Plasma Aß42/Aß40 and phospho-tau217 concentration ratios increase the accuracy of amyloid PET classification in preclinical Alzheimer's disease.

INTRODUCTION: Incorporating blood-based Alzheimer's disease biomarkers such as tau and amyloid beta (Aß) into screening algorithms may improve screening efficiency. METHODS: Plasma Aß, phosphorylated tau (p-tau)181, and p-tau217 concentration levels from AHEAD 3-45 study participants were measured using mass spectrometry. Tau concentration ratios for each proteoform were calculated to normalize for inter-individual differences. Receiver operating characteristic (ROC) curve analysis was performed for each biomarker against amyloid positivity, defined by > 20 Centiloids. Mixture of experts analysis assessed the value of including tau concentration ratios into the existing predictive algorithm for amyloid positron emission tomography status. RESULTS: The area under the receiver operating curve (AUC) was 0.87 for Aß42/Aß40, 0.74 for phosphorylated variant p-tau181 ratio (p-tau181/np-tau181), and 0.92 for phosphorylated variant p-tau217 ratio (p-tau217/np-tau217). The Plasma Predicted Centiloid (PPC), a predictive model including p-tau217/np-tau217, Aß42/Aß40, age, and apolipoprotein E improved AUC to 0.95. DISCUSSION: Including plasma p-tau217/np-tau217 along with Aß42/Aß40 in predictive algorithms may streamline screening preclinical individuals into anti-amyloid clinical trials. CLINICALTRIALS: gov Identifier: NCT04468659 HIGHLIGHTS: The addition of plasma phosphorylated variant p-tau217 ratio (p-tau217/np-tau217) significantly improved plasma biomarker algorithms for identifying preclinical amyloid positron emission tomography positivity. Prediction performance at higher NAV Centiloid levels was improved with p-tau217/np-tau217. All models generated for this study are incorporated into the Plasma Predicted Centiloid (PPC) app for public use.

Clinical validation of the PrecivityAD2 blood test: A mass spectrometry-based test with algorithm combining %p-tau217 and Aß42/40 ratio to identify presence of brain amyloid.

BACKGROUND: With the availability of disease-modifying therapies for Alzheimer's disease (AD), it is important for clinicians to have tests to aid in AD diagnosis, especially when the presence of amyloid pathology is a criterion for receiving treatment. METHODS: High-throughput, mass spectrometry-based assays were used to measure %p-tau217 and amyloid beta (Aß)42/40 ratio in blood samples from 583 individuals with suspected AD (53% positron emission tomography [PET] positive by Centiloid > 25). An algorithm (PrecivityAD2 test) was developed using these plasma biomarkers to identify brain amyloidosis by PET. RESULTS: The area under the receiver operating characteristic curve (AUC-ROC) for %p-tau217 (0.94) was statistically significantly higher than that for p-tau217 concentration (0.91). The AUC-ROC for the PrecivityAD2 test output, the Amyloid Probability Score 2, was 0.94, yielding 88% agreement with amyloid PET. Diagnostic performance of the APS2 was similar by ethnicity, sex, age, and apoE4 status. DISCUSSION: The PrecivityAD2 blood test showed strong clinical validity, with excellent agreement with brain amyloidosis by PET.

Plasma Aß42/Aß40 Ratios in Older People With Human Immunodeficiency Virus.

BACKGROUND: As people with human immunodeficiency virus (HIV) (PWH) age, it remains unclear whether they are at higher risk for age-related neurodegenerative disorders-for example, Alzheimer disease (AD)-and, if so, how to differentiate HIV-associated neurocognitive impairment from AD. We examined a clinically available blood biomarker test for AD (plasma amyloid-ß [Aß] 42/Aß40 ratio) in PWH who were cognitively normal (PWH_CN) or cognitively impaired (PWH_CI) and people without HIV (PWoH) who were cognitively normal (PWoH_CN) or had symptomatic AD (PWoH_AD). METHODS: A total of 66 PWH (age >40 years) (HIV RNA <50 copies/mL) and 195 PWoH provided blood samples, underwent magnetic resonance imaging, and completed a neuropsychological battery or clinical dementia rating scale. Participants were categorized by impairment (PWH_CN, n = 43; PWH_CI, n = 23; PWoH_CN, n = 138; PWoH_AD, n = 57). Plasma Aß42 and Aß40 concentrations were obtained using a liquid chromatography-tandem mass spectrometry method to calculate the PrecivityAD amyloid probability score (APS). The APS incorporates age and apolipoprotein E proteotype into a risk score for brain amyloidosis. Plasma Aß42/Aß40 ratios and APSs were compared between groups and assessed for relationships with hippocampal volumes or cognition and HIV clinical characteristics (PWH only). RESULTS: The plasma Aß42/Aß40 ratio was significantly lower, and the APS higher, in PWoH_AD than in other groups. A lower Aß42/Aß40 ratio and higher APS was associated with smaller hippocampal volumes for PWoH_AD. The Aß42/Aß40 ratio and APS were not associated with cognition or HIV clinical measures for PWH. CONCLUSIONS: The plasma Aß42/Aß40 ratio can serve as a screening tool for AD and may help differentiate effects of HIV from AD within PWH, but larger studies with older PWH are needed.

Characterization of pre-analytical sample handling effects on a panel of Alzheimer's disease-related blood-based biomarkers: Results from the Standardization of Alzheimer's Blood Biomarkers (SABB) working group.

INTRODUCTION: Pre-analytical sample handling might affect the results of Alzheimer's disease blood-based biomarkers. We empirically tested variations of common blood collection and handling procedures. METHODS: We created sample sets that address the effect of blood collection tube type, and of ethylene diamine tetraacetic acid plasma delayed centrifugation, centrifugation temperature, aliquot volume, delayed storage, and freeze-thawing. We measured amyloid beta (Aß)42 and 40 peptides with six assays, and Aß oligomerization-tendency (OAß), amyloid precursor protein (APP)699-711 , glial fibrillary acidic protein (GFAP), neurofilament light (NfL), total tau (t-tau), and phosphorylated tau181. RESULTS: Collection tube type resulted in different values of all assessed markers. Delayed plasma centrifugation and storage affected Aß and t-tau; t-tau was additionally affected by centrifugation temperature. The other markers were resistant to handling variations. DISCUSSION: We constructed a standardized operating procedure for plasma handling, to facilitate introduction of blood-based biomarkers into the research and clinical settings.

PP2A-3 interacts with ACR4 and regulates formative cell division in the Arabidopsis root.

In plants, the generation of new cell types and tissues depends on coordinated and oriented formative cell divisions. The plasma membrane-localized receptor kinase ARABIDOPSIS CRINKLY 4 (ACR4) is part of a mechanism controlling formative cell divisions in the Arabidopsis root. Despite its important role in plant development, very little is known about the molecular mechanism with which ACR4 is affiliated and its network of interactions. Here, we used various complementary proteomic approaches to identify ACR4-interacting protein candidates that are likely regulators of formative cell divisions and that could pave the way to unraveling the molecular basis behind ACR4-mediated signaling. We identified PROTEIN PHOSPHATASE 2A-3 (PP2A-3), a catalytic subunit of PP2A holoenzymes, as a previously unidentified regulator of formative cell divisions and as one of the first described substrates of ACR4. Our in vitro data argue for the existence of a tight posttranslational regulation in the associated biochemical network through reciprocal regulation between ACR4 and PP2A-3 at the phosphorylation level.

C-terminal phosphorylation of NaV1.5 impairs FGF13-dependent regulation of channel inactivation.

Voltage-gated Na+ (NaV) channels are key regulators of myocardial excitability, and Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent alterations in NaV1.5 channel inactivation are emerging as a critical determinant of arrhythmias in heart failure. However, the global native phosphorylation pattern of NaV1.5 subunits associated with these arrhythmogenic disorders and the associated channel regulatory defects remain unknown. Here, we undertook phosphoproteomic analyses to identify and quantify in situ the phosphorylation sites in the NaV1.5 proteins purified from adult WT and failing CaMKIIδc-overexpressing (CaMKIIδc-Tg) mouse ventricles. Of 19 native NaV1.5 phosphorylation sites identified, two C-terminal phosphoserines at positions 1938 and 1989 showed increased phosphorylation in the CaMKIIδc-Tg compared with the WT ventricles. We then tested the hypothesis that phosphorylation at these two sites impairs fibroblast growth factor 13 (FGF13)-dependent regulation of NaV1.5 channel inactivation. Whole-cell voltage-clamp analyses in HEK293 cells demonstrated that FGF13 increases NaV1.5 channel availability and decreases late Na+ current, two effects that were abrogated with NaV1.5 mutants mimicking phosphorylation at both sites. Additional co-immunoprecipitation experiments revealed that FGF13 potentiates the binding of calmodulin to NaV1.5 and that phosphomimetic mutations at both sites decrease the interaction of FGF13 and, consequently, of calmodulin with NaV1.5. Together, we have identified two novel native phosphorylation sites in the C terminus of NaV1.5 that impair FGF13-dependent regulation of channel inactivation and may contribute to CaMKIIδc-dependent arrhythmogenic disorders in failing hearts.

Recommendations for the Generation, Quantification, Storage, and Handling of Peptides Used for Mass Spectrometry-Based Assays.

BACKGROUND: For many years, basic and clinical researchers have taken advantage of the analytical sensitivity and specificity afforded by mass spectrometry in the measurement of proteins. Clinical laboratories are now beginning to deploy these work flows as well. For assays that use proteolysis to generate peptides for protein quantification and characterization, synthetic stable isotope-labeled internal standard peptides are of central importance. No general recommendations are currently available surrounding the use of peptides in protein mass spectrometric assays. CONTENT: The Clinical Proteomic Tumor Analysis Consortium of the National Cancer Institute has collaborated with clinical laboratorians, peptide manufacturers, metrologists, representatives of the pharmaceutical industry, and other professionals to develop a consensus set of recommendations for peptide procurement, characterization, storage, and handling, as well as approaches to the interpretation of the data generated by mass spectrometric protein assays. Additionally, the importance of carefully characterized reference materials-in particular, peptide standards for the improved concordance of amino acid analysis methods across the industry-is highlighted. The alignment of practices around the use of peptides and the transparency of sample preparation protocols should allow for the harmonization of peptide and protein quantification in research and clinical care.

The growing story of (ARABIDOPSIS) CRINKLY 4.

Receptor kinases play important roles in plant growth and development, but only few of them have been functionally characterized in depth. Over the past decade CRINKLY 4 (CR4)-related research has peaked as a result of a newly discovered role of ARABIDOPSIS CR4 (ACR4) in the root. Here, we comprehensively review the available (A)CR4 literature and describe its role in embryo, seed, shoot, and root development, but we also flag an unexpected role in plant defence. In addition, we discuss ACR4 domains and protein structure, describe known ACR4-interacting proteins and substrates, and elaborate on the transcriptional regulation of ACR4 Finally, we address the missing knowledge in our understanding of ACR4 signalling.

Genomic impact of transient low-dose decitabine treatment on primary AML cells.

Acute myeloid leukemia (AML) is characterized by dysregulated gene expression and abnormal patterns of DNA methylation; the relationship between these events is unclear. Many AML patients are now being treated with hypomethylating agents, such as decitabine (DAC), although the mechanisms by which it induces remissions remain unknown. The goal of this study was to use a novel stromal coculture assay that can expand primary AML cells to identify the immediate changes induced by DAC with a dose (100nM) that decreases total 5-methylcytosine content and reactivates imprinted genes (without causing myeloid differentiation, which would confound downstream genomic analyses). Using array-based technologies, we found that DAC treatment caused global hypomethylation in all samples (with a preference for regions with higher levels of baseline methylation), yet there was limited correlation between changes in methylation and gene expression. Moreover, the patterns of methylation and gene expression across the samples were primarily determined by the intrinsic properties of the primary cells, rather than DAC treatment. Although DAC induces hypomethylation, we could not identify canonical target genes that are altered by DAC in primary AML cells, suggesting that the mechanism of action of DAC is more complex than previously recognized.

Associations of sleep duration and daytime sleepiness with plasma amyloid beta and cognitive performance in cognitively unimpaired, middle-aged and older African Americans.

STUDY OBJECTIVES: Given the established racial disparities in both sleep health and dementia risk for African American populations, we assess cross-sectional and longitudinal associations of self-report sleep duration (SRSD) and daytime sleepiness with plasma amyloid beta (Aß) and cognition in an African American (AA) cohort. METHODS: In a cognitively unimpaired sample drawn from the African Americans Fighting Alzheimer's in Midlife (AA-FAiM) study, data on SRSD, Epworth Sleepiness Scale, demographics, and cognitive performance were analyzed. Aß40, Aß42, and the Aß42/40 ratio were quantified from plasma samples. Cross-sectional analyses explored associations between baseline predictors and outcome measures. Linear mixed-effect regression models estimated associations of SRSD and daytime sleepiness with plasma Aß and cognitive performance levels and change over time. RESULTS: One hundred and forty-seven participants comprised the cross-sectional sample. Baseline age was 63.2 ±â€…8.51 years. 69.6% self-identified as female. SRSD was 6.4 ±â€…1.1 hours and 22.4% reported excessive daytime sleepiness. The longitudinal dataset included 57 participants. In fully adjusted models, neither SRSD nor daytime sleepiness is associated with cross-sectional or longitudinal Aß. Associations with level and trajectory of cognitive test performance varied by measure of sleep health. CONCLUSIONS: SRSD was below National Sleep Foundation recommendations and daytime sleepiness was prevalent in this cohort. In the absence of observed associations with plasma Aß, poorer self-reported sleep health broadly predicted poorer cognitive function but not accelerated decline. Future research is necessary to understand and address modifiable sleep mechanisms as they relate to cognitive aging in AA at disproportionate risk for dementia. CLINICAL TRIAL INFORMATION: Not applicable.

Insights into molecular interactions between the juxtamembrane and kinase subdomains of the Arabidopsis Crinkly-4 receptor-like kinase.

Arabidopsis CRINKLY4 (ACR4), a receptor-like kinase required for plant growth and development, possesses an extracellular ligand binding domain, a transmembrane helix, and an intracellular domain (ICD). The ICD contains the juxtamembrane (JMD) and the C-terminal (CTD) subdomains, which flank the core kinase domain (KD), with at least 16 autophosphorylation sites. Phosphorylation sites are often docking sites for the modification-dependent recruitment of interacting proteins that orchestrate many downstream signaling events. In this context, we have specifically probed the role of the two phosphorylation sites Ser(475) and Thr(478) in the JMD using mutagenesis and phage-peptide screening techniques. Thus, naïve and phosphorylated 15-mer peptides derived from the JMD were panned against a 21-amino acid random phage peptide library. The phosphorylated peptide preferentially recognized the consensus sequence LxSLL. This sequence harbors the LxxLL motif, a known protein-protein interaction motif that is also present in the N-terminal lobe of the KD. We demonstrate the binding of JMD peptides to the KD and also show through kinetic analyses of mutants that phosphorylation of Ser(475) and Thr(478) in the JMD is necessary for optimal substrate phosphorylation in vitro. Our experiments suggest that an intramolecular interaction can occur between the JM and the N-terminal lobe of the KD.

Independent study demonstrates amyloid probability score accurately indicates amyloid pathology.

BACKGROUND: The amyloid probability score (APS) is the model read-out of the analytically validated mass spectrometry-based PrecivityAD® blood test that incorporates the plasma Aß42/40 ratio, ApoE proteotype, and age to identify the likelihood of brain amyloid plaques among cognitively impaired individuals being evaluated for Alzheimer's disease. PURPOSE: This study aimed to provide additional independent evidence that the pre-established APS algorithm, along with its cutoff values, discriminates between amyloid positive and negative individuals. METHODS: The diagnostic performance of the PrecivityAD test was analyzed in a cohort of 200 nonrandomly selected Australian Imaging, Biomarker & Lifestyle Flagship Study of Aging (AIBL) study participants, who were either cognitively impaired or healthy controls, and for whom a blood sample and amyloid PET imaging were available. RESULTS: In a subset of the dataset aligned with the Intended Use population (patients aged 60 and older with CDR ≥0.5), the pre-established APS algorithm predicted amyloid PET with a sensitivity of 84.9% (CI: 72.9-92.1%) and specificity of 96% (CI: 80.5-99.3%), exclusive of 13 individuals for whom the test was inconclusive. INTERPRETATION: The study shows individuals with a high APS are more likely than those with a low APS to have abnormal amounts of amyloid plaques and be on an amyloid accumulation trajectory, a dynamic and evolving process characteristic of progressive AD pathology. Exploratory data suggest APS retains its diagnostic performance in healthy individuals, supporting further screening studies in the cognitively unimpaired.

Comparison of plasma and CSF biomarkers in predicting cognitive decline.

OBJECTIVES: Concentrations of amyloid-ß peptides (Aß42/Aß40) and neurofilament light (NfL) can be measured in plasma or cerebrospinal fluid (CSF) and are associated with Alzheimer's disease brain pathology and cognitive impairment. This study directly compared plasma and CSF measures of Aß42/Aß40 and NfL as predictors of cognitive decline. METHODS: Participants were 65 years or older and cognitively normal at baseline with at least one follow-up cognitive assessment. Analytes were measured with the following types of assays: plasma Aß42/Aß40, immunoprecipitation-mass spectrometry; plasma NfL, Simoa; CSF Aß42/Aß40, automated immunoassay; CSF NfL plate-based immunoassay. Mixed effects models evaluated the global cognitive composite score over a maximum of 6 years as predicted by the fluid biomarkers. RESULTS: Analyses included 371 cognitively normal participants, aged 72.7 ± 5.2 years (mean ± standard deviation) with an average length of follow-up of 3.9 ± 1.6 years. Standardized concentrations of biomarkers were associated with annualized cognitive change: plasma Aß42/Aß40, 0.014 standard deviations (95% confidence intervals 0.002 to 0.026); CSF Aß42/Aß40, 0.020 (0.008 to 0.032); plasma Nfl, -0.018 (-0.030 to -0.005); and CSF NfL, -0.024 (-0.036 to -0.012). Power analyses estimated that 266 individuals in each treatment arm would be needed to detect a 50% slowing of decline if identified by abnormal plasma measures versus 229 for CSF measures. INTERPRETATION: Both plasma and CSF measures of Aß42/Aß40 and NfL predicted cognitive decline. A clinical trial that enrolled individuals based on abnormal plasma Aß42/Aß40 and NfL levels would require only a marginally larger cohort than if CSF measures were used.

Assessment of a Plasma Amyloid Probability Score to Estimate Amyloid Positron Emission Tomography Findings Among Adults With Cognitive Impairment.

Importance: The diagnostic evaluation for Alzheimer disease may be improved by a blood-based diagnostic test identifying presence of brain amyloid plaque pathology. Objective: To determine the clinical performance associated with a diagnostic algorithm incorporating plasma amyloid-ß (Aß) 42:40 ratio, patient age, and apoE proteotype to identify brain amyloid status. Design, Setting, and Participants: This cohort study includes analysis from 2 independent cross-sectional cohort studies: the discovery cohort of the Plasma Test for Amyloidosis Risk Screening (PARIS) study, a prospective add-on to the Imaging Dementia-Evidence for Amyloid Scanning study, including 249 patients from 2018 to 2019, and MissionAD, a dataset of 437 biobanked patient samples obtained at screenings during 2016 to 2019. Data were analyzed from May to November 2020. Exposures: Amyloid detected in blood and by positron emission tomography (PET) imaging. Main Outcomes and Measures: The main outcome was the diagnostic performance of plasma Aß42:40 ratio, together with apoE proteotype and age, for identifying amyloid PET status, assessed by accuracy, sensitivity, specificity, and area under the receiver operating characteristic curve (AUC). Results: All 686 participants (mean [SD] age 73.2 [6.3] years; 368 [53.6%] men; 378 participants [55.1%] with amyloid PET findings) had symptoms of mild cognitive impairment or mild dementia. The AUC of plasma Aß42:40 ratio for PARIS was 0.79 (95% CI, 0.73-0.85) and 0.86 (95% CI, 0.82-0.89) for MissionAD. Ratio cutoffs for Aß42:40 based on the Youden index were similar between cohorts (PARIS: 0.089; MissionAD: 0.092). A logistic regression model (LRM) incorporating Aß42:40 ratio, apoE proteotype, and age improved diagnostic performance within each cohort (PARIS: AUC, 0.86 [95% CI, 0.81-0.91]; MissionAD: AUC, 0.89 [95% CI, 0.86-0.92]), and overall accuracy was 78% (95% CI, 72%-83%) for PARIS and 83% (95% CI, 79%-86%) for MissionAD. The model developed on the prospectively collected samples from PARIS performed well on the MissionAD samples (AUC, 0.88 [95% CI, 0.84-0.91]; accuracy, 78% [95% CI, 74%-82%]). Training the LRM on combined cohorts yielded an AUC of 0.88 (95% CI, 0.85-0.91) and accuracy of 81% (95% CI, 78%-84%). The output of this LRM is the Amyloid Probability Score (APS). For clinical use, 2 APS cutoff values were established yielding 3 categories, with low, intermediate, and high likelihood of brain amyloid plaque pathology. Conclusions and Relevance: These findings suggest that this blood biomarker test could allow for distinguishing individuals with brain amyloid-positive PET findings from individuals with amyloid-negative PET findings and serve as an aid for Alzheimer disease diagnosis.

Identification of in vitro autophosphorylation sites and effects of phosphorylation on the Arabidopsis CRINKLY4 (ACR4) receptor-like kinase intracellular domain: insights into conformation, oligomerization, and activity.

Arabidopsis CRINKLY4 (ACR4) is a receptor-like kinase (RLK) that consists of an extracellular domain and an intracellular domain (ICD) with serine/threonine kinase activity. While genetic and cell biology experiments have demonstrated that ACR4 is important in cell fate specification and overall development of the plant, little is known about the biochemical properties of the kinase domain and the mechanisms that underlie the overall function of the receptor. To complement in planta studies of the function of ACR4, we have expressed the ICD in Escherichia coli as a soluble C-terminal fusion to the N-utilization substance A (NusA) protein, purified the recombinant protein, and characterized the enzymatic and conformational properties. The protein autophosphorylates via an intramolecular mechanism, prefers Mn(2+) over Mg(2+) as the divalent cation, and displays typical Michaelis-Menten kinetics with respect to ATP with an apparent K(m) of 6.67 ± 2.07 µM and a V(max) of 1.83 ± 0.18 nmol min(-1) mg(-1). Autophosphorylation is accompanied by a conformational change as demonstrated by circular dichroism, fluorescence spectroscopy, and limited proteolysis with trypsin. Analysis by nanoliquid chromatography and mass spectrometry revealed 16 confirmed sites of phosphorylation at Ser and Thr residues. Sedimentation velocity and gel filtration experiments indicate that the ICD has a propensity to oligomerize and that this property is lost upon autophosphorylation.

The PrecivityAD™ test: Accurate and reliable LC-MS/MS assays for quantifying plasma amyloid beta 40 and 42 and apolipoprotein E proteotype for the assessment of brain amyloidosis.

BACKGROUND: There is an unmet need for an accessible, less invasive, cost-effective method to facilitate clinical trial enrollment and aid in clinical Alzheimer's disease (AD) diagnosis. APOE genotype affects the clearance and deposition of amyloid-beta (Aß) with APOE4 carriers having increased risk while APOE2 alleles appear to be protective. Lower plasma Aß42/40 correlates with brain amyloidosis. In response, C2N has developed the PrecivityAD™ test; plasma LC-MS/MS assays for Aß isoform quantitation and qualitative APOE isoform-specific proteotyping. METHODS: In accord with CLIA standards, we developed and validated assay performance: precision, accuracy, linearity, limit of detection (LoD), interferences. RESULTS: Within-day precision varied from 1.5-3.0% (Aß40) and 2.5-8.4% (Aß42). Total (within-lab) variability was 2.7-7.7% (Aß40) and 3.1-9.5% (Aß42). Aß40 quantitation was linear from 10 to 1780 pg/mL; Aß42 was linear from 2 to 254 pg/mL. LoD was 11 and 2 pg/mL for Aß40 and Aß42, respectively. APOE proteotypes were 100% concordant with genotype, while LoD (fM) was much lower than APOE concentrations observed in plasma (mM). CONCLUSIONS: The PrecivityAD™ assays are precise, accurate, sensitive, and linear over a wide analytical range, free from significant interferences, and suitable for use in the clinical laboratory.

A blood-based diagnostic test incorporating plasma Aß42/40 ratio, ApoE proteotype, and age accurately identifies brain amyloid status: findings from a multi cohort validity analysis.

BACKGROUND: The development of blood-based biomarker tests that are accurate and robust for Alzheimer's disease (AD) pathology have the potential to aid clinical diagnosis and facilitate enrollment in AD drug trials. We developed a high-resolution mass spectrometry (MS)-based test that quantifies plasma Aß42 and Aß40 concentrations and identifies the ApoE proteotype. We evaluated robustness, clinical performance, and commercial viability of this MS biomarker assay for distinguishing brain amyloid status. METHODS: We used the novel MS assay to analyze 414 plasma samples that were collected, processed, and stored using site-specific protocols, from six independent US cohorts. We used receiver operating characteristic curve (ROC) analyses to assess assay performance and accuracy for predicting amyloid status (positive, negative, and standard uptake value ratio; SUVR). After plasma analysis, sites shared brain amyloid status, defined using diverse, site-specific methods and cutoff values; amyloid PET imaging using various tracers or CSF Aß42/40 ratio. RESULTS: Plasma Aß42/40 ratio was significantly (p < 0.001) lower in the amyloid positive vs. negative participants in each cohort. The area under the ROC curve (AUC-ROC) was 0.81 (95% CI = 0.77-0.85) and the percent agreement between plasma Aß42/40 and amyloid positivity was 75% at the optimal (Youden index) cutoff value. The AUC-ROC (0.86; 95% CI = 0.82-0.90) and accuracy (81%) for the plasma Aß42/40 ratio improved after controlling for cohort heterogeneity. The AUC-ROC (0.90; 95% CI = 0.87-0.93) and accuracy (86%) improved further when Aß42/40, ApoE4 copy number and participant age were included in the model. CONCLUSIONS: This mass spectrometry-based plasma biomarker test: has strong diagnostic performance; can accurately distinguish brain amyloid positive from amyloid negative individuals; may aid in the diagnostic evaluation process for Alzheimer's disease; and may enhance the efficiency of enrolling participants into Alzheimer's disease drug trials.

Investigation of Autophosphorylation Sites of Plant Receptor Kinases and Phosphorylation of Interacting Partners.

The optimal kinase activity of plant receptor-like kinases (RLKs) is often regulated by autophosphorylation of specific sites. Many of these phosphorylated residues then serve as recruiting sites for downstream interacting proteins. Therefore, identification of the phosphosites can be an important first step in delineating the signaling network. This chapter describes a protocol for identification of phosphorylated residues by mass spectrometry as well as a protocol to determine if an interacting partner can be phosphorylated in vitro.

Using the CPTAC Assay Portal to Identify and Implement Highly Characterized Targeted Proteomics Assays.

The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as an open-source repository of well-characterized targeted proteomic assays. The portal is designed to curate and disseminate highly characterized, targeted mass spectrometry (MS)-based assays by providing detailed assay performance characterization data, standard operating procedures, and access to reagents. Assay content is accessed via the portal through queries to find assays targeting proteins associated with specific cellular pathways, protein complexes, or specific chromosomal regions. The position of the peptide analytes for which there are available assays are mapped relative to other features of interest in the protein, such as sequence domains, isoforms, single nucleotide polymorphisms, and posttranslational modifications. The overarching goals are to enable robust quantification of all human proteins and to standardize the quantification of targeted MS-based assays to ultimately enable harmonization of results over time and across laboratories.