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Nano LC-MS/MS for the monitoring of hippocampal neuropeptides possibly involved in limbic epilepsy.
Book Contribution - Book Chapter Conference Contribution
Introduction
Neuropeptides are an important and diverse class of neuronal signalling molecules. They seem to play an important role when the central nervous system is challenged, as during epileptic seizures [1,2]. In order to further characterise the neuropeptides involved in the physiopathology of epilepsy, it is essential to monitor their concentration dynamics in the brain. Quantification of neuropeptides in dialysates is challenging due to their low extracellular concentrations (low pM range), their low microdialysis efficiencies, the need for acceptable temporal resolution, the small sample volumes, the complexity of the matrix and the tendency of peptides to stick to glass and polymeric materials [3]. The quantification of neuropeptides in dialysates therefore necessitates the use of very sensitive nano LC-MS/MS methods. In this study the feasibility of quantifying eight neuropeptides, possibly involved in epilepsy, is investigated.
Methods
Microdialysis is used as in vivo sampling technique to investigate the extracellular concentration of neuropeptides in the hippocampus, i.e. the brain region which is the most vulnerable for seizures. Samples (10 ?l) were concentrated at 30 ?l/min on a C18 precolumn (5 mm x 300 ?m id, 5 ?m, PepmapTM, LC Packings-Dionex, The Netherlands) and then back-flushed via a SwitchosTM micro switching module (LC Packings-Dionex) onto a nano C18 column (15 cm x 75 ?m id, PepmapTM, LC Packings-Dionex) at 300 nl/min. Gradient elution was performed using solvent A (water/ACN/FA 98/2/0,1 V/V/V) and solvent B (water/ACN/FA 20/80/0,1 V). The linear LC gradient for separation was 100% A for 5 min (loading period), 0-100% B in 8 min, 100% B for 5 min, 100-0% B in 0,5 min and 100% A for 16,5 min. The outlet of the nano LC system was hyphenated to the nanosource of a Quattro PremierTM triple quadrupole MS (Waters, UK) using distal coated Picotip® nanospray emitters (10 ?m id, New Objective, USA). Detection was performed in ESI positive mode and quantification was executed in selected reaction monitoring (SRM) mode.
Results and discussion
The following neuropeptides were selected for screening, based on known anticonvulsant action and/or their presence in the hippocampus: bradykinin, dynorphin A (1-13), galanin, ghrelin, neuromedin B, neuromedin N, neuropeptide Y and neurotensin.
First, the compound-specific MS/MS parameters of the different peptides are optimised by infusing them directly into the nanosource. The following parameters were obtained: precursor and product ion, cone voltage and collision energy. 2 Injection onto nano LC-MS/MS. All peptides were injected onto the nano LC-MS/MS system using the above described generic gradient method. During this screening, the retention time, peak width and limit of detection were determined. Further optimisation of the LC method is necessary for ghrelin and dynorphin A (1-13) to decrease the peak width. Galanin and neuropeptide Y exhibited good peak shapes when high concentrations were injected (nM-range). However, at lower concentrations (pM-range) these peptides could not me measured. The limit of detection for bradykinin, neurotensin, neuromedin N and neuromedin B was found to be 7.0; 2.6; 1.5 and 23 pM respectively. The washing method needs to be optimised, to minimise the carry-over and to consequently increase the sensitivity of the method. 3 Matrix effects. Because microdialysis samples contain a large amount of salts and ESI is very sensitive to matrix effects, the influence of the matrix is studied. The responses in modified Ringer's solution and in hippocampal dialysate increased respectively with a factor three and six. To correct for these matrix effects, the use of an internal standard is highly recommended. 4 Screening of basal microdialysis samples. A screening was performed in basal microdialysis samples from the hippocampus of the rat. Neuromedin N, neurotensin and ghrelin were detected in a hippocampal dialysate. 5 Neuropeptide changes during seizures. Afterwards, an in vivo microdialysis experiment was performed, in which the basal and picrotoxin-stimulated release of neuromedin N, neurotensin and ghrelin were compared. The concentration of neuromedin N increased significantly during seizures and afterwards returned to baseline. 6 Stability. Finally, fresh collected samples and samples stored at 4°C were compared to investigate the stability of peptides in dialysates. The response of neuromedin N decreased with approximately 50 % after 4 hours of storage at 4°C. In order to avoid degradation of the peptides, dialysates need to be analysed immediately after collection. Conclusion: These preliminary results show that, after further optimisation and validation, the quantification of the selected neuropeptides with nano LC-MS/MS is feasible. This will help to characterise the neuropeptide systems involved in the pathophysiology of TLE.
Neuropeptides are an important and diverse class of neuronal signalling molecules. They seem to play an important role when the central nervous system is challenged, as during epileptic seizures [1,2]. In order to further characterise the neuropeptides involved in the physiopathology of epilepsy, it is essential to monitor their concentration dynamics in the brain. Quantification of neuropeptides in dialysates is challenging due to their low extracellular concentrations (low pM range), their low microdialysis efficiencies, the need for acceptable temporal resolution, the small sample volumes, the complexity of the matrix and the tendency of peptides to stick to glass and polymeric materials [3]. The quantification of neuropeptides in dialysates therefore necessitates the use of very sensitive nano LC-MS/MS methods. In this study the feasibility of quantifying eight neuropeptides, possibly involved in epilepsy, is investigated.
Methods
Microdialysis is used as in vivo sampling technique to investigate the extracellular concentration of neuropeptides in the hippocampus, i.e. the brain region which is the most vulnerable for seizures. Samples (10 ?l) were concentrated at 30 ?l/min on a C18 precolumn (5 mm x 300 ?m id, 5 ?m, PepmapTM, LC Packings-Dionex, The Netherlands) and then back-flushed via a SwitchosTM micro switching module (LC Packings-Dionex) onto a nano C18 column (15 cm x 75 ?m id, PepmapTM, LC Packings-Dionex) at 300 nl/min. Gradient elution was performed using solvent A (water/ACN/FA 98/2/0,1 V/V/V) and solvent B (water/ACN/FA 20/80/0,1 V). The linear LC gradient for separation was 100% A for 5 min (loading period), 0-100% B in 8 min, 100% B for 5 min, 100-0% B in 0,5 min and 100% A for 16,5 min. The outlet of the nano LC system was hyphenated to the nanosource of a Quattro PremierTM triple quadrupole MS (Waters, UK) using distal coated Picotip® nanospray emitters (10 ?m id, New Objective, USA). Detection was performed in ESI positive mode and quantification was executed in selected reaction monitoring (SRM) mode.
Results and discussion
The following neuropeptides were selected for screening, based on known anticonvulsant action and/or their presence in the hippocampus: bradykinin, dynorphin A (1-13), galanin, ghrelin, neuromedin B, neuromedin N, neuropeptide Y and neurotensin.
First, the compound-specific MS/MS parameters of the different peptides are optimised by infusing them directly into the nanosource. The following parameters were obtained: precursor and product ion, cone voltage and collision energy. 2 Injection onto nano LC-MS/MS. All peptides were injected onto the nano LC-MS/MS system using the above described generic gradient method. During this screening, the retention time, peak width and limit of detection were determined. Further optimisation of the LC method is necessary for ghrelin and dynorphin A (1-13) to decrease the peak width. Galanin and neuropeptide Y exhibited good peak shapes when high concentrations were injected (nM-range). However, at lower concentrations (pM-range) these peptides could not me measured. The limit of detection for bradykinin, neurotensin, neuromedin N and neuromedin B was found to be 7.0; 2.6; 1.5 and 23 pM respectively. The washing method needs to be optimised, to minimise the carry-over and to consequently increase the sensitivity of the method. 3 Matrix effects. Because microdialysis samples contain a large amount of salts and ESI is very sensitive to matrix effects, the influence of the matrix is studied. The responses in modified Ringer's solution and in hippocampal dialysate increased respectively with a factor three and six. To correct for these matrix effects, the use of an internal standard is highly recommended. 4 Screening of basal microdialysis samples. A screening was performed in basal microdialysis samples from the hippocampus of the rat. Neuromedin N, neurotensin and ghrelin were detected in a hippocampal dialysate. 5 Neuropeptide changes during seizures. Afterwards, an in vivo microdialysis experiment was performed, in which the basal and picrotoxin-stimulated release of neuromedin N, neurotensin and ghrelin were compared. The concentration of neuromedin N increased significantly during seizures and afterwards returned to baseline. 6 Stability. Finally, fresh collected samples and samples stored at 4°C were compared to investigate the stability of peptides in dialysates. The response of neuromedin N decreased with approximately 50 % after 4 hours of storage at 4°C. In order to avoid degradation of the peptides, dialysates need to be analysed immediately after collection. Conclusion: These preliminary results show that, after further optimisation and validation, the quantification of the selected neuropeptides with nano LC-MS/MS is feasible. This will help to characterise the neuropeptide systems involved in the pathophysiology of TLE.
Book: Belgian Society for Neuroscience
Series: Belgian Society for Neuroscience
Publication year:2011
Keywords:Nano LC-MS/MS, Neuropeptides, Epilepsy