Nanoscale characterization of spider venom peptides by high resolution LC-MS/MS analysis

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In-depth, comprenhensive mapping of the human seminal plasma proteome by a novel, iterative LC/MS/MS/database search workflow

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Human platelets generate phospholipid-esterified prostaglandins via cyclooxygenase-1 that are inhibited by low dose aspirin supplementation

Oxidized phospholipids (oxPLs) generated nonenzymatically display pleiotropic biological actions in inflammation. Their generation by cellular cyclooxygenases (COXs) is currently unknown. To determine whether platelets generate prostaglandin (PG)-containing oxPLs, then characterize their structures and mechanisms of formation, we applied precursor scanning-tandem mass spectrometry to lipid extracts of agonist-activated human platelets. Thrombin, collagen, or ionophore activation stimulated generation of families of PGs comprising PGE2 and D2 attached to four phosphatidylethanolamine (PE) phospholipids (16:0p/, 18:1p/, 18:0p/, and 18:0a/). They formed within 2 to 5 min of activation in a calcium, phospholipase C, p38 MAP kinases, MEK1, cPLA2, and src tyrosine kinase-dependent manner (28.1 ± 2.3 pg/2 × 108 platelets). Unlike free PGs, they remained cell associated, suggesting an autocrine mode of action. Their generation was inhibited by in vivo aspirin supplementation (75 mg/day) or in vitro COX-1 blockade. Inhibitors of fatty acyl reesterification blocked generation significantly, while purified COX-1 was unable to directly oxidize PE in vitro. This indicates that they form in platelets via rapid esterification of COX-1 derived PGE2/D2 into PE. In summary, COX-1 in human platelets acutely mediates membrane phospholipid oxidation via formation of PG-esterified PLs in response to pathophysiological agonists

Potential of sterol analysis by liquid chromatography-tandem mass spectrometry for the prenatal diagnosis of Smith-Lemli-Opitz syndrome

BACKGROUND: Smith-Lemli-Opitz syndrome (SLOS) is a severe disorder of cholesterol synthesis classically diagnosed prenatally by GC-MS analysis of sterols in amniotic fluid. In recognition of the current move towards tandem mass spectrometry (MS/MS) methodologies we present prototype LC-MS/MS methods for the accurate diagnosis of the disorder METHODS: 3β-Hydroxysterols in amniotic fluid are oxidised with cholesterol oxidase to their corresponding 3-ketones, which are then derivatised with Girard P (GP) hydrazine in a “one-pot” reaction. The resulting GP-hydrazones give an improved response in electrospray (ES)-MS/MS due to the presence of a charged quaternary nitrogen and are analysed by reversed-phase LC-ES-MS/MS. Both capillary LC-MS/MS and conventional LC-MS/MS formats are suitable, and the method is also applicable to paper absorbed blood spots. RESULTS: In a double blind analysis of 18 amniotic fluid samples comprising 6 SLOS and 12 controls, the 7+8-dehydrocholesterol (7+8-DHC) to cholesterol ratio was found to lie below 0.02 (range, 0.00 - 0.02: mean ± SD, 0.01 ± 0.007) in all control samples (intra assay variation 5.91%), and above 0.20 (range, 0.20 - 1.13: mean ± SD, 0.79 ± 0.35) in SLOS (intra assay variation 4.56%), corresponding to a difference in ratios between the two groups of a factor of at least 10. The limit of quantification was equivalent to 2 nL of amniotic fluid injected on-column. CONCLUSION: Here we describe a “proof-of-concept” study for the prenatal diagnosis of SLOS. However, further developments will be necessary to automate sample handling and reduce chromatographic time in order to allow the methodology to be used for pre- and postnatal diagnosis

Triple Quadruple Mass Spectromery-Based Peptide Assays Using Intelligent SRM (iSRM)

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