Synaptic dysfunction is a key pathogenic event in neurodegenerative and psychiatric diseases. Cerebrospinal fluid (CSF) biomarkers reflecting synaptic integrity would be highly valuable tools to monitor synaptic dysfunction directly in patients. We have recently developed mass spectrometry based CSF-assays for the pre-synaptic proteins SNAP-25 and synaptotagmin-1 and employed them in the study of neurodegenerative and psychiatric diseases.
The assays have undergone several improvements in sensitivity and through-put which will be discussed. Briefly, immunoprecipitation of SNAP-25, SYT-1 or SNAP-25/SYT-1 was performed on a KingFisher™ Duo or KingFisher™ Flex Purification System. Isotopically labeled standards were added and the enriched proteins were digested with trypsin or trypsin/Lys-C. MS based quantification was performed either with LC-PRM-MS on a Q Exactive/Ultimate 3000 system (Thermo Fisher Scientific) or LC-MRM-MS on a 6495 Triple Quadrupole LC/MS system (Agilent Technologies).
CSF SNAP-25 and SYT-1 concentrations in AD compared to controls were found to be significantly higher also at the very early stages of the disease. Interestingly, a longer soluble form of SNAP-25 provides improved differentiation between the different groups of patients and controls than all soluble SNAP-25. A study on patients with small cell lung cancer indicate that SNAP-25 is expressed both in presynaptic and postsynaptic parts of the neuron and that CSF levels of the different forms mirror different populations of the SNAP-25. The assay has also been used to monitor secretion of SNAP-25/SYT-1 during cortical neuronal differentiation of human induced pluripotent stem cells. Recently the combined SNAP-25/SYT-1 assay has been employed in a study of more than 2000 patients from several neurodegenerative diseases.
We conclude that IP-MS can be employed in clinical studies of neurodegenerative and psychiatric diseases with sufficient number of patients to reveal biologically relevant results.