BSA can be precipitated overnight. The enzymatic digestion is more efficient in a small volume of 50 µL, with a trypsin:protein ratio of 1:25. However, owing to pellet solubilization issues, the enzymatic digestion must be performed in a minimum volume of 100 µL. A commercially available recombinant podocin was used to choose the most sensitive tryptic peptides and to optimize the UPLC-MS/MS method. We have been able to analyze 5 specific tryptic peptides from the podocin. After the MRM experiment, the doubly-charged precursor ion at m/z 671.4 with the singly-charged fragment ion at m/z 831.4 seem to be the most promising transitions for the absolute quantification of podocin. We are currently developing the UPLC-MS/MS method, optimizing the parameters to obtain the best transition signals in MRM for the podocin peptides, and working on the use of internal standards before validating the method. Analysis of patients dried urine filter paper samples from the three groups of patients will follow.
Conclusion
The ultimate goal of this project is to facilitate the diagnosis of patients affected with Fabry disease, preeclampsia and chronic kidney diseases, and to provide better management and monitoring over time. A technological transfer of this method to the clinical field is expected.
References & Acknowledgements:
1. W. Kriz et al., The podocyte’s response to stress: the enigma of foot process effacement, Am J Physiol Renal Physiol, 304 : F333-F347 (2013).
2. P. Jaakko et T. Karl, New insights into the role of podocytes in proteinuria, Nature reviews Nephrology. 5: 8, 463-468 (2009).
3. G. Eknoyan, N. Lameire et al., Kidney Disease: Improving Global Outcomes (KDIGO): Clinical practice guideline for the evaluation and management of chronic kidney disease, Kidney Int Suppl. 3:1 (2013).
4. I. Craici, S. Wagner, K. Bailey & al., Podocyturia predates proteinuria and clinical features of preeclampsia: Longitudinal prospective study, Hypertension; 61:1289-1296 (2013).
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