Our understanding of Alzheimer's disease (AD) pathophysiology remains incomplete. Here we used quantitative mass spectrometry and coexpression network analysis to conduct the largest proteomic study ...thus far on AD. A protein network module linked to sugar metabolism emerged as one of the modules most significantly associated with AD pathology and cognitive impairment. This module was enriched in AD genetic risk factors and in microglia and astrocyte protein markers associated with an anti-inflammatory state, suggesting that the biological functions it represents serve a protective role in AD. Proteins from this module were elevated in cerebrospinal fluid in early stages of the disease. In this study of >2,000 brains and nearly 400 cerebrospinal fluid samples by quantitative proteomics, we identify proteins and biological processes in AD brains that may serve as therapeutic targets and fluid biomarkers for the disease.
The complicated cellular and biochemical changes that occur in brain during Alzheimer's disease are poorly understood. In a previous study we used an unbiased label-free quantitative mass ...spectrometry-based proteomic approach to analyze these changes at a systems level in post-mortem cortical tissue from patients with Alzheimer's disease (AD), asymptomatic Alzheimer's disease (AsymAD), and controls. We found modules of co-expressed proteins that correlated with AD phenotypes, some of which were enriched in proteins identified as risk factors for AD by genetic studies.
The amount of information that can be obtained from such systems-level proteomic analyses is critically dependent upon the number of proteins that can be quantified across a cohort. We report here a new proteomic systems-level analysis of AD brain based on 6,533 proteins measured across AD, AsymAD, and controls using an analysis pipeline consisting of isobaric tandem mass tag (TMT) mass spectrometry and offline prefractionation.
Our new TMT pipeline allowed us to more than double the depth of brain proteome coverage. This increased depth of coverage greatly expanded the brain protein network to reveal new protein modules that correlated with disease and were unrelated to those identified in our previous network. Differential protein abundance analysis identified 350 proteins that had altered levels between AsymAD and AD not caused by changes in specific cell type abundance, potentially reflecting biochemical changes that are associated with cognitive decline in AD. RNA binding proteins emerged as a class of proteins altered between AsymAD and AD, and were enriched in network modules that correlated with AD pathology. We developed a proteogenomic approach to investigate RNA splicing events that may be altered by RNA binding protein changes in AD. The increased proteome depth afforded by our TMT pipeline allowed us to identify and quantify a large number of alternatively spliced protein isoforms in brain, including AD risk factors such as BIN1, PICALM, PTK2B, and FERMT2. Many of the new AD protein network modules were enriched in alternatively spliced proteins and correlated with molecular markers of AD pathology and cognition.
Further analysis of the AD brain proteome will continue to yield new insights into the biological basis of AD.
The biological processes that are disrupted in the Alzheimer's disease (AD) brain remain incompletely understood. In this study, we analyzed the proteomes of more than 1,000 brain tissues to reveal ...new AD-related protein co-expression modules that were highly preserved across cohorts and brain regions. Nearly half of the protein co-expression modules, including modules significantly altered in AD, were not observed in RNA networks from the same cohorts and brain regions, highlighting the proteopathic nature of AD. Two such AD-associated modules unique to the proteomic network included a module related to MAPK signaling and metabolism and a module related to the matrisome. The matrisome module was influenced by the APOE ε4 allele but was not related to the rate of cognitive decline after adjustment for neuropathology. By contrast, the MAPK/metabolism module was strongly associated with the rate of cognitive decline. Disease-associated modules unique to the proteome are sources of promising therapeutic targets and biomarkers for AD.
Robust and accessible biomarkers that can capture the heterogeneity of Alzheimer's disease and its diverse pathological processes are urgently needed. Here, we undertook an investigation of ...Alzheimer's disease cerebrospinal fluid (CSF) and plasma from the same subjects (n=18 control, n=18 AD) using three different proteomic platforms-SomaLogic SomaScan, Olink proximity extension assay, and tandem mass tag-based mass spectrometry-to assess which protein markers in these two biofluids may serve as reliable biomarkers of AD pathophysiology observed from unbiased brain proteomics studies. Median correlation of overlapping protein measurements across platforms in CSF (r~0.7) and plasma (r~0.6) was good, with more variability in plasma. The SomaScan technology provided the most measurements in plasma. Surprisingly, many proteins altered in AD CSF were found to be altered in the opposite direction in plasma, including important members of AD brain co-expression modules. An exception was SMOC1, a key member of the brain matrisome module associated with amyloid-β deposition in AD, which was found to be elevated in both CSF and plasma. Protein co-expression analysis on greater than 7000 protein measurements in CSF and 9500 protein measurements in plasma across all proteomic platforms revealed strong changes in modules related to autophagy, ubiquitination, and sugar metabolism in CSF, and endocytosis and the matrisome in plasma. Cross-platform and cross-biofluid proteomics represents a promising approach for AD biomarker development.
Native chemical ligation of unprotected peptide segments involves reaction between a peptide-α-thioester and a cysteine-peptide, to yield a product with a native amide bond at the ligation site. ...Peptide-α-thioalkyl esters are commonly used because of their ease of preparation. These thioalkyl esters are rather unreactive so the ligation reaction is catalyzed by in situ transthioesterification with thiol additives. The most common thiol catalysts used to date have been either a mixture of thiophenol/benzyl mercaptan, or the alkanethiol MESNA. Despite the use of these thiol catalysts, ligation reactions typically take 24−48 h. To gain insight into the mechanism of native chemical ligaton and in order to find a better catalyst, we investigated the use of a number of thiol compounds. Substituted thiophenols with pK a > 6 were found to best combine the ability to exchange rapidly and completely with thioalkyl esters, and to then act as effective leaving groups in reaction of the peptide-thioester with the thiol side chain of a cysteine-peptide. A highly effective and practical catalyst was (4-carboxylmethyl)thiophenol (‘MPAA'), a nonmalodorous, water-soluble thiol. Use of MPAA gave an order of magnitude faster reaction in model studies of native chemical ligation and in the synthesis of a small protein, turkey ovomucoid third domain (OMTKY3). MPAA should find broad use in native chemical ligation and in the total synthesis of proteins.
Alzheimer's disease (AD) is the most frequent and costly neurodegenerative disorder. Although diverse lines of evidence suggest that the amyloid precursor protein (APP) is involved in its causation, ...the precise mechanisms remain unknown and no treatments are available to prevent or halt the disease. A favorite hypothesis has been that APP contributes to AD pathogenesis through the cerebral accumulation of the amyloid-β peptide (Aβ), which is derived from APP through sequential proteolytic cleavage by BACE1 and γ-secretase. However, inhibitors of these enzymes have failed in clinical trials despite clear evidence for target engagement.
To further elucidate the roles of APP and its metabolites in AD pathogenesis, we analyzed transgenic mice overexpressing wildtype human APP (hAPP) or hAPP carrying mutations that cause autosomal dominant familial AD (FAD), as well as App knock-in mice that do not overexpress hAPP but have two mouse App alleles with FAD mutations and a humanized Aβ sequence.
Although these lines of mice had marked differences in cortical and hippocampal levels of APP, APP C-terminal fragments, soluble Aβ, Aβ oligomers and age-dependent amyloid deposition, they all developed cognitive deficits as well as non-convulsive epileptiform activity, a type of network dysfunction that also occurs in a substantive proportion of humans with AD. Pharmacological inhibition of BACE1 effectively reduced levels of amyloidogenic APP C-terminal fragments (C99), soluble Aβ, Aβ oligomers, and amyloid deposits in transgenic mice expressing FAD-mutant hAPP, but did not improve their network dysfunction and behavioral abnormalities, even when initiated at early stages before amyloid deposits were detectable.
hAPP transgenic and App knock-in mice develop similar pathophysiological alterations. APP and its metabolites contribute to AD-related functional alterations through complex combinatorial mechanisms that may be difficult to block with BACE inhibitors and, possibly, also with other anti-Aβ treatments.
Despite being twice as likely to get Alzheimer's disease (AD), African Americans have been grossly underrepresented in AD research. While emerging evidence indicates that African Americans with AD ...have lower cerebrospinal fluid (CSF) levels of Tau compared to Caucasians, other differences in AD CSF biomarkers have not been fully elucidated. Here, we performed unbiased proteomic profiling of CSF from African Americans and Caucasians with and without AD to identify both common and divergent AD CSF biomarkers.
Multiplex tandem mass tag-based mass spectrometry (TMT-MS) quantified 1,840 proteins from 105 control and 98 AD patients of which 100 identified as Caucasian while 103 identified as African American. We used differential protein expression and co-expression approaches to assess how changes in the CSF proteome are related to race and AD. Co-expression network analysis organized the CSF proteome into 14 modules associated with brain cell-types and biological pathways. A targeted mass spectrometry method, selected reaction monitoring (SRM), with heavy labeled internal standards was used to measure a panel of CSF module proteins across a subset of African Americans and Caucasians with or without AD. A receiver operating characteristic (ROC) curve analysis assessed the performance of each protein biomarker in differentiating controls and AD by race.
Consistent with previous findings, the increase of Tau levels in AD was greater in Caucasians than in African Americans by both immunoassay and TMT-MS measurements. CSF modules which included 14-3-3 proteins (YWHAZ and YWHAG) demonstrated equivalent disease-related elevations in both African Americans and Caucasians with AD, whereas other modules demonstrated more profound disease changes within race. Modules enriched with proteins involved with glycolysis and neuronal/cytoskeletal proteins, including Tau, were more increased in Caucasians than in African Americans with AD. In contrast, a module enriched with synaptic proteins including VGF, SCG2, and NPTX2 was significantly lower in African Americans than Caucasians with AD. Following SRM and ROC analysis, VGF, SCG2, and NPTX2 were significantly better at classifying African Americans than Caucasians with AD.
Our findings provide insight into additional protein biomarkers and pathways reflecting underlying brain pathology that are shared or differ by race.
Proteomic studies for Alzheimer's disease (AD) are instrumental in identifying AD pathways but often focus on single tissues and sporadic AD cases. Here, we present a proteomic study analyzing 1305 ...proteins in brain tissue, cerebrospinal fluid (CSF), and plasma from patients with sporadic AD,
risk variant carriers, patients with autosomal dominant AD (ADAD), and healthy individuals. We identified 8 brain, 40 CSF, and 9 plasma proteins that were altered in individuals with sporadic AD, and we replicated these findings in several external datasets. We identified a proteomic signature that differentiated
variant carriers from both individuals with sporadic AD and healthy individuals. The proteins associated with sporadic AD were also altered in patients with ADAD, but with a greater effect size. Brain-derived proteins associated with ADAD were also replicated in additional CSF samples. Enrichment analyses highlighted several pathways, including those implicated in AD (calcineurin and Apo E), Parkinson's disease (α-synuclein and LRRK2), and innate immune responses (SHC1, ERK-1, and SPP1). Our findings suggest that combined proteomics across brain tissue, CSF, and plasma can be used to identify markers for sporadic and genetically defined AD.
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.
Alzheimer's disease (AD) lacks protein biomarkers reflective of its diverse underlying pathophysiology, hindering diagnostic and therapeutic advancements. Here, we used integrative proteomics to ...identify cerebrospinal fluid (CSF) biomarkers representing a wide spectrum of AD pathophysiology. Multiplex mass spectrometry identified ~3500 and ~12,000 proteins in AD CSF and brain, respectively. Network analysis of the brain proteome resolved 44 biologically diverse modules, 15 of which overlapped with the CSF proteome. CSF AD markers in these overlapping modules were collapsed into five protein panels representing distinct pathophysiological processes. Synaptic and metabolic panels were decreased in AD brain but increased in CSF, while glial-enriched myelination and immunity panels were increased in brain and CSF. The consistency and disease specificity of panel changes were confirmed in >500 additional CSF samples. These panels also identified biological subpopulations within asymptomatic AD. Overall, these results are a promising step toward a network-based biomarker tool for AD clinical applications.
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