Mass Spectrometry-Based Quantification of Tau in Human Cerebrospinal Fluid Using a Complementary Tryptic Peptide Standard.

Here, we report a method for the generation of complementary tryptic (CompTryp) isotope-labeled peptide standards for the relative and absolute quantification of proteins by mass spectrometry (MS). These standards can be digested in parallel with either trypsin (Tryp-C) or trypsin-N (Tryp-N), to generate peptides that significantly overlap in primary sequence having C- and N-terminal arginine and lysine residues, respectively. As a proof of concept, an isotope-labeled CompTryp standard was synthesized for Tau, a well-established biomarker in Alzheimer's disease (AD), which included both N- and C-terminal heavy isotope-labeled (15N and 13C) arginine residues and flanking amino acid sequences to monitor proteolytic digestion. Despite having the exact same mass, the N- and C-terminal heavy Tau peptides are distinguishable by retention time and MS/MS fragmentation profiles. The isotope-labeled Tau CompTryp standard was added to human cerebrospinal fluid (CSF) followed by parallel digestion with Tryp-N and Tryp-C. The native and isotope-labeled peptide pairs were quantified by parallel reaction monitoring (PRM) in a single assay. Notably, both tryptic peptides were effective at quantifying Tau in human CSF, and both showed a significant difference in CSF Tau levels between AD and controls. Treating these CompTryp Tau peptide measurements as independent replicates also improved the coefficient of variation and correlation with Tau immunoassays. More broadly, we propose that CompTryp standards can be generated for any protein of interest, providing an efficient method to improve the robustness and reproducibility for MS analysis of clinical and research samples.

Recombinant High-Mobility Group Box 1 (rHMGB1) Promotes NRF2-Independent Mitochondrial Fusion through CXCR4/PSMB5-Mediated Drp1 Degradation in Endothelial Cells.

Mitochondrial dynamics plays an important role in maintaining normal endothelial cell function and in the pathogenesis of cardiovascular disease. It is not identified whether high-mobility group box 1 (HMGB1), a representative damage-associated molecular pattern (DAMP) molecule, could influence mitochondrial dynamics in endothelial cells. The objective of this study is to clarify the effect of HMGB1 on mitochondrial dynamics in endothelial cells and the underlying mechanism. EA.hy926 human endothelial cells were incubated with recombinant HMGB1 (rHMGB1); mitochondrial morphology was observed with a confocal microscope and transmission electron microscope (TEM). The expression of dynamin-related protein 1 (Drp1), Mitofusin 1 (Mfn1), Mitofusin 2 (Mfn2), Optic atrophy 1 (Opa1), phosphatase and tensin homolog- (PTEN-) induced kinase 1 (PINK1), NOD-like receptor 3 (NLRP3), caspase 1, cleaved caspase 1, 20S proteasome subunit beta 5 (PSMB5), and antioxidative master nuclear factor E2-related factor 2 (NRF2) and the concentration of interleukin 1ß (IL-1ß) were determined. Specific inhibitors C29, TAK-242, FPS-ZM1, AMD3100, and epoxomicin were used to block toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4), receptor for advanced glycation end products (RAGE), C-X-C-chemokine receptor 4 (CXCR4), and PSMB5, respectively. siRNAs were used to silence the expression of NRF2. rHMGB1 promoted mitochondrial fusion in endothelial cells, while no significant proinflammatory effects were found. The expression of mitochondrial fission protein Drp1 and phosphorylated subtypes p-Drp1-S616 and p-Drp1-S637 were all downregulated; no significant expression changes of PINK1 and Mfn1, Mfn2, and Opa1 were found. Inhibition of CXCR4 but not TLR4, RAGE, or TLR2 reversed rHMGB1-induced Drp1 downregulation and mitochondrial fusion. Interestingly, inhibition of TLR4 with TAK-242 promoted Drp1 downregulation and mitochondrial fusion. rHMGB1 increased the expression of NRF2 and PSMB5; inhibition of PSMB5 but not silencing NRF2 abolished rHMGB1-induced Drp1 downregulation and mitochondrial fusion. These results indicate that rHMGB1 promotes NRF2 independent mitochondrial fusion via CXCR4/PSMB5 pathway-mediated Drp1 proteolysis. rHMGB1 may influence mitochondrial and endothelial function through this effect on mitochondrial dynamics.

Identification of Conserved Proteomic Networks in Neurodegenerative Dementia.

Data-driven analyses are increasingly valued in modern medicine. We integrate quantitative proteomics and transcriptomics from over 1,000 post-mortem brains from six cohorts representing Alzheimer's disease (AD), asymptomatic AD, progressive supranuclear palsy (PSP), and control patients from the Accelerating Medicines Partnership - Alzheimer's Disease consortium. We define robust co-expression trajectories related to disease progression, including early neuronal, microglial, astrocyte, and immune response modules, and later mRNA splicing and mitochondrial modules. The majority of, but not all, modules are conserved at the transcriptomic level, including module C3, which is only observed in proteome networks and enriched in mitogen-activated protein kinase (MAPK) signaling. Genetic risk enriches in modules changing early in disease and indicates that AD and PSP have distinct causal biological drivers at the pathway level, despite aspects of similar pathology, including synaptic loss and glial inflammatory changes. The conserved, high-confidence proteomic changes enriched in genetic risk represent targets for drug discovery.

Effects of APOE Genotype on Brain Proteomic Network and Cell Type Changes in Alzheimer's Disease.

Polymorphic alleles in the apolipoprotein E (APOE) gene are the main genetic determinants of late-onset Alzheimer's disease (AD) risk. Individuals carrying the APOE E4 allele are at increased risk to develop AD compared to those carrying the more common E3 allele, whereas those carrying the E2 allele are at decreased risk for developing AD. How ApoE isoforms influence risk for AD remains unclear. To help fill this gap in knowledge, we performed a comparative unbiased mass spectrometry-based proteomic analysis of post-mortem brain cortical tissues from pathologically-defined AD or control cases of different APOE genotypes. Control cases (n = 10) were homozygous for the common E3 allele, whereas AD cases (n = 24) were equally distributed among E2/3, E3/3, and E4/4 genotypes. We used differential protein expression and co-expression analytical approaches to assess how changes in the brain proteome are related to APOE genotype. We observed similar levels of amyloid-ß, but reduced levels of neurofibrillary tau, in E2/3 brains compared to E3/3 and E4/4 AD brains. Weighted co-expression network analysis revealed 33 modules of co-expressed proteins, 12 of which were significantly different by APOE genotype in AD. The modules that were significantly different by APOE genotype were associated with synaptic transmission and inflammation, among other biological processes. Deconvolution and analysis of brain cell type changes revealed that the E2 allele suppressed homeostatic and disease-associated cell type changes in astrocytes, microglia, oligodendroglia, and endothelia. The E2 allele-specific effect on brain cell type changes was validated in a separate cohort of 130 brains. Our systems-level proteomic analyses of AD brain reveal alterations in the brain proteome and brain cell types associated with allelic variants in APOE, and suggest further areas for investigation into the upstream mechanisms that drive ApoE-associated risk for AD.

A proteomic network approach across the ALS-FTD disease spectrum resolves clinical phenotypes and genetic vulnerability in human brain.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases with overlap in clinical presentation, neuropathology, and genetic underpinnings. The molecular basis for the overlap of these disorders is not well established. We performed a comparative unbiased mass spectrometry-based proteomic analysis of frontal cortical tissues from postmortem cases clinically defined as ALS, FTD, ALS and FTD (ALS/FTD), and controls. We also included a subset of patients with the C9orf72 expansion mutation, the most common genetic cause of both ALS and FTD Our systems-level analysis of the brain proteome integrated both differential expression and co-expression approaches to assess the relationship of these differences to clinical and pathological phenotypes. Weighted co-expression network analysis revealed 15 modules of co-expressed proteins, eight of which were significantly different across the ALS-FTD disease spectrum. These included modules associated with RNA binding proteins, synaptic transmission, and inflammation with cell-type specificity that showed correlation with TDP-43 pathology and cognitive dysfunction. Modules were also examined for their overlap with TDP-43 protein-protein interactions, revealing one module enriched with RNA-binding proteins and other causal ALS genes that increased in FTD/ALS and FTD cases. A module enriched with astrocyte and microglia proteins was significantly increased in ALS cases carrying the C9orf72 mutation compared to sporadic ALS cases, suggesting that the genetic expansion is associated with inflammation in the brain even without clinical evidence of dementia. Together, these findings highlight the utility of integrative systems-level proteomic approaches to resolve clinical phenotypes and genetic mechanisms underlying the ALS-FTD disease spectrum in human brain.