Hybrid-DIA: intelligent data acquisition integrates targeted and discovery proteomics to analyze phospho-signaling in single spheroids.

Achieving sufficient coverage of regulatory phosphorylation sites by mass spectrometry (MS)-based phosphoproteomics for signaling pathway reconstitution is challenging, especially when analyzing tiny sample amounts. To address this, we present a hybrid data-independent acquisition (DIA) strategy (hybrid-DIA) that combines targeted and discovery proteomics through an Application Programming Interface (API) to dynamically intercalate DIA scans with accurate triggering of multiplexed tandem mass spectrometry (MSx) scans of predefined (phospho)peptide targets. By spiking-in heavy stable isotope labeled phosphopeptide standards covering seven major signaling pathways, we benchmark hybrid-DIA against state-of-the-art targeted MS methods (i.

Temporal trends in radiometrically dated sediment cores from English lakes show polybrominated diphenyl ethers correlate with brominated but not mixed bromo/chloro dioxins and furans.

This paper reports concentrations between ~1950 and present, of polybrominated diphenyl ethers (PBDEs) and polybrominated dibenzo-p-dioxins and furans (PBDD/Fs), in radiometrically-dated sediment cores from three English lakes. Mixed bromo/chloro dibenzo-p-dioxins and furans (PXDD/Fs) were measured in two of the same lakes. Concentrations of PXDD/Fs decreased over time to the present.

First insight into human extrahepatic metabolism of flame retardants: Biotransformation of EH-TBB and Firemaster-550 components by human skin subcellular fractions.

2-ethylhexyl-2,3,4,5-tetrabromobenzoate (EH-TBB) and a mixture of EH-TBB, Bis(2-ethylhexyl)tetrabromphthalate (BEH-TEBP) and Triphenyl phosphate (TPhP), prepared in a ratio similar to the Firemaster-550™ (FM550) flame retardant formulation, were exposed to human skin subcellular fractions (S9) to evaluate their dermal in vitro metabolism for the first time. After 60 min of incubation, tetrabromobenzoic acid (TBBA) and diphenyl phosphate (DPhP) were identified as the major metabolites of EH-TBB and TPhP, respectively using UPLC-Q-Exactive Orbitrap™-MS analysis. Dermal biotransformation of EH-TBB and TPhP was catalyzed by skin carboxylesterases rather than CYP450 enzymes, while no stable metabolites could be identified for BEH-TEBP.

Legacy PBDEs and NBFRs in sediments of the tidal River Thames using liquid chromatography coupled to a high resolution accurate mass Orbitrap mass spectrometer.

Surface sediment samples (n = 45) were collected along a 110 km transect of the river Thames in October 2011, starting from Teddington Lock out through the industrial area of London to the southern North Sea. Several legacy and novel brominated flame retardants (NBFRs) were analysed, including 13 polybrominated diphenyl ethers (PBDEs) (congeners 17, 28, 47, 99, 100, 153, 154, 183, 196, 197, 206, 207 and 209), hexabromocyclododecane (HBCDDs), tetrabromobisphenol A (TBBPA), hexabromobenzene (HBB), 2,4,6-tribromophenol (TBP), 2-ethylhexyl 2,3,4,5-tetrabromobenzoate (EH-TBB or TBB), bis(2-ethylhexyl) tetrabromophthalate (BEH-TEBP or TBPH), 1,2-bis(2,4,6-tribromophenoxy)ethane (BTBPE), decabromodiphenyl ethane (DBDPE), pentabromoethylbenzene (PBEB), anti/syn-dechlorane plus (a/s-DP), 2,2',4,4',5,5'-hexabromobiphenyl (BB153) and α-,β-1,2-dibromo-4-(1,2-dibromoethyl) cyclohexane (α-,β-DBE-DBCH or TBECH). A novel analysis method based on liquid chromatographic separation, followed by high resolution accurate mass detection using the Orbitrap platform was used for quantification.

Biotransformation of the Flame Retardant 1,2-Dibromo-4-(1,2-dibromoethyl)cyclohexane (TBECH) in Vitro by Human Liver Microsomes.

The technical mixture of 1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane (TBECH or DBE-DBCH) and the pure β-TBECH isomer were subjected to in vitro biotransformation by human liver microsomes (HLM). After 60 min of incubation, 5 potential metabolites of TBECH were identified in microsomal assays of both the TBECH mixture and β-TBECH using ultraperformance liquid chromatography-Q-Exactive Orbitrap mass spectrometry. These include mono- and dihydroxylated TBECH and mono- and dihydroxylated TriBECH as well as an α-oxidation metabolite bromo-(1,2-dibromocyclohexyl)-acetic acid.

Building ProteomeTools based on a complete synthetic human proteome.

We describe ProteomeTools, a project building molecular and digital tools from the human proteome to facilitate biomedical research. Here we report the generation and multimodal liquid chromatography-tandem mass spectrometry analysis of >330,000 synthetic tryptic peptides representing essentially all canonical human gene products, and we exemplify the utility of these data in several applications. The resource (available at http://www.

Targeted proteomic quantification on quadrupole-orbitrap mass spectrometer.

There is an immediate need for improved methods to systematically and precisely quantify large sets of peptides in complex biological samples. To date protein quantification in biological samples has been routinely performed on triple quadrupole instruments operated in selected reaction monitoring mode (SRM), and two major challenges remain. Firstly, the number of peptides to be included in one survey experiment needs to be increased to routinely reach several hundreds, and secondly, the degree of selectivity should be improved so as to reliably discriminate the targeted analytes from background interferences.

High precision measurement and fragmentation analysis for metabolite identification.

The degree of precision in measuring accurate masses in LC MS/MS-based metabolomics experiments is a determinant in the successful identification of the metabolites present in the original extract. Using the methods described here, complex broccoli extracts containing hundreds of small-molecule compounds (mass range 100-1,400 Da) can be profiled at resolutions up to 100,000 (full width half maximum, FWHM), useful for accurate and sensitive relative quantification experiments. Using external instrument calibration, analyte masses can be measured with high (sub-ppm to a maximum of 2 ppm) accuracy, leading to compound identifications based on elemental composition analysis.

Sensitive determination of prohibited drugs in dried blood spots (DBS) for doping controls by means of a benchtop quadrupole/Orbitrap mass spectrometer.

In the present study, a new type of mass spectrometer combining a quadrupole mass filter, a higher collision dissociation (HCD) cell and an Orbitrap detector, was evaluated for the analysis of dried blood spots (DBS) in doping controls. DBS analysis is characterized by the necessity to detect prohibited compounds in sub-nanogram-per-milliliter levels with high identification capacity. After extraction of DBS with an organic solvent and liquid chromatographic separation (using a regular C18-RP-analytical UHPLC-column) of target analytes, mass spectrometry is performed with a high-resolution full scan in positive and negative mode by means of electrospray ionisation.

Top-down lipidomic screens by multivariate analysis of high-resolution survey mass spectra.

Direct profiling of total lipid extracts on a hybrid LTQ Orbitrap mass spectrometer by high-resolution survey spectra clusters species of 11 major lipid classes into 7 groups, which are distinguished by their sum compositions and could be identified by accurately determined masses. Rapid acquisition of survey spectra was employed as a "top-down" screening tool that, together with the computational method of principal component analysis, revealed pronounced perturbations in the abundance of lipid precursors within the entire series of experiments. Altered lipid precursors were subsequently identified either by accurately determined masses or by in-depth MS/MS characterization that was performed on the same instrument.

Collision-induced dissociation pathways of yeast sphingolipids and their molecular profiling in total lipid extracts: a study by quadrupole TOF and linear ion trap-orbitrap mass spectrometry.

The yeast Saccharomyces cerevisiae synthesizes three classes of sphingolipids: inositolphosphoceramides (IPCs), mannosyl-inositolphosphoceramides (MIPCs), and mannosyl-diinositolphosphoceramides (M(IP)2C). Tandem mass spectrometry of their molecular anions on a hybrid quadrupole time-of-flight (QqTOF) instrument produced fragments of inositol-containing head groups, which were specific for each lipid class. MS(n) analysis performed on a hybrid linear ion trap-orbitrap (LTQ Orbitrap) mass spectrometer with better than 3 ppm mass accuracy identified fragment ions specific for the amide-linked fatty acid and the long chain base moieties in individual molecular species.