Skyline is a freely available, open‐source Windows client application for accelerating targeted proteomics experimentation, with an emphasis on the proteomics and mass spectrometry community as users ...and as contributors. This review covers the informatics encompassed by the Skyline ecosystem, from computationally assisted targeted mass spectrometry method development, to raw acquisition file data processing, and quantitative analysis and results sharing.
Data-independent acquisition (DIA) is an emerging mass spectrometry (MS)-based technique for unbiased and reproducible measurement of protein mixtures. DIA tandem mass spectrometry spectra are often ...highly multiplexed, containing product ions from multiple cofragmenting precursors. Detecting peptides directly from DIA data is therefore challenging; most DIA data analyses require spectral libraries. Here we present PECAN (http://pecan.maccosslab.org), a library-free, peptide-centric tool that robustly and accurately detects peptides directly from DIA data. PECAN reports evidence of detection based on product ion scoring, which enables detection of low-abundance analytes with poor precursor ion signal. We demonstrate the chromatographic peak picking accuracy and peptide detection capability of PECAN, and we further validate its detection with data-dependent acquisition and targeted analyses. Lastly, we used PECAN to build a plasma proteome library from DIA data and to query known sequence variants.
We examined whether plasma β-amyloid (Aβ)42/Aβ40, as measured by a high-precision assay, accurately diagnosed brain amyloidosis using amyloid PET or CSF p-tau181/Aβ42 as reference standards.
Using an ...immunoprecipitation and liquid chromatography-mass spectrometry assay, we measured Aβ42/Aβ40 in plasma and CSF samples from 158 mostly cognitively normal individuals that were collected within 18 months of an amyloid PET scan.
Plasma Aβ42/Aβ40 had a high correspondence with amyloid PET status (receiver operating characteristic area under the curve AUC 0.88, 95% confidence interval CI 0.82-0.93) and CSF p-tau181/Aβ42 (AUC 0.85, 95% CI 0.79-0.92). The combination of plasma Aβ42/Aβ40, age, and
ε4 status had a very high correspondence with amyloid PET (AUC 0.94, 95% CI 0.90-0.97). Individuals with a negative amyloid PET scan at baseline and a positive plasma Aβ42/Aβ40 (<0.1218) had a 15-fold greater risk of conversion to amyloid PET-positive compared to individuals with a negative plasma Aβ42/Aβ40 (
= 0.01).
Plasma Aβ42/Aβ40, especially when combined with age and
ε4 status, accurately diagnoses brain amyloidosis and can be used to screen cognitively normal individuals for brain amyloidosis. Individuals with a negative amyloid PET scan and positive plasma Aβ42/Aβ40 are at increased risk for converting to amyloid PET-positive. Plasma Aβ42/Aβ40 could be used in prevention trials to screen for individuals likely to be amyloid PET-positive and at risk for Alzheimer disease dementia.
This study provides Class II evidence that plasma Aβ42/Aβ40 levels accurately determine amyloid PET status in cognitively normal research participants.
The extracellular buildup of amyloid beta (Aβ) plaques in the brain is a hallmark of Alzheimer's disease (AD). Detection of Aβ pathology is essential for AD diagnosis and for identifying and ...recruiting research participants for clinical trials evaluating disease-modifying therapies. Currently, AD diagnoses are usually made by clinical assessments, although detection of AD pathology with positron emission tomography (PET) scans or cerebrospinal fluid (CSF) analysis can be used by specialty clinics. These measures of Aβ aggregation, e.g. plaques, protofibrils, and oligomers, are medically invasive and often only available at specialized medical centers or not covered by medical insurance, and PET scans are costly. Therefore, a major goal in recent years has been to identify blood-based biomarkers that can accurately detect AD pathology with cost-effective, minimally invasive procedures.To assess the performance of plasma Aβ assays in predicting amyloid burden in the central nervous system (CNS), this review compares twenty-one different manuscripts that used measurements of 42 and 40 amino acid-long Aβ (Aβ42 and Aβ40) in plasma to predict CNS amyloid status. Methodologies that quantitate Aβ42 and 40 peptides in blood via immunoassay or immunoprecipitation-mass spectrometry (IP-MS) were considered, and their ability to distinguish participants with amyloidosis compared to amyloid PET and CSF Aβ measures as reference standards was evaluated. Recent studies indicate that some IP-MS assays perform well in accurately and precisely measuring Aβ and detecting brain amyloid aggregates.
To determine the diagnostic accuracy of a plasma Aβ42/Aβ40 assay in classifying amyloid PET status across global research studies using samples collected by multiple centers that utilize different ...blood collection and processing protocols.
Plasma samples (n = 465) were obtained from 3 large Alzheimer disease (AD) research cohorts in the United States (n = 182), Australia (n = 183), and Sweden (n = 100). Plasma Aβ42/Aβ40 was measured by a high precision immunoprecipitation mass spectrometry (IPMS) assay and compared to the reference standards of amyloid PET and CSF Aβ42/Aβ40.
In the combined cohort of 465 participants, plasma Aβ42/Aβ40 had good concordance with amyloid PET status (receiver operating characteristic area under the curve AUC 0.84, 95% confidence interval CI 0.80-0.87); concordance improved with the inclusion of
ε4 carrier status (AUC 0.88, 95% CI 0.85-0.91). The AUC of plasma Aβ42/Aβ40 with CSF amyloid status was 0.85 (95% CI 0.78-0.91) and improved to 0.93 (95% CI 0.89-0.97) with
ε4 status. These findings were consistent across the 3 cohorts, despite differences in protocols. The assay performed similarly in both cognitively unimpaired and impaired individuals.
Plasma Aβ42/Aβ40 is a robust measure for detecting amyloid plaques and can be utilized to aid in the diagnosis of AD, identify those at risk for future dementia due to AD, and improve the diversity of populations enrolled in AD research and clinical trials.
This study provides Class II evidence that plasma Aβ42/Aβ40, as measured by a high precision IPMS assay, accurately diagnoses brain amyloidosis in both cognitively unimpaired and impaired research participants.
Quantitative analysis of fatty acids (FAs) is an important area of analytical biochemistry. Ultra high sensitivity FA analysis usually is done with gas chromatography of pentafluorobenzyl esters ...coupled to an electron-capture detector. With the popularity of electrospray ionization (ESI) mass spectrometers coupled to liquid chromatography, it would be convenient to develop a method for ultra high sensitivity FA detection using this equipment. Although FAs can be analyzed by ESI in negative ion mode, this method is not very sensitive. In this study, we demonstrate a new method of FA analysis based on conversion of the carboxylic acid to an amide bearing a permanent positive charge, N-(4-aminomethylphenyl)pyridinium (AMPP) combined with analysis on a reverse-phase liquid chromatography column coupled to an ESI mass spectrometer operating in positive ion mode. This leads to an ∼60,000-fold increase in sensitivity compared with the same method carried out with underivatized FAs. The new method is about 10-fold more sensitive than the existing method of gas chromatography/electron-capture mass spectrometry of FA pentafluorobenzyl esters. Furthermore, significant fragmentation of the precursor ions in the nontag portion improves analytical specificity. We show that a large number of FA molecular species can be analyzed with this method in complex biological samples such as mouse serum.
Introduction
Sleep deprivation increases cerebrospinal fluid (CSF) amyloid beta (Aβ) and tau levels; however, sleep's effect on Aβ and tau in plasma is unknown.
Methods
In a cross‐over design, CSF Aβ ...and tau concentrations were measured in five cognitively normal individuals who had blood and CSF collected every 2 hours for 36 hours during sleep‐deprived and normal sleep control conditions.
Results
Aβ40, Aβ42, unphosphorylated tau threonine181 (T181), unphosphorylated tau threonine‐217 (T217), and phosphorylated T181 (pT181) concentrations increased ∼35% to 55% in CSF and decreased ∼5% to 15% in plasma during sleep deprivation. CSF/plasma ratios of all Alzheimer's disease (AD) biomarkers increased during sleep deprivation while the CSF/plasma albumin ratio, a measure of blood–CSF barrier permeability, decreased. CSF and plasma Aβ42/40, pT181/T181, and pT181/Aβ42 ratios were stable longitudinally in both groups.
Discussion
These findings show that sleep loss alters some plasma AD biomarkers by lowering brain clearance mechanisms and needs to be taken into account when interpreting individual plasma AD biomarkers but not ratios.
Mitochondrial DNA (mtDNA) is a highly potent inflammatory trigger and is reportedly found outside the cells in blood in various pathologies. Platelets are abundant in blood where they promote ...hemostasis. Although lacking a nucleus, platelets contain functional mitochondria. On activation, platelets produce extracellular vesicles known as microparticles. We hypothesized that activated platelets could also release their mitochondria. We show that activated platelets release respiratory-competent mitochondria, both within membrane-encapsulated microparticles and as free organelles. Extracellular mitochondria are found in platelet concentrates used for transfusion and are present at higher levels in those that induced acute reactions (febrile nonhemolytic reactions, skin manifestations, and cardiovascular events) in transfused patients. We establish that the mitochondrion is an endogenous substrate of secreted phospholipase A2 IIA (sPLA2-IIA), a phospholipase otherwise specific for bacteria, likely reflecting the ancestral proteobacteria origin of mitochondria. The hydrolysis of the mitochondrial membrane by sPLA2-IIA yields inflammatory mediators (ie, lysophospholipids, fatty acids, and mtDNA) that promote leukocyte activation. Two-photon microscopy in live transfused animals revealed that extracellular mitochondria interact with neutrophils in vivo, triggering neutrophil adhesion to the endothelial wall. Our findings identify extracellular mitochondria, produced by platelets, at the midpoint of a potent mechanism leading to inflammatory responses.
•When activated and in platelet storage bags, platelets release respiratory-competent mitochondria, a recognized damage-associated molecular pattern.•Mitochondria, descendant of Rickettsia prowazekii, serve as substrate for bactericidal sPLA2-IIA to promote inflammation.
Objective
In Alzheimer's disease, hyperphosphorylated tau is associated with formation of insoluble paired helical filaments that aggregate as neurofibrillary tau tangles and are associated with ...neuronal loss and cognitive symptoms. Dual orexin receptor antagonists decrease soluble amyloid‐β levels and amyloid plaques in mouse models overexpressing amyloid‐β, but have not been reported to affect tau phosphorylation. In this randomized controlled trial, we tested the acute effect of suvorexant, a dual orexin receptor antagonist, on amyloid‐β, tau, and phospho‐tau.
Methods
Thirty‐eight cognitively unimpaired participants aged 45 to 65 years were randomized to placebo (N = 13), suvorexant 10 mg (N = 13), and suvorexant 20 mg (N = 12). Six milliliters of cerebrospinal fluid were collected via an indwelling lumbar catheter every 2 hours for 36 hours starting at 20:00. Participants received placebo or suvorexant at 21:00. All samples were processed and measured for multiple forms of amyloid‐β, tau, and phospho‐tau via immunoprecipitation and liquid chromatography‐mass spectrometry.
Results
The ratio of phosphorylated‐tau‐threonine‐181 to unphosphorylated‐tau‐threonine‐181, a measure of phosphorylation at this tau phosphosite, decreased ~10% to 15% in participants treated with suvorexant 20 mg compared to placebo. However, phosphorylation at tau‐serine‐202 and tau‐threonine‐217 were not decreased by suvorexant. Suvorexant decreased amyloid‐β ~10% to 20% compared to placebo starting 5 hours after drug administration.
Interpretation
In this study, suvorexant acutely decreased tau phosphorylation and amyloid‐β concentrations in the central nervous system. Suvorexant is approved by the US Food and Drug Administration to treatment insomnia and may have potential as a repurposed drug for the prevention of Alzheimer's disease, however, future studies with chronic treatment are needed. ANN NEUROL 2023;94:27–40
Combined liquid chromatography−electrospray ionization-tandem mass spectrometry (LC−ESI-MS/MS) is a powerful method for the analysis of oxygenated metabolites of polyunsaturated fatty acids including ...eicosanoids. Here we describe the synthesis of a new derivatization reagent N-(4-aminomethylphenyl)pyridinium (AMPP) that can be coupled to eicosanoids via an amide linkage in quantitative yield. Conversion of the carboxylic acid of eicosanoids to a cationic AMPP amide improves sensitivity of detection by 10- to 20-fold compared to negative mode electrospray ionization detection of underivatized analytes. This charge reversal derivatization allows detection of cations rather than anions in the electrospray ionization mass spectrometer, which enhances sensitivity. Another factor is that AMPP amides undergo considerable collision-induced dissociation in the analyte portion rather than exclusively in the cationic tag portion, which allows isobaric derivatives to be distinguished by tandem mass spectrometry, and this further enhances sensitivity and specificity. This simple derivatization method allows prostaglandins, thromboxane B2, leukotriene B4, hydroxyeicosatetraenoic acid isomers, and arachidonic acid to be quantified in complex biological samples with limits of quantification in the 200−900 fg range. One can anticipate that the AMPP derivatization method can be extended to other carboxylic acid analytes for enhanced sensitivity detection.
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