Targeted mass spectrometry to quantify brain-derived cerebrospinal fluid biomarkers in Alzheimer’s disease

Abstract

Introduction: Alzheimer's disease (AD) is the most common cause of dementia, characterized by progressive cognitive decline. Protein biomarkers of AD brain pathology, including β-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. Method: We 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. Results: Despite 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/β-amyloid ratio. Notably, SMOC1 levels in postmortem brain are highly

An Integrative Multi-omics Approach Reveals New Central Nervous System Pathway Alterations in Alzheimer’s Disease

“…Single modality feature selection: We rst used Elastic-net regression, to identify molecules associated with individual biomarkers of CSF AD pathology without considering any possible interactions between different 'omics modalities. This approach identi ed several proteins (SPARC-related modular calciumbinding protein 1, brain acid soluble protein 1, neuromodulin, pyruvate kinase PKM, thymosin beta-10, 14-3-3 protein zeta/delta, and fructose-bisphosphate aldolase A) in strong accordance with recent studies of the AD CSF proteome (16,28). The zeta/delta isoform of protein 14-3-3 was associated with Aβ1-42,…”

Integrated proteomics reveals brain-based cerebrospinal fluid biomarkers in asymptomatic and symptomatic Alzheimer’s disease

“…To progress toward clinical MS-based assay development, future directions will also require large-scale biomarker validation using targeted quantitation methods, such as selective or parallel reaction monitoring ( 62 ). We recently validated many of the CSF protein changes described here using parallel reaction monitoring ( 63 ). Several prioritized panel targets were quantified with marked precision, including YWHAZ, ALDOA, and SMOC1, which map to our synaptic, metabolic, and inflammatory panels, respectively ( 63 ).…”

“…We recently validated many of the CSF protein changes described here using parallel reaction monitoring ( 63 ). Several prioritized panel targets were quantified with marked precision, including YWHAZ, ALDOA, and SMOC1, which map to our synaptic, metabolic, and inflammatory panels, respectively ( 63 ). Data-independent acquisition (DIA) and other MS-based strategies may also prove useful for target validation.…”

Systems-based proteomics to resolve the biology of Alzheimer’s disease beyond amyloid and tau

“…Several studies have successfully used targeted MS strategies to quantify Aβ, tau, and APOE protein levels in CSF with similar diseaseassociation efficacy to that of traditional ELISA assays [189][190][191]. More recently, targeted MS was applied to a more diverse panel of promising AD CSF biomarkers reflecting a much wider array of disease pathophysiologies [192]. In this study, Zhou and colleagues used PRM to quantify a panel of brain-derived proteins significantly altered in a discovery-driven proteomic analysis of AD CSF.…”

“…Protein targets mapping to synaptic, metabolism, and neuroinflammation modules in brain [ 96 ] were quantified with high precision, showcasing their potential for future biomarker translation. SMOC1, YWHAZ, ALDOA, and MAP1B emerged as biomarkers that could best discriminate between individuals with AD and non-AD cognitive impairment, correlating well with Aβ and tau levels [ 192 ]. Overall, these results further illustrate the utility of targeted MS strategies to significantly advance AD biomarker discovery toward network-based protein assays with multiple clinical uses in disease (Fig.…”