Post Mortem Proteomic Characterization of Human Subgenual Prefrontal Cortex in Tobacco Abuse and PTSD

Significance
We propose developing a novel strategy for protein identification and quantification in post mortem brain tissue of nicotine addicted individuals with Posttraumatic Stress Disorder (PTSD). Standard, single shot label free proteomic approaches do not interrogate the proteome at sufficient depth to see differences arising from low abundance mRNA transcripts. Offline fractionation of peptides allows for increased rates of protein identification, but quantification of proteins becomes a serious technical challenge with post fractionation. It is known that important proteomic changes are occurring at the level of low abundance proteins and so our focus is on developing technologies to identify these changes. By combining tandem mass tag labeling with offline fractionation, we can benefit from the increased depth of proteome coverage gained by fractionation, while using the TMT tags to allow between sample peptide quantification.

As protein forms the final output of the central dogma, it is important to develop proteomic technologies that move towards matching the sensitivity of RNA-based approaches. We have previously identified numerous mRNA changes in our PTSD cohorts, making integration of the transcriptome and proteome an important goal of this study. Our current post-mortem repository allows for a unique and powerful analysis of the human subgenual prefrontal cortex (PFC), not only measuring the protein changes occurring in PTSD smokers versus PTSD nonsmokers but also across cohorts. In addition to being the first systematic examination of proteomic changes occurring in nicotine dependent versus non-dependent PFC, this study will also be the first to examine proteomic changes in PTSD brain.

Specific Aim 1
In Aim1, we will assess the potential of using fractionated tandem mass tagged peptides from post mortem PFC in LC/MS-MS to detect changes in protein abundance. To increase the sensitivity of quantifying low abundance peptides, we propose using tandem mass tag (TMT) peptide labeling in combination with offline sample fractionation. These tags are designed to ensure that identical peptides labeled with different TMTs co-migrate throughout offline fractionation and online LC. These TMTs permit simultaneous determination of both the identity and relative abundance of peptides using a collision induced dissociation (CID)-based analysis method.

Specific Aim 2
In Aim 2, we will identify the proteomic changes in subgenual PFC of smokers with PTSD versus nonsmokers with PTSD. Building upon Aim 1, we will use fractionated TMT-LC-MS/MS to identify changes to the subgenual PFC proteome. A subset of our PTSD (~50%) and control cohorts (~33%) were using tobacco at the time of death. This will allow us to directly interrogate the changes in the cortical proteome of smoking subjects with PTSD versus nonsmoking PTSD patients. Additionally, we will be able to evaluate the proteomic changes in PFC in smoking and nonsmoking subjects with no mental health history.