ObjectiveWe 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.MethodsUsing 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.ResultsPlasma 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 APOE 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 (p = 0.01).ConclusionsPlasma Aβ42/Aβ40, especially when combined with age and APOE 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.Classification of evidenceThis study provides Class II evidence that plasma Aβ42/Aβ40 levels accurately determine amyloid PET status in cognitively normal research participants.

Expression of the full set of human and mouse groups I, II, V, X, and XII secreted phospholipases A 2 (sPLA 2 s) in Escherichia coli and insect cells has provided pure recombinant enzymes for detailed comparative interfacial kinetic and binding studies. The set of mammalian sPLA 2 s display dramatically different sensitivity to dithiothreitol. The specific activity for the hydrolysis of vesicles of differing phospholipid composition by these enzymes varies by up to 4 orders of magnitude, and yet all enzymes display similar catalytic site specificity toward phospholipids with different polar head groups. Discrimination between sn-2 polyunsaturated versus saturated fatty acyl chains is <6-fold. These enzymes display apparent dissociation constants for activation by calcium in the 1-225 M range, depending on the phospholipid substrate. Analysis of the inhibition by a set of 12 active site-directed, competitive inhibitors reveals a large variation in the potency among the mammalian sPLA 2 s, with Me-Indoxam being the most generally potent sPLA 2 inhibitor. A dramatic correlation exists between the ability of the sPLA 2 s to hydrolyze phosphatidylcholine-rich vesicles efficiently in vitro and the ability to release arachidonic acid when added exogenously to mammalian cells; the group V and X sPLA 2 s are uniquely efficient in this regard.

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.

Increased lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ) activity is associated with increased risk of cardiac events, but it is not known whether Lp-PLA 2 is a causative agent. Here we show that selective inhibition of Lp-PLA 2 with darapladib reduced development of advanced coronary atherosclerosis in diabetic and hypercholesterolemic swine. Darapladib markedly inhibited plasma and lesion Lp-PLA 2 activity and reduced lesion lysophosphatidylcholine content. Analysis of coronary gene expression showed that darapladib exerted a general anti-inflammatory action, substantially reducing the expression of 24 genes associated with macrophage and T lymphocyte functioning. Darapladib treatment resulted in a considerable decrease in plaque area and, notably, a markedly reduced necrotic core area and reduced medial destruction, resulting in fewer lesions with an unstable phenotype. These data show that selective inhibition of Lp-PLA 2 inhibits progression to advanced coronary atherosclerotic lesions and confirms a crucial role of vascular inflammation independent from hypercholesterolemia in the development of lesions implicated in the pathogenesis of myocardial infarction and stroke.Atherosclerosis, the most common cause of myocardial infarction, stroke and cardiovascular death, is an inflammatory-immunomodulatory disease 1,2 . A key early step in its development is the accumulation and subsequent oxidation of low-density lipoproteins COMPETING INTERESTS STATEMENTThe authors declare competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/naturemedicine/. Lp-PLA 2 , also known as platelet-activating factor acetylhydrolase or type VIIA PLA 2 , is a calcium-independent phospholipase A 2 . In humans, Lp-PLA 2 is secreted by leukocytes and is associated with circulating LDL and macrophages in atherosclerotic plaques. Although some have hypothesized that Lp-PLA 2 has a protective role in atherosclerotic lesion development 9,10 , the preponderance of recent data suggests that Lp-PLA 2 has an active role in atherosclerotic development and progression [11][12][13] . Elevated circulating Lp-PLA 2 activity predicts increased cardiovascular risk 14 . A proatherogenic role for Lp-PLA 2 has been postulated on the basis of its ability to generate two key proinflammatory mediators, lysophosphatidylcholine (LPC) and oxidized nonesterified fatty acids (oxNEFAs), through the cleavage of oxidized or polar phospholipids generated during LDL oxidation 15,16 . Evidence exists for a regulatory role of these proinflammatory lipids, particularly of LPC 12,13,17 , in promoting atherosclerotic plaque development that can ultimately lead to the formation of a necrotic core. These steps include recruitment and activation of leukocytes 12,18 , induction of apoptosis 12,19 and impaired removal of dead cells 20,21 . The demonstration that Lp-PLA 2 is highly upregulated in macrophages undergoing apoptosis within the necrotic core and fibrous cap of vulnerable and ruptured plaques, ...

Arachidonic acid metabolites, the eicosanoids, are key mediators of allergen-induced airway inflammation and remodeling in asthma. The availability of free arachidonate in cells for subsequent eicosanoid biosynthesis is controlled by phospholipase A2s (PLA2s), most notably cytosolic PLA2-α. 10 secreted PLA2s (sPLA2s) have also been identified, but their function in eicosanoid generation is poorly understood. We investigated the role of group X sPLA2 (sPLA2-X), the sPLA2 with the highest in vitro cellular phospholipolysis activity, in acute and chronic mouse asthma models in vivo. The lungs of sPLA2-X−/− mice, compared with those of sPLA2-X+/+ littermates, had significant reduction in ovalbumin-induced infiltration by CD4+ and CD8+ T cells and eosinophils, goblet cell metaplasia, smooth muscle cell layer thickening, subepithelial fibrosis, and levels of T helper type 2 cell cytokines and eicosanoids. These data direct attention to sPLA2-X as a novel therapeutic target for asthma.

Mammals contain 9 -10 secreted phospholipases A 2 (sPLA 2 s) that display widely different affinities for membranes, depending on the phospholipid composition. The much higher enzymatic activity of human group X sPLA 2 (hGX) compared with human group IIA sPLA 2 (hGIIA) on phosphatidylcholine (PC)-rich vesicles is due in large part to the higher affinity of the former enzyme for such vesicles; this result also holds when vesicles contain cholesterol and sphingomyelin. The inclusion of anionic phosphatidylserine in PC vesicles dramatically enhances interfacial binding and catalysis of hGIIA but not of hGX. This is the result of the large number of lysine and arginine residues scattered over the entire surface of hGIIA, which cause the enzyme to form a supramolecular aggregate with multiple vesicles. Thus, high affinity binding of hGIIA to anionic vesicles is a complex process and cannot be attributed to a few basic residues on its interfacial binding surface, as is also evident from mutagenesis studies. The main reason hGIIA binds poorly to PC-rich vesicles is that it lacks a tryptophan residue on its interfacial binding surface, a residue that contributes to the high affinity binding of hGX to PC-rich vesicles. Results show that the lag in the onset of hydrolysis of PC vesicles by hGIIA is due in part to the poor affinity of this enzyme for these vesicles. Binding affinity of hGIIA, hGX, and their mutants to PC-rich vesicles is well correlated to the ability of these enzymes to act on the PC-rich outer plasma membrane of mammalian cells.

Platelets are anucleated blood elements highly potent at generating extracellular vesicles (EVs) called microparticles (MPs). Whereas EVs are accepted as an important means of intercellular communication, the mechanisms underlying platelet MP internalization in recipient cells are poorly understood. Our lipidomic analyses identified 12(S)-hydroxyeicosatetranoic acid [12(S)-HETE] as the predominant eicosanoid generated by MPs. Mechanistically, 12(S)-HETE is produced through the concerted activity of secreted phospholipase A2 IIA (sPLA2-IIA), present in inflammatory fluids, and platelet-type 12-lipoxygenase (12-LO), expressed by platelet MPs. Platelet MPs convey an elaborate set of transcription factors and nucleic acids, and contain mitochondria. We observed that MPs and their cargo are internalized by activated neutrophils in the endomembrane system via 12(S)-HETE. Platelet MPs are found inside neutrophils isolated from the joints of arthritic patients, and are found in neutrophils only in the presence of sPLA2-IIA and 12-LO in an in vivo model of autoimmune inflammatory arthritis. Using a combination of genetically modified mice, we show that the coordinated action of sPLA2-IIA and 12-LO promotes inflammatory arthritis. These findings identify 12(S)-HETE as a trigger of platelet MP internalization by neutrophils, a mechanism highly relevant to inflammatory processes. Because sPLA2-IIA is induced during inflammation, and 12-LO expression is restricted mainly to platelets, these observations demonstrate that platelet MPs promote their internalization in recipient cells through highly regulated mechanisms.

In mass spectrometry-based shogun proteomics, data-independent acquisition (DIA) is an emerging technique for unbiased and reproducible measurement of protein mixtures. Without targeting a specific precursor ion, DIA MS/MS spectra are often highly multiplexed, containing product ions from multiple co-fragmenting precursors. Thus, detecting peptides directly from DIA data is challenging; most DIA data analyses require spectral libraries. Here we present a new library-free, peptide-centric tool PECAN that detects peptides directly from DIA data. PECAN reports evidence of detection based on product ion scoring, enabling detection of low abundance analytes with poor precursor ion signal. We benchmarked PECAN with chromatographic peak picking accuracy and peptide detection capability. We further validated PECAN detection with data-dependent acquisition and targeted analyses. Last, we used PECAN to build a library from DIA data and to query sequence variants. Together, these results show that PECAN detects peptides robustly and accurately from DIA data without using a library.