Liquid Chromatography-Selected Reaction Monitoring/Mass Spectrometry (LC-SRM/MS) is a robust and high-throughput technique useful for selective and sensitive detection of molecules smaller than 1000 Da. The key advantages of using highly sensitive liquid chromatography interfaced to a triple-stage quadrupole mass spectrometer is that it dramatically reduces assay development times for target proteins and has all the necessary features for small molecule applications. It is especially useful in the clinical and pharmaceutical setting for the quantitation of low-abundance proteins in limited samples.1 The LC-SRM/MS method employs the principle of surrogacy wherein a proteolytic peptide produced from the enzymatic digestion of a protein is used to infer the concentration of the protein, assuming a 1:1 molar ratio between the peptide and the protein.
The LC-SRM/MS method has emerged as a valuable tool to examine synaptic trafficking events in postmortem brain tissue, thereby enabling scientists to further investigate the etiology of neuropsychiatric disorders. Neuropsychiatric illnesses are believed to result from aberrant synaptic signaling, which involves a multitude of proteins that are clustered in a microdomain-specific manner. Exploring the proteomes of synaptic microdomains in postmortem brain tissue could therefore potentially unlock the mystery behind disorders such as autism, depression, schizophrenia, etc., and facilitate discovery of novel therapies to these conditions. Thus, it is essential to have the ability to assess protein expression and trafficking events in postmortem brain tissue with high precision and accuracy.
MacDonald et al.,2 have successfully used LC-SRM/MS to quantify synaptic proteins in subcellular fractions prepared from postmortem human brain tissue using a stable-isotope labeled neuronal proteome internal standard. By using protein standards generated from stable isotope labeling in mammals (SILAM) mouse brain, the group carried out multiplexed targeted quantitative analysis of synaptic protein trafficking in mammalian brain samples. This method represents a significant advance over existing methodologies for quantifying proteins that use either synthetic stable isotope-labeled peptide standards or isobaric tags for relative and absolute quantitation in terms of sensitivity, reproducibility, and cost-effectiveness. The group reported quantification of 189 neuronal proteins using the TSQ Vantage triple stage quadrupole mass spectrometer (Thermo Scientific) interfaced to a 2Dnano LC and a CaptiveSpray source.2 Further, bioinformatic analyses indicate that this method is capable of quantifying thousands of additional neuronal proteins in many model systems.
A significant discovery reported by the group is that synaptic proteins are enriched in microdomains in a function- and family-specific manner.2 For example, the postsynaptic density (PSD) fraction is highly enriched in kinases and scaffolding proteins, such as PSD-95, which are responsible for the amplification and propagation of neurotransmitter signals in the PSD. The synaptic vesicles, on the other hand, are highly enriched for soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complex proteins, responsible for fusing vesicles to the presynaptic membrane for neurotransmitter release. This groundbreaking work demonstrates that biochemical fractionation combined with liquid-chromatography-SRM-based mass spectrometry and SILAM-brain internal standards is a powerful tool for investigating the protein composition of synaptic microdomains in the postmortem brain tissue. This approach has broad applications for neuropathological studies in humans and other model systems.
References
1. Rauh, M. (2012) ‘LC-MS/MS for protein and peptide quantification in clinical chemistry‘ Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences, 883-884, (pp. 59-67)
2. MacDonald, M., et al. (2012) ‘Biochemical fractionation and stable isotope dilution liquid chromatography-mass spectrometry for targeted and microdomain specific protein quantification in human postmortem brain tissue‘, Molecular and Cellular Proteomics, 11 (12),
(pp. 1670-1681)
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