Organosulfate Formation in Biogenic Secondary Organic Aerosol

Organosulfates of isoprene, α-pinene, and β-pinene have recently been identified in both laboratory-generated and ambient secondary organic aerosol (SOA). In this study, the mechanism and ubiquity of organosulfate formation in biogenic SOA is investigated by a comprehensive series of laboratory photooxidation (i.e., OH-initiated oxidation) and nighttime oxidation (i.e., NO3-initiated oxidation under dark conditions) experiments using nine monoterpenes (α-pinene, β-pinene, d-limonene, l-limonene, α-terpinene, γ-terpinene, terpinolene, Δ3-carene, and β-phellandrene) and three monoterpenes (α-pinene, d-limonene, and l-limonene), respectively. Organosulfates were characterized using liquid chromatographic techniques coupled to electrospray ionization combined with both linear ion trap and high-resolution time-of-flight mass spectrometry. Organosulfates are formed only when monoterpenes are oxidized in the presence of acidified sulfate seed aerosol, a result consistent with prior work. Archived laboratory-generated isoprene SOA and ambient filter samples collected from the southeastern U.S. were reexamined for organosulfates. By comparing the tandem mass spectrometric and accurate mass measurements collected for both the laboratory-generated and ambient aerosol, previously uncharacterized ambient organic aerosol components are found to be organosulfates of isoprene, α-pinene, β-pinene, and limonene-like monoterpenes (e.g., myrcene), demonstrating the ubiquity of organosulfate formation in ambient SOA. Several of the organosulfates of isoprene and of the monoterpenes characterized in this study are ambient tracer compounds for the occurrence of biogenic SOA formation under acidic conditions. Furthermore, the nighttime oxidation experiments conducted under highly acidic conditions reveal a viable mechanism for the formation of previously identified nitrooxy organosulfates found in ambient nighttime aerosol samples. We estimate that the organosulfate contribution to the total organic mass fraction of ambient aerosol collected from K-puszta, Hungary, a field site with a similar organosulfate composition as that found in the present study for the southeastern U.S., can be as high as 30%

A unique bacteriohopanetetrol stereoisomer of marine anammox

Anaerobic ammonium oxidation (anammox) is a major process of bioavailable nitrogen removal from marine systems. Previously, a bacteriohopanetetrol (BHT) isomer, with unknown stereochemistry, eluting later than BHT using high performance liquid chromatography (HPLC), was detected in ‘Ca. Scalindua profunda’ and proposed as a biomarker for anammox in marine paleo-environments. However, the utility of this BHT isomer as an anammox biomarker is hindered by the fact that four other, non-anammox bacteria are also known to produce a late-eluting BHT stereoisomer. The stereochemistry in Acetobacter pasteurianus, Komagataeibacter xylinus and Frankia sp. was known to be 17β, 21β(H), 22R, 32R, 33R, 34R (BHT-34R). The stereochemistry of the late-eluting BHT in Methylocella palustris was unknown. To determine if marine anammox bacteria produce a unique BHT isomer, we studied the BHT distributions and stereochemistry of known BHT isomer producers and of previously unscreened marine (‘Ca. Scalindua brodeae’) and freshwater (‘Ca. Brocadia sp.’) anammox bacteria using HPLC and gas chromatographic (GC) analysis of acetylated BHTs and ultra high performance liquid chromatography (UHPLC)-high resolution mass spectrometry (HRMS) analysis of non-acetylated BHTs. The 34R stereochemistry was confirmed for the BHT isomers in Ca. Brocadia sp. and Methylocella palustris. However, ‘Ca. Scalindua sp.’ synthesise a stereochemically distinct BHT isomer, with still unconfirmed stereochemistry (BHT-x). Only GC analysis of acetylated BHT and UHPLC analysis of non-acetylated BHT distinguished between late-eluting BHT isomers. Acetylated BHT-x and BHT-34R co-elute by HPLC. As BHT-x is currently only known to be produced by ‘Ca. Scalindua spp.’, it may be a biomarker for marine anammox

Targeted online liquid chromatography electron capture dissociation mass spectrometry for the localization of sites of in vivo phosphorylation in human Sprouty2

We demonstrate a strategy employing collision-induced dissociation for phosphopeptide discovery, followed by targeted electron capture dissociation (ECD) for site localization. The high mass accuracy and low background noise of the ECD mass spectra allow facile sequencing of coeluting isobaric phosphopeptides, with up to two isobaric phosphopeptides sequenced from a single mass spectrum. In contrast to the previously described neutral loss of dependent ECD method, targeted ECD allows analysis of both phosphotyrosine peptides and lower abundance phosphopeptides. The approach was applied to phosphorylation analysis of human Sprouty2, a regulator of receptor tyrosine kinase signaling. Fifteen sites of phosphorylation were identified, 11 of which are novel

Identifying metabolites by integrating metabolome databases with mass spectrometry cheminformatics.

Novel metabolites distinct from canonical pathways can be identified through the integration of three cheminformatics tools: BinVestigate, which queries the BinBase gas chromatography-mass spectrometry (GC-MS) metabolome database to match unknowns with biological metadata across over 110,000 samples; MS-DIAL 2.0, a software tool for chromatographic deconvolution of high-resolution GC-MS or liquid chromatography-mass spectrometry (LC-MS); and MS-FINDER 2.0, a structure-elucidation program that uses a combination of 14 metabolome databases in addition to an enzyme promiscuity library. We showcase our workflow by annotating N-methyl-uridine monophosphate (UMP), lysomonogalactosyl-monopalmitin, N-methylalanine, and two propofol derivatives

Characterization of polar organosulfates in secondary organic aerosol from the unsaturated aldehydes 2-E-pentenal, 2-E-hexenal, and 3-Z-hexenal

We show in the present study that the unsaturated aldehydes 2-E-pentenal, 2-E-hexenal, and 3-Z-hexenal are biogenic volatile organic compound (BVOC) precursors for polar organosulfates with molecular weights (MWs) 230 and 214, which are also present in ambient fine aerosol from a forested site, i.e., K-puszta, Hungary. These results complement those obtained in a previous study showing that the green leaf aldehyde 3-Z-hexenal serves as a precursor for MW 226 organosulfates. Thus, in addition to isoprene, the green leaf volatiles (GLVs) 2-E-hexenal and 3-Z-hexenal, emitted due to plant stress (mechanical wounding or insect attack), and 2-E-pentenal, a photolysis product of 3-Z-hexenal, should be taken into account for secondary organic aerosol and organosulfate formation. Polar organosulfates are of climatic relevance because of their hydrophilic properties and cloud effects. Extensive use was made of organic mass spectrometry (MS) and detailed interpretation of MS data (i.e., ion trap MS and accurate mass measurements) to elucidate the chemical structures of the MW 230, 214 and 170 organosulfates formed from 2-E-pentenal and indirectly from 2-E-hexenal and 3-Z-hexenal. In addition, quantum chemical calculations were performed to explain the different mass spectral behavior of 2,3-dihydroxypentanoic acid sulfate derivatives, where only the isomer with the sulfate group at C-3 results in the loss of SO3. The MW 214 organosulfates formed from 2-E-pentenal are explained by epoxidation of the double bond in the gas phase and sulfation of the epoxy group with sulfuric acid in the particle phase through the same pathway as that proposed for 3-sulfooxy-2-hydroxy-2-methylpropanoic acid from the isoprene-related alpha,beta-unsaturated aldehyde methacrolein in previous work (Lin et al., 2013). The MW 230 organosulfates formed from 2-E-pentenal are tentatively explained by a novel pathway, which bears features of the latter pathway but introduces an additional hydroxyl group at the C-4 position. Evidence is also presented that the MW 214 positional isomer, 2-sulfooxy-3-hydroxypentanoic acid, is unstable and decarboxylates, giving rise to 1-sulfooxy-2-hydroxybutane, a MW 170 organosulfate. Furthermore, evidence is obtained that lactic acid sulfate is generated from 2-E-pentenal. This chemistry could be important on a regional and local scale where GLV emissions such as from grasses and cereal crops are substantial

Frequency of occurrence of novel milk protein variants in a New Zealand dairy cattle study population : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry, Massey University

Since the discovery of genetic polymorphism within milk protein genes, a considerable volume of research has been published relating milk protein genetic variants and milk production properties. Polymorphism of milk proteins can result in two effects: (a) changes in the biological and physico-chemical properties of systems containing the variant protein, (b) changes in the synthesis level of variant proteins. As a result several studies of milk protein variants have identified phenotypes which may be commercially advantageous for specific products. Currently employed methods to determine milk protein phenotypes are generally limited to electrophoretic techniques. The gel electrophoretic techniques commonly used are able to detect most milk protein variants that differ by their net electrical charge. However single amino acid substitutions that result in a change in net charge account for only 25% of the possible substitutions that could occur. The remaining 75% of potential variants are the result of a neutral residue substituted by another neutral residue - a 'silent' variant. Thus it is likely that some substitutions, and hence genetic variants have gone undetected in the past. The purpose of this study was to develop new methods for determining the phenotype of milk proteins, and to determine the frequency of occurrence of silent or other novel variants in a New Zealand dairy cattle study population. Polyacrylamide gel electrophoresis (PAGE), free zone capillary electrophoresis (CE), peptide mapping by reverse-phase HPLC and electrospray mass spectrometry (ESI-MS) were used in the characterisation of milk proteins purified from 109 individual dairy cows. Three different PAGE systems were used. Alkaline-urea PAGE enabled the detection of α si-casin variants B and C, β-casein variants group A (variants A1, A2 and A3) and B, and K-casein variants A and B in the study population. Beta-casein variants A1, A2 and A3 were subsequently resolved in an acid-urea PAGE system. The whey proteins were very poorly resolved in PAGE systems containing urea. Alpha-lactalbumin A, and β-lactoglobulin (β-LG) variants A and B were resolved in a non-denaturing 'native' PAGE system. The frequencies of the various milk protein variants corresponded closely to figures previously published. A free zone CE method that is able to resolve β-LG variants A, B and C was used to check the phenotype of purified β-LG samples. Three samples previously typed as β-LG BB were subsequently determined to be β-LG CC; one sample typed as β-LG BB was re-assigned as β-LG BC. This highlighted the limitations of PAGE systems for the detection of known variants. Tryptic hydrolysis of purified casein proteins and β-LG, followed by reverse-phase HPLC separation of the resultant peptides was used to create peptide 'maps' of the hydrolysis products. Differences in peptide maps were noted between protein variants. The differences corresponded to peptides containing a substitution site. All samples analysed in this way contained more peptide peaks than expected. Analysis revealed that some were the result of incomplete digestion; others the result of chymotryptic-like cleavages. No aberrant peptide maps, indicative of a silent mutation, were detected. Purified casein proteins and β-LG were subjected to ESI-MS for mass analysis. The mass of each protein species was determined as follows: Protein Average mass Std. dev. as1-CN B-8P 23614.9 Da 1.2 Da as2-CN A-11P 25228.9 Da 1.5 Da β-CN A1-5P 24023.9 Da 3.1 Da β-CN A2-5P 23983.5 Da 1.8 Da β-CN B-5P 24092.6 Da n.d. k-CN A-1P 19038.8 Da 1.5 Da k-CN B-1P 19003.8 Da n.d. β-LG A 18362.6 Da 1.0 Da β-LG B 18277.0 Da 0.9 Da β-LG C 18287.2 Da 0.6 Da In all cases the experimentally determined mass corresponded to the mass calculated from published primary sequences of milk protein variants. In addition to the expected β-LG variant in each mass spectrum, additional species were detected differing from the mass of the β-LG species by increments of approximately 324 Da. Although less pronounced, the +324 Da molecular weight species were also detected in a sample of β-LG purchased from the Sigma Chemical Company. The additional species were also detected in whey prepared by ultra-centrifugation, although at a much lower level. The 324 Da molecular weight adducts observed in ESI-MS spectra of purified β-LG are consistent with an addition of a lactosyl residue to the protein. The observation that these species remain after heat denaturation, reduction and RP-HPLC treatment suggest that the linkage is covalent. Lactulosyl-lysine is known to form in milk products during some processing conditions, particularly during heating. The observation of these glycated species in gently treated, unheated milk suggests that glycation may occur to some extent in the udder of the cow. The association of the 324 Da molecule with β-LG does not alter the charge, molecular weight or hydrophobicity sufficiently to be detected by PAGE. CE or RP-HPLC

Trapped ion mobility spectrometry and PASEF enable in-depth lipidomics from minimal sample amounts

A comprehensive characterization of the lipidome from limited starting material remains very challenging. Here we report a high-sensitivity lipidomics workflow based on nanoflow liquid chromatography and trapped ion mobility spectrometry (TIMS). Taking advantage of parallel accumulation-serial fragmentation (PASEF), we fragment on average 15 precursors in each of 100 ms TIMS scans, while maintaining the full mobility resolution of co-eluting isomers. The acquisition speed of over 100 Hz allows us to obtain MS/MS spectra of the vast majority of isotope patterns. Analyzing 1 mu L of human plasma, PASEF increases the number of identified lipids more than three times over standard TIMS-MS/MS, achieving attomole sensitivity. Building on high intra- and inter-laboratory precision and accuracy of TIMS collisional cross sections (CCS), we compile 1856 lipid CCS values from plasma, liver and cancer cells. Our study establishes PASEF in lipid analysis and paves the way for sensitive, ion mobility-enhanced lipidomics in four dimensions

Impact of glucuronide interferences on therapeutic drug monitoring of posaconazole by tandem mass spectrometry

Background: Posaconazole is a novel antifungal drug for oral application intended especially for therapy of invasive mycoses. Due to variable gastrointestinal absorption, adverse side effects, and suspected drug-drug interactions, therapeutic drug monitoring (TDM) of posaconazole is recommended. Method: A fast ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantification of posaconazole with a run-time <3 min was developed and compared to a LC-MS/MS method and HPLC method with fluorescence detection. Results: During evaluation of UPLC-MS/MS, two earlier eluting peaks were observed in the MRM trace of posaconazole. This was only seen in patient samples, but not in spiked calibrator samples. Comparison with LC-MS/MS disclosed a significant bias with higher concentrations measured by LC-MS/MS, while UPLC-MS/MS showed excellent agreement with the commercially available HPLC method. In the LC-MS/MS procedure, comparably wide and left side shifted peaks were noticed. This could be ascribed to in-source fragmentation of conjugate metabolites during electrospray ionisation. Precursor and product ion scans confirmed the assumption that the additional compounds are posaconazole glucuronides. Reducing the cone voltage led to disappearance of the glucuronide peaks. Slight modification of the LC-MS/MS method enabled separation of the main interference, leading to significantly reduced deviation. Conclusions: These results highlight the necessity to reliably eliminate interference from labile drug metabolites for correct TDM results, either by sufficient separation or selective MS conditions. The presented UPLC-MS/MS method provides a reliable and fast assay for TDM of posaconazole. Clin Chem Lab Med 2010; 48:1723-31

An in silico MS/MS library for automatic annotation of novel FAHFA lipids.

BackgroundA new lipid class named 'fatty acid esters of hydroxyl fatty acids' (FAHFA) was recently discovered in mammalian adipose tissue and in blood plasma and some FAHFAs were found to be associated with type 2 diabetes. To facilitate the automatic annotation of FAHFAs in biological specimens, a tandem mass spectra (MS/MS) library is needed. Due to the limitation of the commercial available standard compounds, we proposed building an in silico MS/MS library to extend the coverage of molecules.ResultsWe developed a computer-generated library with 3267 tandem mass spectra (MS/MS) for 1089 FAHFA species. FAHFA spectra were generated based on authentic standards with negative mode electrospray ionization and 10, 20, and 40 V collision induced dissociation at 4 spectra/s as used in in ultra-high performance liquid chromatography-QTOF mass spectrometry studies. However, positional information of the hydroxyl group is only obtained either at lower QTOF spectra acquisition rates of 1 spectrum/s or at the MS(3) level in ion trap instruments. Therefore, an additional set of 4290 fragment-rich MS/MS spectra was created to enable distinguishing positional FAHFA isomers. The library was generated based on ion fragmentations and ion intensities of FAHFA external reference standards, developing a heuristic model for fragmentation rules and extending these rules to large swaths of computer-generated structures of FAHFAs with varying chain lengths, degrees of unsaturation and hydroxyl group positions. Subsequently, we validated the new in silico library by discovering several new FAHFA species in egg yolk, showing that this library enables high-throughput screening of FAHFA lipids in various biological matrices.ConclusionsThe developed library and templates are freely available for commercial or noncommercial use at http://fiehnlab.ucdavis.edu/staff/yanma/fahfa-lipid-library. This in silico MS/MS library allows users to annotate FAHFAs from accurate mass tandem mass spectra in an easy and fast manner with NIST MS Search or PepSearch software. The developing template is provided for advanced users to modify the parameters and export customized libraries according to their instrument features. Graphical abstractExample of experimental and in silico MS/MS spectra for FAHFA lipids

A New synthesis of triazolo[4,5-g]quinolines and unexpected ring reduced products by treatment with hydrazine hydrate

A new synthesis of the linear heterocycle 4-chloro-1H-triazolo[4,5-g]quinoline by reduction of the novel compound 4-chloro-1H-triazolo[4,5-g]quinoline-1-oxide is reported. Treatment of the latter with hydrazine hydrate in ethanol in a sealed steel vessel in the presence or not of palladised charcoal, under various conditions of both time and temperature, afforded some derivatives of both ring reduction and ring construction