Dataset Information

Domain-specific Quantification of Prion Protein in Cerebrospinal Fluid by Targeted Mass Spectrometry.

ABSTRACT: Therapies currently in preclinical development for prion disease seek to lower prion protein (PrP) expression in the brain. Trials of such therapies are likely to rely on quantification of PrP in cerebrospinal fluid (CSF) as a pharmacodynamic biomarker and possibly as a trial endpoint. Studies using PrP ELISA kits have shown that CSF PrP is lowered in the symptomatic phase of disease, a potential confounder for reading out the effect of PrP-lowering drugs in symptomatic patients. Because misfolding or proteolytic cleavage could potentially render PrP invisible to ELISA even if its concentration were constant or increasing in disease, we sought to establish an orthogonal method for CSF PrP quantification. We developed a multi-species targeted mass spectrometry method based on multiple reaction monitoring (MRM) of nine PrP tryptic peptides quantified relative to an isotopically labeled recombinant protein standard for human samples, or isotopically labeled synthetic peptides for nonhuman species. Analytical validation experiments showed process replicate coefficients of variation below 15%, good dilution linearity and recovery, and suitable performance for both CSF and brain homogenate and across humans as well as preclinical species of interest. In n = 55 CSF samples from individuals referred to prion surveillance centers with rapidly progressive dementia, all six human PrP peptides, spanning the N- and C-terminal domains of PrP, were uniformly reduced in prion disease cases compared with individuals with nonprion diagnoses. Thus, lowered CSF PrP concentration in prion disease is a genuine result of the disease process and not an artifact of ELISA-based measurement. As a result, dose-finding studies for PrP lowering drugs may need to be conducted in presymptomatic at-risk individuals rather than in symptomatic patients. We provide a targeted mass spectrometry-based method suitable for preclinical quantification of CSF PrP as a tool for drug development.

SUBMITTER: Minikel EV

PROVIDER: S-EPMC6885701 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Domain-specific Quantification of Prion Protein in Cerebrospinal Fluid by Targeted Mass Spectrometry.

Minikel Eric Vallabh EV Kuhn Eric E Cocco Alexandra R AR Vallabh Sonia M SM Hartigan Christina R CR Reidenbach Andrew G AG Safar Jiri G JG Raymond Gregory J GJ McCarthy Michael D MD O'Keefe Rhonda R Llorens Franc F Zerr Inga I Capellari Sabina S Parchi Piero P Schreiber Stuart L SL Carr Steven A SA

Molecular & cellular proteomics : MCP 20190926 12

Therapies currently in preclinical development for prion disease seek to lower prion protein (PrP) expression in the brain. Trials of such therapies are likely to rely on quantification of PrP in cerebrospinal fluid (CSF) as a pharmacodynamic biomarker and possibly as a trial endpoint. Studies using PrP ELISA kits have shown that CSF PrP is lowered in the symptomatic phase of disease, a potential confounder for reading out the effect of PrP-lowering drugs in symptomatic patients. Because misfold ...[more]

PMID: 31558565

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Project description:IntroductionAlzheimer's disease (AD) is the most common cause of dementia, characterized by progressive cognitive decline. Protein biomarkers of AD brain pathology, including ?-amyloid and Tau, are reflected in cerebrospinal fluid (CSF), yet the identification of additional biomarkers linked to other brain pathophysiologies remains elusive. We recently reported a multiplex tandem-mass tag (TMT) CSF proteomic analysis of nearly 3000 proteins, following depletion of highly abundant proteins and off-line fractionation, across control and AD cases. Of these, over 500 proteins were significantly increased or decreased in AD, including markers reflecting diverse biological functions in brain. Here, we use a targeted mass spectrometry (MS) approach, termed parallel reaction monitoring (PRM), to quantify select CSF biomarkers without pre-depletion or fractionation to assess the reproducibility of our findings and the specificity of changes for AD versus other causes of cognitive impairment.MethodWe nominated 41 proteins (94 peptides) from the TMT CSF discovery dataset, representing a variety of brain cell-types and biological functions, for label-free PRM analysis in a replication cohort of 88 individuals that included 20 normal controls, 37 clinically diagnosed AD cases and 31 cases with non-AD cognitive impairment. To control for technical variables, isotopically labeled synthetic heavy peptide standards were added into each of the 88 CSF tryptic digests. Furthermore, a peptide pool, representing an equivalent amount of peptide from all samples, was analyzed (n?=?10) across each batch. Together, this approach enabled us to assess both the intra- and inter-sample differences in peptide signal response and retention time.ResultsDespite differences in sample preparation, quantitative MS approaches and patient samples, 25 proteins, including Tau, had a consistent and significant change in AD in both the discovery and replication cohorts. Validated CSF markers with low coefficient of variation included the protein products for neuronal/synaptic (GDA, GAP43, SYN1, BASP1, YWHAB, YWHAZ, UCHL1, STMN1 and MAP1B), glial/inflammation (SMOC1, ITGAM, CHI3L1, SPP1, and CHIT1) and metabolic (PKM, ALDOA and FABP3) related genes. Logistical regression analyses revealed several proteins with high sensitivity and specificity for classifying AD cases from controls and other non-AD dementias. SMOC1, YWHAZ, ALDOA and MAP1B emerged as biomarker candidates that could best discriminate between individuals with AD and non-AD cognitive impairment as well as Tau/?-amyloid ratio. Notably, SMOC1 levels in postmortem brain are highly correlated with AD pathology even in the preclinical stage of disease, indicating that CSF SMOC1 levels reflect underlying brain pathology specific for AD.ConclusionCollectively these findings highlight the utility of targeted MS approaches to quantify biomarkers associated with AD that could be used for monitoring disease progression, stratifying patients for clinical trials and measuring therapeutic response.

Project description:BACKGROUND:Depression affects approximately 7.1% of the United States population every year and has an annual economic burden of over $210 billion dollars. Several recent studies have sought to investigate the pathophysiology of depression utilizing focused cerebrospinal fluid (CSF) and serum analysis. Inflammation and metabolic dysfunction have emerged as potential etiological factors from these studies. A dysregulation in the levels of inflammatory proteins such as IL-12, TNF, IL-6 and IFN-? have been found to be significantly correlated with depression. METHODS:CSF samples were obtained from 15 patients, seven with major depressive disorder and eight age- and gender-matched non-psychiatric controls. CSF protein profiles were obtained using quantitative mass spectrometry. The data were analyzed by Progenesis QI proteomics software to identify significantly dysregulated proteins. The results were subjected to bioinformatics analysis using the Ingenuity Pathway Analysis suite to obtain unbiased mechanistic insight into biologically relevant interactions and pathways. RESULTS:Several dysregulated proteins were identified. Bioinformatics analysis indicated that the potential disorder/disease pathways include inflammatory response, metabolic disease and organismal injury. Molecular and cellular functions that were affected include cellular compromise, cell-to-cell signaling & interaction, cellular movement, protein synthesis, and cellular development. The major canonical pathway that was upregulated was acute phase response signaling. Endogenous upstream regulators that may influence dysregulation of proinflammatory molecules associated with depression are interleukin-6 (IL-6), signal transducer and activator of transcription 3 (STAT3), oncostatin M, PR domain zinc finger protein 1 (PRDM1), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A). CONCLUSIONS:The proteome profiling data in this report identifies several potential biological functions that may be involved in the pathophysiology of major depressive disorder. Future research into how the differential expression of these proteins is involved in the etiology and severity of depression will be important.

Dataset Information

Domain-specific Quantification of Prion Protein in Cerebrospinal Fluid by Targeted Mass Spectrometry.

Publications

Domain-specific Quantification of Prion Protein in Cerebrospinal Fluid by Targeted Mass Spectrometry.

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