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Inter-regional proteomic profiling of the human brain using optimized protein extraction method from formalin-fixed tissue to identify signaling pathways

ABSTRACT: Proteomics offers vast potential to study the molecular regulation of the human brain. Formalin fixation is a common method for preserving human tissue, however, presents challenges for proteomic analysis. In this study we compared the efficiency of two different protein extraction buffers on three post-mortem, formalin-fixed human brains. Equal amounts of extracted proteins were subjected to in-gel tryptic digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein, peptide sequence and peptide group identifications, protein abundance and gene ontology pathways were analyzed. Protein extraction was superior using lysis buffer containing Tris (hydroxymethyl) aminomethane hydrochloride, sodium dodecyl sulfate, sodium deoxycholate, and Triton X-100 (TrisHCl, SDS, SDC, Triton X-100), which was then used for inter-regional analysis. Pre-frontal, motor, temporal, and occipital cortex tissue was analyzed by label free quantification (LFQ) proteomics, Ingenuity Pathway Analysis and PANTHERdb. Inter-regional analysis of the human brain revealed differential enrichment of proteins. We found similarly activated cellular signaling pathways, suggesting commonalities in the molecular regulation of neuroanatomically-linked brain functions. Overall, we developed an optimized, robust, and efficient method for protein extraction from formalin-fixed, human brain tissue for in-depth LFQ proteomics. We further demonstrate that this method is suitable for quick analysis of molecular signaling pathways in the human brain.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Brain

SUBMITTER: Albert Lee

LAB HEAD: Albert Lee

PROVIDER: PXD039808 | Pride | 2023-02-22

REPOSITORIES: Pride

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Project description:Alzheimer’s disease (AD) is a chronic ageing related neurodegenerative disease which is characterized by loss of synapses and neurons in the vulnerable brain regions. Expression perturbations of amyloid β (Aβ) and tau protein in the brain are two hallmarks of AD. Aβ is abnormally generated from amyloid precursor protein (APP) which is broadly distributed in different brain regions including the hippocampus and cortex. It is believed that increased Aβ expression plays a causative role in the early stage of AD pathology. Aβ protein interacts with the signaling pathways that control the phosphorylation of the microtubule-associated protein tau, which eventually disrupts the neuronal circuitry as well as network connectivity leading to neurodegenerative processes observed in AD. In addition, substantial molecular and neurodegenerative changes occur in the initial stage of AD even before the cognitive symptoms are evident, which makes the early diagnosis of AD vital to any timely disease stabilization and treatment. However, despite myriad efforts and substantial progress in the field to decipher the molecular mechanisms of disease onset and its progression, specific causes underlying AD pathology remain ill-defined. There is an urgent need to identify novel mechanism based interventional approaches that can stop, or slow down, the progression of AD. Therefore, it is important to improve the knowledge of the early AD and have a better understanding of the underlying molecular mechanisms induced by Aβ. In this study, we performed comparative quantitative proteomics on different brain regions of 2.5 months old APP/PS1 mice (hippocampus, frontal cortex, parietal cortex and cerebellum) in order to investigate the early stage impact of AD. Although over 5000 proteins were identified in all regions, the proteome response across regions was greatly varied. As expected, the greatest proteome perturbation was detected in the hippocampus and frontal cortex (AD-susceptible brain regions), compared to only 155 changed proteins in the cerebellum (less vulnerable region to AD). Increased expression of APP protein was identified in all brain regions. The expression of the majority of the other proteins between hippocampus and cortex areas was not similar, highlighting differential effects of the disease on different brain regions. A series of AD associated markers and pathways were identified as overexpressed in the hippocampus including glutamatergic synapse, GABAergic synapse, retrograde endocannabinoid signaling, long-term potentiation, and calcium signaling. In contrast, the expression of same proteins and pathways was negatively regulated in frontal and parietal cortex regions. Additionally, an increased expression of proteins associated with the oxidative phosphorylation pathway in hippocampus was not evident in the two cortices. Interestingly, an increased expression of proteins involved with myelination, neurofilament cytoskeleton organization, and glutathione metabolism was identified in cortex areas, while these were reduced in the hippocampus. The results obtained from this study highlight important information on brain region specific protein expression changes occurring in the early stages of AD.

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Inter-regional proteomic profiling of the human brain using optimized protein extraction method from formalin-fixed tissue to identify signaling pathways

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