Multiple MS Research Articles

Multiple stage MS in analysis of plasma, serum, urine and in vitro samples relevant to clinical and forensic toxicology.

This paper reviews MS approaches applied to metabolism studies, structure elucidation and qualitative or quantitative screening of drugs (of abuse) and/or their metabolites. Applications in clinical and forensic toxicology were included using blood plasma or serum, urine, in vitro samples, liquids, solids or plant material. Techniques covered are liquid chromatography coupled to low-resolution and high-resolution multiple stage mass analyzers. Only PubMed listed studies published in English between January 2008 and January 2015 were considered. Approaches are discussed focusing on sample preparation and mass spectral settings. Comments on advantages and limitations of these techniques complete the review.

Metabolic profiling for the identification of Huntington biomarkers by on-line solid-phase extraction capillary electrophoresis mass spectrometry combined with advanced data analysis tools.

In this work, an untargeted metabolomic approach based on sensitive analysis by on-line solid-phase extraction capillary electrophoresis mass spectrometry (SPE-CE-MS) in combination with multivariate data analysis is proposed as an efficient method for the identification of biomarkers of Huntington's disease (HD) progression in plasma. For this purpose, plasma samples from wild-type (wt) and HD (R6/1) mice of different ages (8, 12, and 30 weeks), were analyzed by C18 -SPE-CE-MS in order to obtain the characteristic electrophoretic profiles of low molecular mass compounds. Then, multivariate curve resolution alternating least squares (MCR-ALS) was applied to the multiple full scan MS datasets. This strategy permitted the resolution of a large number of metabolites being characterized by their electrophoretic peaks and their corresponding mass spectra. A total number of 29 compounds were relevant to discriminate between wt and HD plasma samples, as well as to follow-up the HD progression. The intracellular signaling was found to be the most affected metabolic pathway in HD mice after 12 weeks of birth, when mice already showed motor coordination deficiencies and cognitive decline. This fact agreed with the atrophy and dysfunction of specific neurons, loss of several types of receptors, and changed expression of neurotransmitters.

Determination of Dufulin Residue in Vegetables, Rice, and Tobacco Using Liquid Chromatography with Tandem Mass Spectrometry.

A rapid and accurate LC/MS/MS method using positive electrospray ionization was established for the determination of residues of the novel plant antiviral agent dufulin in samples of tobacco leaf (dry), tomato, cucumber, and rice. Samples were extracted with acetonitrile; cleaned up by dispersive SPE using primary secondary amine, C18, and graphitized carbon black sorbents; separated on a C18 column; and confirmed by multiple reaction monitoring mode MS with a matrix effect of -21.5-19.6%. The method showed satisfactory linearity (R2≥0.9912) for the target compound. The LOD and the LOQ were 0.05 and 0.15 μg/kg, respectively. The mean recoveries from four matrixes varied from 71.9 to 93.6% with intraday RSD in the range of 2.9 to 9.0% and interday RSD 6.9 to 15.2%. The method was successfully applied for analysis of dufulin in actual trial samples.

Chemical Characterization of Urate Hydroperoxide, A Pro-oxidant Intermediate Generated by Urate Oxidation in Inflammatory and Photoinduced Processes.

Urate hydroperoxide is a strong oxidant generated by the combination of urate free radical and superoxide. The formation of urate hydroperoxide as an intermediate in urate oxidation is potentially responsible for the pro-oxidant effects of urate in inflammatory disorders, protein degradation, and food decomposition. To understand the molecular mechanisms that sustain the harmful effects of urate in inflammatory and oxidative stress related conditions, we report a detailed structural characterization and reactivity of urate hydroperoxide toward biomolecules. Urate hydroperoxide was synthesized by photo-oxidation and by a myeloperoxidase/hydrogen peroxide/superoxide system. Multiple reaction monitoring (MRM) and MS(3) ion fragmentation revealed that urate hydroperoxide from both sources has the same chemical structure. Urate hydroperoxide has a maximum absorption at 308 nm, ε308nm = 6.54 ± 0.38 × 10(3) M(-1) cm(-1). This peroxide decays spontaneously with a rate constant of k = 2.80 ± 0.18 × 10(-4) s(-1) and a half-life of 41 min at 22 °C. Urate hydroperoxide undergoes electrochemical reduction at potential values less negative than -0.5 V (versus Ag/AgCl). When incubated with taurine, histidine, tryptophan, lysine, methionine, cysteine, or glutathione, urate hydroperoxide reacted only with methionine, cysteine, and glutathione. The oxidation of these molecules occurred by a two-electron mechanism, generating the alcohol, hydroxyisourate. No adduct between cysteine or glutathione and urate hydroperoxide was detected. The second-order rate constant for the oxidation of glutathione by urate hydroperoxide was 13.7 ± 0.8 M(-1) s(-1). In conclusion, the oxidation of sulfur-containing biomolecules by urate hydroperoxide is likely to be a mechanism by which the pro-oxidant and damaging effects of urate are mediated in inflammatory and photo-oxidizing processes.

Two novel tyrosine-containing peptides (Tyr(4)) of the adipokinetic hormone family in beetles of the families Coccinellidae and Silphidae.

Novel members of the adipokinetic hormone family of peptides have been identified from the corpora cardiaca (CC) of two species of beetles representing two families, the Silphidae and the Coccinellidae. A crude CC extract (0.3 gland equivalents) of the burying beetle, Nicrophorus vespilloides, was active in mobilizing trehalose in a heterologous assay using the cockroach Periplaneta americana, whereas the CC extract (0.5 gland equivalents) of the ladybird beetle, Harmonia axyridis, exhibited no hypertrehalosemic activity. Primary sequences of one adipokinetic hormone from each species were elucidated by liquid chromatography coupled to electrospray mass spectrometry (LC-MS). The multiple MS(N) electrospray mass data revealed an octapeptide with an unusual tyrosine residue at position 4 for each species: pGlu-Leu-Thr-Tyr-Ser-Thr-Gly-Trp amide for N. vespilloides (code-named Nicve-AKH) and pGlu-Ile-Asn-Tyr-Ser-Thr-Gly-Trp amide for H. axyridis (code-named Harax-AKH). Assignment of the correct sequences was confirmed by synthesis of the peptides and co-elution in reversed-phase high-performance liquid chromatography with fluorescence detection or by LC-MS. Moreover, synthetic peptides were shown to be active in the heterologous cockroach assay system, but Harax-AKH only at a dose of 30 pmol, which explains the negative result with the crude CC extract. It appears that the tyrosine residue at position 4 can be used as a diagnostic feature for certain beetle adipokinetic peptides, because this feature has not been found in another order other than Coleoptera.

Combination of CE-MS and advanced chemometric methods for high-throughput metabolic profiling.

In this work, an untargeted approach based on capillary electrophoresis-mass spectrometry (CE-MS) in combination with multivariate data analyses is proposed as a high-throughput general methodology for metabolomic studies. First, total ion electropherograms (TIEs) were considered for exploratory and classification purposes by means of principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). Then, multivariate curve resolution alternating least squares (MCR-ALS) was applied to the multiple full scan MS data sets. This strategy permitted the resolution of a large number of metabolites being characterized by their electrophoretic peaks and their corresponding mass spectra. The proposed approach allowed solving additional electrophoretic issues, such as background noise contributions, low signal-to-noise ratios, asymmetric peaks and migration time shifts. The usefulness of the proposed methodology is demonstrated in a comparative study of the metabolic profiles from baker's yeast (Saccharomyces cerevisiae) samples cultured at two temperatures, 30°C and 37°C. A total number of 80 metabolites were relevant to yeast samples differentiation at the two temperatures and almost 50 of them were tentatively identified based on their accurate experimental molecular mass. The results show that changes in amino acid, nucleotide and lipid metabolic pathways participated in the acclimatization of yeast cells to grow at 37°C.

Utrophin suppresses low frequency oscillations and coupled gating of mechanosensitive ion channels in dystrophic skeletal muscle

An absence of utrophin in muscle from mdx mice prolongs the open time of single mechanosensitive channels. On a time scale much longer than the duration of individual channel activations, genetic depletion of utrophin produces low frequency oscillations of channel open probability. Oscillatory channel opening occurred in the dystrophin/utrophin mutants, but was absent in wild-type and mdx fibers. By contrast, small conductance channels showed random gating behavior when present in the same patch. Applying a negative pressure to a patch on a DKO fiber produced a burst of mode II activity, but channels subsequently closed and remained silent for tens of seconds during the maintained pressure stimulus. In addition, simultaneous opening of multiple MS channels could be frequently observed in recordings from patches on DKO fibers, but only rarely in wild-type and mdx muscle. A model which accounts for the single-channel data is proposed in which utrophin acts as gating spring which maintains the mechanical stability a caveolar-like compartment. The state of this compartment is suggested to be dynamic; its continuity with the extracellular surface varying over seconds to minutes. Loss of the mechanical stability of this compartment contributes to pathogenic Ca2+ entry through MS channels in Duchenne dystrophy.

Rapid and simultaneous analysis of three molecular sea surface temperature proxies and application to sediments from the Sea of Marmara

Reconstructing ocean temperature values is a major target in paleoceanography and climate research. However, most temperature proxies are organism-based and thus suffer from an “ecological bias”. Multiproxy approaches can potentially overcome this bias but typically require more investment in time and resources, while being susceptible to errors induced by sample preparation steps necessary before analysis. Three lipid-based temperature proxies are widely used: UK′37 (based on long chain alkenones from phytoplanktonic haptophytes), TEX86 [based on glycerol dialkyl glycerol tetraethers (GDGTs) from pelagic archaea] and LDI (based on long chain diols from phytoplanktonic eustigmatophytes). So far, separate analytical methods, including gas chromatography (GC) and liquid chromatography (LC), have been used to determine these proxies. Here we present a sensitive method for determining all three in a single normal phase high performance LC–atmospheric pressure chemical ionization mass spectrometry (NP-HPLC–APCI-MS) analysis. Each of the long chain alkenones and long chain diols was separated and unambiguously identified from the accurate masses and characteristic fragmentation during multiple stage MS analysis (MS2). Comparison of conventional GC and HPLC–MS methods showed similar results for UK′37 and LDI, respectively, using diverse environmental samples and an Emiliania huxleyi culture. Including the three sea surface temperature (SST) proxies; the NP-HPLC–APCI-MS method in fact allows simultaneous determination of nine paleoenvironmental proxies. The extent to which the ecology of the source organisms (ecological bias) influences lipid composition and thereby the reconstructed temperature values was demonstrated by applying the new method to a sediment core from the Sea of Marmara, covering the last 21kyr BP. Reconstructed SST values differed considerably between the proxies for the Last Glacial Maximum (LGM) and the period of Sapropel S1 formation at ca. 10kyr BP, whereas the trends during the late Holocene were similar. Changes in the composition of alkenone-producing species at the transition from the LGM to the Bølling/Allerød (B/A) were inferred from unreasonably high UK′37-derived SST values (ca. 20°C) during the LGM. We ascribe discrepancies between the reconstructed temperature records during S1 deposition to habitat change, e.g. a different depth due to changes in nutrient availability.

Therapeutic strategies for multiple sclerosis: Current data

At present, the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have approved drugs for Relapsing Remitting Multiple Sclerosis (RRMS) and Secondary Progressive MS (SPMS). In this paper, modern therapeutic strategies for MS are reviewed. A comprehensive research in MEDLINE, PUBMED, and SCIENCEDIRECT databases using two Boolean phrases {i.e. 1. [(Multiple Sclerosis) and (Disease Modifying Therapies)] and 2. [(Multiple Sclerosis) and (Neuroprotective Therapies)]} yielded more than 1,5000 articles in total. Therefore, for the purposes of this paper, articles written within the last decade were considered for review.

PPAR α and PPAR γ Polymorphisms as risk factors for Dyslipidemia in a Chinese han population

BackgroundThe PPAR α and PPAR γ are the key messengers responsible for the translation of nutritional stimuli into changes for the expression of genes, particularly genes involved in lipid metabolism. However, the associations between PPAR α / γ polymorphisms and lipid serum levels in the general population were rarely studied, and the conclusions were conflicting. The objective was to investigate the associations of the PPAR α and PPAR γ polymorphisms with dyslipidemia.Methods820 subjects were randomly selected from the Prevention of Multiple Metabolic Disorders and MS in Jiangsu Province cohort populations. The logistic regression model was used to examine the association between these polymorphisms and dyslipidemia. SNPstats was used to explore the haplotype association analyses.ResultsIn the codominant and log-additive models, rs1800206, rs1805192 and rs3856806 were all associated with dyslipidemia (P < 0.005). When the most common haplotype L-G (established by rs1800206, rs4253778) was treated as the reference group, the V-G haplotype was associated with dyslipidemia (P < 0.001), higher TC and TG levels (P < 0.01). Moreover, when compared to Pro-C haplotype (established by rs1805192, rs3856806), the Pro-T, Ala-C, Ala-T haplotypes were associated with dyslipidemia (p < 0.001). A-T haplotype was associated with higher TC levels, (p < 0.01), and the P-T, A-C, A-T haplotypes were associated with higher TG levels (p < 0.01).ConclusionsPPAR α and PPAR γ polymorphisms and haplotypes may be the genetic risk factors for dyslipidemia.

Biochemical characterization of a new β-1,3-galactosyltransferase WbuP from Escherichia coli O114 that catalyzes the second step in O-antigen repeating-unit

In this study, synthetic acceptor substrate GlcNAc alpha-PO3-PO3-(CH2)11-O-phenyl (GlcNAc-PP-PhU) was employed in glycosyl transferase assays to characterize the WbuP galactosyltransferase activity. This activity was time- and enzyme concentration-dependent. The optimal enzyme activity was observed at pH 6.5 and 25°C. The enzyme requires Mn2+ ions for maximal activity and detergents in the assay did not increase glycosyltransfer activity. The enzyme was shown to be specific for the UDP-Gal donor substrate. Kinetic parameters were determined for UDP-Gal, and GlcNAc-PP-PhU. The enzyme product was determined to have a β-1,3-linkage using strategies based on exoglycosidase digestion combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) as well as collision-induced dissociation electrospray ionization ion trap multiple tandem MS (CID-ESI-IT-MSn). Our results conclusively demonstrate that the wbuP gene of Escherichia coli O114 encodes a UDP-Gal: GlcNAc α-pyrophosphate-lipid β-1,3-Gal-transferase that transfers the second sugar moiety in the assembly of the O114 repeating unit.

Rapid and sensitive MRM‐based mass spectrometry approach for systematically exploring ganglioside‐protein interactions

Gangliosides are ubiquitous components of cell membranes. Their interactions with bacterial toxins and membrane-associated proteins (e.g. receptor tyrosine kinases) have important roles in the regulation of multiple cellular functions. Currently, an effective approach for measuring ganglioside-protein interactions especially in a large-scale fashion is largely missing. To this end, we report a facile MS-based approach to explore gangliosides extracted from cells and measure their interactions with protein of interest globally. We optimized a two-step protocol for extracting total gangliosides from cells within 2 h. Easy-to-use magnetic beads conjugated with a protein of interest were used to capture interacting gangliosides. To measure ganglioside-protein interaction on a global scale, we applied a high-sensitive LC-MS system, containing hydrophilic interaction LC separation and multiple reaction monitoring-based MS for ganglioside detection. Sensitivity for ganglioside GM1 is below 100 pg, and the whole analysis can be done in 20 min with isocratic elution. To measure ganglioside interactions with soluble vascular endothelial growth factor receptor 1 (sFlt1), we extracted and readily detected 36 species of gangliosides from perivascular retinal pigment epithelium cells across eight different classes. Twenty-three ganglioside species have significant interactions with sFlt1 as compared with IgG control based on p value cutoff <0.05. These results show that the described method provides a rapid and high-sensitive approach for systematically measuring ganglioside-protein interactions.

Association of Peroxisome Proliferator-Activated Receptor ^|^alpha;/^|^delta;/^|^gamma; With Obesity, and Gene^|^ndash;Gene Interaction, in the Chinese Han Population

BackgroundWe investigated the association of 10 single-nucleotide polymorphisms (SNPs) in the peroxisome proliferator-activated receptors (PPARs) with obesity and the additional role of gene–gene interaction.MethodsParticipants were recruited within the framework of the Prevention of Multiple Metabolic Disorders and MS in Jiangsu Province cohort population survey of an urban community in China. In total, 820 subjects (513 nonobese adults, 307 obese adults) were randomly selected, and no individuals were consanguineous. Ten SNPs (rs135539, rs4253778, rs1800206, rs2016520, rs9794, rs10865710, rs1805192, rs709158, rs3856806, and rs4684847) were genotyped and analyzed.ResultsAfter covariate adjustment, minor alleles of rs2016520 in PPARδ and rs10865170 in PPARγ were associated with lower BMI (P < 0.01 for all). Generalized multifactor dimensionality reduction analysis showed significant gene–gene interaction among rs2016520, rs9794, and rs10865170 in 3-dimensional models (P = 0.0010); prediction accuracy was 0.6011 and cross-validation consistency was 9/10. It also showed significant gene–gene interaction between rs2016520 and rs10865170 in all 2-dimensional models (P = 0.0010); prediction accuracy was 0.6072 and cross-validation consistency was 9/10.Conclusionsrs2016520 and rs10865170 were associated with lower obesity risk. In addition, interaction was identified among rs2016520, rs9794, and rs10865170 in obesity.

Association and interaction of peroxisome proliferator-activated receptor α with low high-density lipoprotein hyperlipidemia and with peroxisome proliferator-activated receptor γ

To investigate the association of ten single nucleotide polymorphisms (SNPs) in the peroxisome proliferator-activated receptors (α, δ, γ) with low high-density lipoprotein-cholesterol (HDL-C) hyperlipidemia and the additional role of a gene-gene interactions among the 10 SNPs. Participants were recruited under the framework of the PMMJS (Prevention of Multiple Metabolic Disorders and MS in Jiangsu Province) cohort populations survey, in the urban community of Jiangsu province, China. 820 subjects (579 normal HDL-C, 241 low HDL-C) were randomly selected, with one of them related to each other. Ten SNPs (rs135539, rs4253778, rs1800206, rs2016520, rs9794, rs10865710, rs1805192, rs709158, rs3856806, rs4684847) were selected from the HapMap database, which covered PPARα, PPARδ and PPARγ. Logistic regression model was used to examine the association between ten SNPs in the PPARs and low HDL-C. Odds ratios (OR) and 95% confident interval (95%CI) were calculated. Interactions were explored by using the method of Generalized Multifactor Dimensionality Reduction (GMDR). After adjusting the factors as age, sex, smoking status, occupational physical activity, high-fat diet as well as low-fiber diet, both rs135539 and rs1800206 were significantly associated with the incidence of low HDL-C, with the OR (95% CI) values as 1.46 (1.07 - 1.99) and 0.62 (0.42 - 0.90). No statistically significant difference was found between other SNPs and the occurrence of low HDL-C. Data from GMDR analysis showed significant gene-gene interaction among rs135539, rs4253778 of PPARα and rs10865710, rs3856806, rs709158 and rs4684847 of PPAR γ (P = 0.0107). PPARα rs135539 was associated with the occurrence of low HDL-C, and had interacted with rs4253778, rs10865710, rs3856806, rs709158 and rs4684847.

Association between peroxisome proliferator-activated receptors gene polymorphism and essential hypertension

To investigate the association between ten single nucleotide polymorphism (SNP) in the peroxisome proliferator-activated receptor (PPAR) α/δ/γ and essential hypertension (EH). Participants were recruited within the framework of a cohort populations survey from the PMMJS (Prevention of Multiple Metabolic Disorders and MS in Jiangsu Province) which was conducted in the urban community of Jiangsu province from 1999 to 2007. Eight hundred and twenty subjects (551 non-hypertensive subjects, 269 hypertensive subjects) were randomly selected but were not related to each other. Ten SNP (rs135539, rs1800206, rs4253778 of PPARα; rs2016520, rs9794 of PPARδ; rs10865710, rs1805192, rs4684847, rs709158 and rs3856806 of PPARγ) were selected from the HapMap database. χ(2) test was used to determine whether the whole population was in H-W genetic equilibrium. SHEsis software was used to examine the relations of SNP and linkage equilibrium. Logistic regression model was used to examine the association between ten SNP in the PPAR and EH. Difference on the distribution of four SNP genotypes including rs1800206, rs9794, rs10865710 and rs4684847 between high blood pressure and non-high blood pressure group, high systolic blood pressure (SBP) and normal SBP group, high diastolic blood pressure (DBP) and normal DBP group was significant (P < 0.05). After adjusting factors as age, sex, body mass index, fasting plasma glucose, high density lipoprotein cholesterol-C, high-fat diet and compared with wild-type gene carriers, the OR (95%CI) of objects with rs1800206 V allele appeared in high blood pressure, high SBP and high DBP were 0.60 (0.41 - 0.89), 0.57 (0.37 - 0.88) and 0.61 (0.39 - 0.96), respectively. The OR (95%CI) of objects with G allele of rs9794 were 0.63 (0.46 - 0.87), 0.51 (0.36 - 0.73) and 0.68 (0.47 - 1.01). The OR (95%CI) of objects with G allele of rs10865710 were 1.62 (1.19 - 2.20), 1.59 (1.14 - 2.22) and 1.53 (1.07 - 2.18), respectively. While the OR (95%CI) of objects with rs4684847 T allele were 1.42 (1.04 - 1.94), 1.38 (1.03 - 1.92) and 1.37 (1.00 - 1.88), respectively. The four SNPs including rs1800206 of PPARα, rs9794 of PPARδ and rs4684847, rs10865710 of PPARγ influenced high blood pressure, high SBP and high DBP to different degrees.

THE HELIUM CONTENT OF GLOBULAR CLUSTERS: NGC 6121 (M4)

He has been proposed as a key element to interpret the observed multiple MS, SGB, and RGB, as well as the complex horizontal branch (HB) morphology. Stars belonging to the bluer part of the HB, are thought to be more He rich (\Delta Y=0.03 or more) and more Na-rich/O-poor than those located in the redder part. This hypothesis was only partially confirmed in NGC 6752, where stars of the redder zero-age HB showed a He content of Y=0.25+-0.01, fully compatible with the primordial He content of the Universe, and were all Na-poor/O-rich. Here we study hot blue HB (BHB) stars in the GC NGC 6121 (M4) to measure their He plus O/Na content. We observed 6 BHB stars using the UVES@VLT2 spectroscopic facility. In addition to He, O, Na, and Fe abundances were estimated. Stars turned out to be all Na-rich and O-poor and to have a homogeneous enhanced He content with a mean value of Y=0.29+-0.01(random)+-0.01(systematic). The high He content of blue HB stars in M4 is also confirmed by the fact that they are brighter than red HB stars (RHB). Theoretical models suggest the BHB stars are He-enhanced by \Delta Y=0.02-0.03 with respect to the RHB stars. The whole sample of stars has a metallicity of [Fe/H]=-1.06+-0.02 (internal error). This is a rare direct measurement of the (primordial) He abundance for stars belonging to the Na-rich/O-poor population of GC stars in a temperature regime where the He content is not altered by sedimentation or extreme mixing as suggested for the hottest, late helium flash HB stars. Our results support theoretical predictions that the Na-rich/O-poor population is also more He-rich than the Na-poor/O-rich generation and that a leading contender for the 2^{nd} parameter is the He abundance.

Analytical method for the accurate determination of tricothecenes in grains using LC-MS/MS: a comparison between MRM transition and MS3 quantitation

The current food crisis demands unambiguous determination of mycotoxin contamination in staple foods to achieve safer food for consumption. This paper describes the first accurate LC-MS/MS method developed to analyze tricothecenes in grains by applying multiple reaction monitoring (MRM) transition and MS(3) quantitation strategies in tandem. The tricothecenes are nivalenol, deoxynivalenol, deoxynivalenol-3-glucoside, fusarenon X, 3-acetyl-deoxynivalenol, 15-acetyldeoxynivalenol, diacetoxyscirpenol, and HT-2 and T-2 toxins. Acetic acid and ammonium acetate were used to convert the analytes into their respective acetate adducts and ammonium adducts under negative and positive MS polarity conditions, respectively. The mycotoxins were separated by reversed-phase LC in a 13.5-min run, ionized using electrospray ionization, and detected by tandem mass spectrometry. Analyte-specific mass-to-charge (m/z) ratios were used to perform quantitation under MRM transition and MS(3) (linear ion trap) modes. Three experiments were made for each quantitation mode and matrix in batches over 6 days for recovery studies. The matrix effect was investigated at concentration levels of 20, 40, 80, 120, 160, and 200 μg kg(-1) (n = 3) in 5 g corn flour and rice flour. Extraction with acetonitrile provided a good overall recovery range of 90-108% (n = 3) at three levels of spiking concentration of 40, 80, and 120 μg kg(-1). A quantitation limit of 2-6 μg kg(-1) was achieved by applying an MRM transition quantitation strategy. Under MS(3) mode, a quantitation limit of 4-10 μg kg(-1) was achieved. Relative standard deviations of 2-10% and 2-11% were reported for MRM transition and MS(3) quantitation, respectively. The successful utilization of MS(3) enabled accurate analyte fragmentation pattern matching and its quantitation, leading to the development of analytical methods in fields that demand both analyte specificity and fragmentation fingerprint-matching capabilities that are unavailable under MRM transition.

Identification of alternative splice variants in Aspergillus flavus through comparison of multiple tandem MS search algorithms

BackgroundDatabase searching is the most frequently used approach for automated peptide assignment and protein inference of tandem mass spectra. The results, however, depend on the sequences in target databases and on search algorithms. Recently by using an alternative splicing database, we identified more proteins than with the annotated proteins in Aspergillus flavus. In this study, we aimed at finding a greater number of eligible splice variants based on newly available transcript sequences and the latest genome annotation. The improved database was then used to compare four search algorithms: Mascot, OMSSA, X! Tandem, and InsPecT.ResultsThe updated alternative splicing database predicted 15833 putative protein variants, 61% more than the previous results. There was transcript evidence for 50% of the updated genes compared to the previous 35% coverage. Database searches were conducted using the same set of spectral data, search parameters, and protein database but with different algorithms. The false discovery rates of the peptide-spectrum matches were estimated < 2%. The numbers of the total identified proteins varied from 765 to 867 between algorithms. Whereas 42% (1651/3891) of peptide assignments were unanimous, the comparison showed that 51% (568/1114) of the RefSeq proteins and 15% (11/72) of the putative splice variants were inferred by all algorithms. 12 plausible isoforms were discovered by focusing on the consensus peptides which were detected by at least three different algorithms. The analysis found different conserved domains in two putative isoforms of UDP-galactose 4-epimerase.ConclusionsWe were able to detect dozens of new peptides using the improved alternative splicing database with the recently updated annotation of the A. flavus genome. Unlike the identifications of the peptides and the RefSeq proteins, large variations existed between the putative splice variants identified by different algorithms. 12 candidates of putative isoforms were reported based on the consensus peptide-spectrum matches. This suggests that applications of multiple search engines effectively reduced the possible false positive results and validated the protein identifications from tandem mass spectra using an alternative splicing database.

Mass spectrometric identification of novel lysine acetylation sites in huntingtin.

Huntingtin (Htt) is a protein with a polyglutamine stretch in the N-terminus and expansion of the polyglutamine stretch causes Huntington's disease (HD). Htt is a multiple domain protein whose function has not been well characterized. Previous reports have shown, however, that post-translational modifications of Htt such as phosphorylation and acetylation modulate mutant Htt toxicity, localization, and vesicular trafficking. Lysine acetylation of Htt is of particular importance in HD as this modification regulates disease progression and toxicity. Treatment of mouse models with histone deacetylase inhibitors ameliorates HD-like symptoms and alterations in acetylation of Htt promotes clearance of the protein. Given the importance of acetylation in HD and other diseases, we focused on the systematic identification of lysine acetylation sites in Htt23Q (1-612) in a cell culture model using mass spectrometry. Myc-tagged Htt23Q (1-612) overexpressed in the HEK 293T cell line was immunoprecipitated, separated by SDS-PAGE, digested and subjected to high performance liquid chromatography tandem MS analysis. Five lysine acetylation sites were identified, including three novel sites Lys-178, Lys-236, Lys-345 and two previously described sites Lys-9 and Lys-444. Antibodies specific to three of the Htt acetylation sites were produced and confirmed the acetylation sites in Htt. A multiple reaction monitoring MS assay was developed to compare quantitatively the Lys-178 acetylation level between wild-type Htt23Q and mutant Htt148Q (1-612). This report represents the first comprehensive mapping of lysine acetylation sites in N-terminal region of Htt.

Application of complementary mass spectrometric techniques to the identification of ketoprofen phototransformation products

Ketoprofen (KP) is a nonsteroidal anti-inflammatory drug, which during UV irradiation rapidly transforms into benzophenone derivatives. Such transformation products may occur after topical application of KP, which is then exposed to sunlight resulting in a photo-allergic reaction. These reactions are mediated by the benzophenone moiety independently of the amount of allergen. The same reactions will also occur during wastewater or drinking water treatment albeit their effect in the aqueous environment is yet to be ascertained. In addition, only a few such transformation products have been recognised. To enable the detection and structural elucidation of the widest range of KP transformation products, this study applies complementary chromatographic and mass spectrometric techniques including gas chromatography coupled to single quadrupole or ion trap mass spectrometry and liquid chromatography hyphenated with quadrupole-time-of-flight mass spectrometry. Based on structural information gained in tandem and multiple MS experiments, and on highly accurate molecular mass measurements, chemical structures of 22 transformation products are proposed and used to construct an overall breakdown pathway. Among the identified transformation products all but two compounds retained the benzophenone moiety--a result, which raises important issues concerning the possible toxic synergistic effects of KP and its transformation products. These findings trigger further research into water treatment technologies that would limit their entrance into environmental or drinking waters.