The Post-Synaptic Density of Human Postmortem Brain Tissues: An Experimental Study Paradigm for Neuropsychiatric Illnesses

Recent molecular genetics studies have suggested various trans-synaptic processes for pathophysiologic mechanisms of neuropsychiatric illnesses. Examination of pre- and post-synaptic scaffolds in the brains of patients would greatly aid further investigation, yet such an approach in human postmortem tissue has yet to be tested. We have examined three methods using density gradient based purification of synaptosomes followed by detergent extraction (Method 1) and the pH based differential extraction of synaptic membranes (Methods 2 and 3). All three methods separated fractions from human postmortem brains that were highly enriched in typical PSD proteins, almost to the exclusion of pre-synaptic proteins. We examined these fractions using electron microscopy (EM) and verified the integrity of the synaptic membrane and PSD fractions derived from human postmortem brain tissues. We analyzed protein composition of the PSD fractions using two dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) and observed known PSD proteins by mass spectrometry. Immunoprecipitation and immunoblot studies revealed that expected protein-protein interactions and certain posttranscriptional modulations were maintained in PSD fractions. Our results demonstrate that PSD fractions can be isolated from human postmortem brain tissues with a reasonable degree of integrity. This approach may foster novel postmortem brain research paradigms in which the stoichiometry and protein composition of specific microdomains are examined

The absolute quantification of focal adhesion kinase activation loop phosphorylation by liquid chromatography-mass spectrometry: A biomarker of Src kinase activity

Non-receptor tyrosine kinases Src and focal adhesion kinase (FAK) serve together as a signaling complex. The binding of Src to FAK activates both kinases, and results in the phosphorylation of FAK on key tyrosine residues, including FAK Y576 and Y577. Our initial experiments developed a mass spectrometry method to monitor all known sites of tyrosine phosphorylation critical to the function and signaling of FAK, including phosphorylated Y397 (pY397), pY407, Y 576, pY577, pY861 and pY925 . This technique was able to identify sites of phosphorylation on FAK as well as qualitatively differentiate between autocatalytic and Src-induced phosphorylation events. Interestingly, Y577 was unique among all sites monitored in that it was phosphorylated only in the presence of Src. The results of these discovery experiments prompted the development of a more focused approach, in which it was hypothesized that Src activity could be monitored by accurately measuring the activation loop phosphorylation of FAK. To further explore this hypothesis a method allowing for the absolute quantification of phosphorylation on the kinase activation loop of FAK by stable isotope dilution liquid chromatography/tandem mass spectrometry was developed. An isotopically labeled and phosphorylated FAK protein standard was synthesized and used to control for loss during immunoprecipitation of FAK. This approach allowed for two different phosphorylated forms of FAK, together with the non-phosphorylated form to be quantified both in vitro and in cell culture systems after immunoprecipitation. Following the development and rigorous validation of the methodology for absolute quantification of FAK activation loop Y576 and Y577 phosphorylation, it was used to quantify FAK activation loop phosphorylation in a cellular model of elevated Src activity and following global inhibition of tyrosine phosphatases. The results of cellular global phosphatase inhibition with pervanadate and in vitro de-phosphorylation of recombinant FAK revealed that Src phosphorylation on Y576 and Y577 was counterbalanced by cellular phosphatase activity. The usefulness of using bisphosphorylated pY576/Y577 -FAK as a biomarker of Src activity was further demonstrated by experiments that showed Src inhibition with AZD0530 prohibited pY576/pY 577-FAK formation in a dose dependant manner both in vitro and in cell culture

The absolute quantification of focal adhesion kinase activation loop phosphorylation by liquid chromatography-mass spectrometry: A biomarker of Src kinase activity

Non-receptor tyrosine kinases Src and focal adhesion kinase (FAK) serve together as a signaling complex. The binding of Src to FAK activates both kinases, and results in the phosphorylation of FAK on key tyrosine residues, including FAK Y576 and Y577. Our initial experiments developed a mass spectrometry method to monitor all known sites of tyrosine phosphorylation critical to the function and signaling of FAK, including phosphorylated Y397 (pY397), pY407, Y 576, pY577, pY861 and pY925 . This technique was able to identify sites of phosphorylation on FAK as well as qualitatively differentiate between autocatalytic and Src-induced phosphorylation events. Interestingly, Y577 was unique among all sites monitored in that it was phosphorylated only in the presence of Src. The results of these discovery experiments prompted the development of a more focused approach, in which it was hypothesized that Src activity could be monitored by accurately measuring the activation loop phosphorylation of FAK. To further explore this hypothesis a method allowing for the absolute quantification of phosphorylation on the kinase activation loop of FAK by stable isotope dilution liquid chromatography/tandem mass spectrometry was developed. An isotopically labeled and phosphorylated FAK protein standard was synthesized and used to control for loss during immunoprecipitation of FAK. This approach allowed for two different phosphorylated forms of FAK, together with the non-phosphorylated form to be quantified both in vitro and in cell culture systems after immunoprecipitation. Following the development and rigorous validation of the methodology for absolute quantification of FAK activation loop Y576 and Y577 phosphorylation, it was used to quantify FAK activation loop phosphorylation in a cellular model of elevated Src activity and following global inhibition of tyrosine phosphatases. The results of cellular global phosphatase inhibition with pervanadate and in vitro de-phosphorylation of recombinant FAK revealed that Src phosphorylation on Y576 and Y577 was counterbalanced by cellular phosphatase activity. The usefulness of using bisphosphorylated pY576/Y577 -FAK as a biomarker of Src activity was further demonstrated by experiments that showed Src inhibition with AZD0530 prohibited pY576/pY 577-FAK formation in a dose dependant manner both in vitro and in cell culture

Accurate quantitation of dystrophin protein in human skeletal muscle using mass spectrometry

Quantitation of human dystrophin protein in muscle biopsies is a clinically relevant endpoint for both diagnosis and response to dystrophin-replacement therapies for dystrophinopathies. A robust and accurate assay would enable the use of dystrophin as a surrogate biomarker, particularly in exploratory Phase 2 trials. Currently available methods to quantitate dystrophin rely on immunoblot or immunohistochemistry methods that are not considered robust. Here we present a mass spectrometry based approach to accurately quantitate dystrophin protein in a total protein extract from human muscle biopsies. Our approach uses a combination of stable isotope labeled dystrophin as a spike-in standard, gel electrophoresis and high precision mass spectrometry to detect and quantitate multiple peptides of dystrophin within a complex protein mixture. The method was found highly reproducible and linear over a wide dynamic range, detecting as low as 5% of dystrophin relative to the normal amount in healthy individuals