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.e., SureQuant) using EGF-stimulated HeLa cells and find the quantitative accuracy and sensitivity to be comparable while hybrid-DIA also profiles the global phosphoproteome. To demonstrate the robustness, sensitivity, and biomedical potential of hybrid-DIA, we profile chemotherapeutic agents in single colon carcinoma multicellular spheroids and evaluate the phospho-signaling difference of cancer cells in 2D vs 3D culture.

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. To our knowledge, this is the first report of temporal trends of PXDD/Fs in the environment. In contrast, concentrations of PBDEs increased towards the present and were significantly correlated (R = 0.88-0.98; p < 0.05) with concentrations of PBDFs in all three lakes. These observations suggest that the sources of PXDD/Fs are not related to PBDEs and differ from those of PBDFs. We also report for the first time the presence of octabromodibenzofuran (OBDF) in the two most recent core slices at one lake. The source of OBDF in these samples is unclear. While OBDF has been reported previously as a significant contaminant of some commercial formulations of Deca-BDE, it is also present in Octa-BDE products and in emissions from a variety of combustion activities. Overall, while the positive correlation between PBDEs and PBDFs suggests increased use of PBDEs has contributed substantially to environmental contamination with PBDFs; examination of PBDF homologue patterns implies emissions from combustion activities are likely also important.

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. Metabolite formation rates of EH-TBB as individual compound and as a component of FM550 fitted the Michaelis-Menten model, while no steady state could be reached for TPhP under experimental conditions. Estimated maximum metabolic rate (Vmax) for TBBA formation upon exposure to FM550 was lower than Vmax for EH-TBB (1.08 and 15.2 pmol min-1 mg protein-1, respectively). This indicates dermal metabolism would contribute less to the clearance of EH-TBB body burden than hepatic metabolism (Vmax = 644 pmol min-1 mg protein-1). Implications for human exposure include EH-TBB accumulation in skin tissue and human exposure to dermal metabolic products, which may have different toxicokinetic and toxicodynamic parameters than parent flame retardants.

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-,ß-1,2-dibromo-4-(1,2-dibromoethyl) cyclohexane (α-,ß-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. Results revealed that BDE-209 had the highest concentrations (<0.1 to 540 µg kg-1 dw) and detection frequency, accounting for 95% of all PBDE congeners measured. Indicative evidence of debromination of the PentaBDE technical mixture was observed through elevated relative abundance of BDE-28 in sediment compared to the Penta-BDE formulation. NBFRs were detected at comparable levels to PBDEs (excluding BDE-209), which indicates increasing use of the former. Spatial trend analysis showed that samples from industrial areas had significantly higher concentrations of Σ12PBDEs, ΣHBCDDs, TBBPA, BEH-TEBP, BTBPE and TBP. Three locations showed high concentrations of HBCDDs with diastereomer patterns comparable to the technical mixture, which indicate recent input sources to the sediment.

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 ß-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 ß-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. Our results indicate potential hepatic biotransformation of TBECH via cyctochrome P450-catalyzed hydroxylation, debromination, and α-oxidation. Kinetic studies revealed that the formation of monohydroxy-TBECH, dihydroxy-TBECH, and monohydroxy-TriBECH were best fitted to a Michaelis-Menten enzyme kinetic model. Respective estimated Vmax values (maximum metabolic rate) for these metabolites were 11.8 ± 4, 0.6 ± 0.1, and 10.1 ± 0.8 pmol min-1 mg protein-1 in TBECH mixture and 4992 ± 1340, 14.1 ± 4.9, and 66.1 ± 7.3 pmol min-1 mg protein-1 in ß-TBECH. This indicates monohydroxy-TBECH as the major metabolite of TBECH by in vitro HLM-based assay. The estimated in vitro intrinsic clearance (Clint) of TBECH mixture was slower (P < 0.05) than that of pure ß-TBECH. While the formation of monohydroxy-TBECH may reduce the bioaccumulation potential and provide a useful biomarker for monitoring TBECH exposure, further studies are required to fully understand the levels and toxicological implications of the identified metabolites.

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.proteometools.org) will be extended to >1 million peptides, and all data will be shared with the community via ProteomicsDB and ProteomeXchange.

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 resolution and accurate mass (HR/AM) analysis on the recently developed Q-Exactive mass spectrometer can potentially address these issues. This instrument presents a unique configuration: it is constituted of an orbitrap mass analyzer equipped with a quadrupole mass filter as the front-end for precursor ion mass selection. This configuration enables new quantitative methods based on HR/AM measurements, including targeted analysis in MS mode (single ion monitoring) and in MS/MS mode (parallel reaction monitoring). The ability of the quadrupole to select a restricted m/z range allows one to overcome the dynamic range limitations associated with trapping devices, and the MS/MS mode provides an additional stage of selectivity. When applied to targeted protein quantification in urine samples and benchmarked with the reference SRM technique, the quadrupole-orbitrap instrument exhibits similar or better performance in terms of selectivity, dynamic range, and sensitivity. This high performance is further enhanced by leveraging the multiplexing capability of the instrument to design novel acquisition methods and apply them to large targeted proteomic studies for the first time, as demonstrated on 770 tryptic yeast peptides analyzed in one 60-min experiment. The increased quality of quadrupole-orbitrap data has the potential to improve existing protein quantification methods in complex samples and address the pressing demand of systems biology or biomarker evaluation studies.

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. Unambiguous identification of four analytes (citric acid, chlorogenic acid, phenylalanine, and UDP-D: -glucose) is used to validate the performance of the different MS/MS fragmentation regimes. Identifications are carried out either via resonance excitation collision induced dissociation (CID) or via higher energy collision dissociation (HCD) experiments, and validated by infrared multiphoton dissociation (IRMPD) fragmentation of standards. Such results, obtained on both hybrid and non-hybrid systems from metabolite profiling and identification experiments, provide evidence that the strategies selected can be successfully applied to other LC-MS based projects for plant metabolomic studies.

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. Single-product ion mass spectra are acquired using the data-dependent analysis mode (employing an inclusion list) for previously selected precursors of known prohibited compounds with fixed retention time ranges. Besides, a sensitive screening in a targeted approach, non-targeted analysis for retrospective data evaluation is thus possible. The chosen experimental design enables the determination of various drugs from different classes with one generic sample preparation which is shown for 26 selected model compounds (Δ(9)-tetrahydrocannabinol (THC), tetrahydrocannabinol-9-carboxylic acid (THC-COOH), methylhexaneamine, methylphenidate, cocaine, nikethamide, 3,4-methylenedioxyamphetamine, N-methyl-3,4-methylenedioxyamphetamine, strychnine, mesocarb, salbutamol, formoterol, clenbuterol, metandienone, stanozolol, bisoprolol, propranolol, metoprolol, anastrazole, clomiphene, exemestane, dexamethasone, budesonide, selective androgen receptor modulator (SARM) S4 (andarine), SARM S1, hydrochlorothiazide). Generally, only qualitative result interpretation was focussed upon, but for target analytes with deuterium-labelled internal standards (salbutamol, clenbuterol, cocaine, dexamethasone, THC-COOH and THC) quantitative analysis was also possible. Especially the most challenging analytes, THC and its carboxy metabolite, were detected in DBS at relevant concentrations (<0.5 ng/mL) using targeted HCD experiments. The method was validated for the parameters: specificity, linearity (0-20 ng/mL), precision (<25%), recovery (mean 60%), limit of detection/quantification, ion suppression, stability and accuracy (80-120%). Six isotope-labelled analogues used as internal standards facilitate a quantitative result interpretation which is of utmost importance especially for in-competition drug sports testing.

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. Hence, the sensitivity, throughput and robustness of lipidomics screens were improved without compromising the accuracy and specificity of molecular species identification. The top-down lipidomics strategy lends itself for high-throughput screens complementing ongoing functional genomics efforts.

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. By selecting m/z of class-specific fragment ions for multiple precursor ion scanning, we profiled yeast sphingolipids in total lipid extracts on a QqTOF mass spectrometer. Thus, a combination of QqTOF and LTQ Orbitrap mass spectrometry lends itself to rapid, comprehensive and structure-specific profiling of the molecular composition of sphingolipids and glycerophospholipids in important model organisms, such as fungi and plants.