Improving gene annotation using peptide mass spectrometry

Annotation of protein-coding genes is a key goal of genome sequencing projects. In spite of tremendous recent advances in computational gene finding, comprehensive annotation remains a challenge. Peptide mass spectrometry is a powerful tool for researching the dynamic proteome and suggests an attractive approach to discover and validate protein-coding genes. We present algorithms to construct and efficiently search spectra against a genomic database, with no prior knowledge of encoded proteins. By searching a corpus of 18.5 million tandem mass spectra (MS/MS) from human proteomic samples, we validate 39,000 exons and 11,000 introns at the level of translation. We present translation-level evidence for novel or extended exons in 16 genes, confirm translation of 224 hypothetical proteins, and discover or confirm over 40 alternative splicing events. Polymorphisms are efficiently encoded in our database, allowing us to observe variant alleles for 308 coding SNPs. Finally, we demonstrate the use of mass spectrometry to improve automated gene prediction, adding 800 correct exons to our predictions using a simple rescoring strategy. Our results demonstrate that proteomic profiling should play a role in any genome sequencing project.

Statistical discrimination of black gel pen inks analysed by laser desorption/ionization mass spectrometry

Pearson correlation coefficients were applied for the objective comparison of 30 black gel pen inks analysed by laser desorption ionization mass spectrometry (LDI-MS). The mass spectra were obtained for ink lines directly on paper using positive and negative ion modes at several laser intensities. This methodology has the advantage of taking into account the reproducibility of the results as well as the variability between spectra of different pens. A differentiation threshold could thus be selected in order to avoid the risk of false differentiation. Combining results from positive and negative mode yielded a discriminating power up to 85%, which was better than the one obtained previously with other optical comparison methodologies. The technique also allowed discriminating between pens from the same brand.

Quantification of glucuronidated and sulfated steroids in human urine by ultra-high pressure liquid chromatography quadrupole time-of-flight mass spectrometry.

The urinary steroid profile is constituted by anabolic androgenic steroids, including testosterone and its relatives, that are extensively metabolized into phase II sulfated or glucuronidated steroids. The use of liquid chromatography coupled to mass spectrometry (LC-MS) is an issue for the direct analysis of conjugated steroids, which can be used as urinary markers of exogenous steroid administration in doping analysis, without hydrolysis of the conjugated moiety. In this study, a sensitive and selective ultra high-pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometer (UHPLC-QTOF-MS) method was developed to quantify major urinary metabolites simultaneously after testosterone intake. The sample preparation of the urine (1 mL) was performed by solid-phase extraction on Oasis HLB sorbent using a 96-well plate format. The conjugated steroids were analyzed by UHPLC-QTOF-MS(E) with a single-gradient elution of 36 min (including re-equilibration time) in the negative electrospray ionization mode. MS(E) analysis involved parallel alternating acquisitions of both low- and high-collision energy functions. The method was validated and applied to samples collected from a clinical study performed with a group of healthy human volunteers who had taken testosterone, which were compared with samples from a placebo group. Quantitative results were also compared to GC-MS and LC-MS/MS measurements, and the correlations between data were found appropriate. The acquisition of full mass spectra over the entire mass range with QTOF mass analyzers gives promise of the opportunity to extend the steroid profile to a higher number of conjugated steroids.

A statistical methodology for the comparison of blue gel pen inks analyzed by laser desorption/ionization mass spectrometry

A statistical methodology for the objective comparison of LDI-MS mass spectra of blue gel pen inks was evaluated. Thirty-three blue gel pen inks previously studied by RAMAN were analyzed directly on the paper using both positive and negative mode. The obtained mass spectra were first compared using relative areas of selected peaks using the Pearson correlation coefficient and the Euclidean distance. Intra-variability among results from one ink and inter-variability between results from different inks were compared in order to choose a differentiation threshold minimizing the rate of false negative (i.e. avoiding false differentiation of the inks). This yielded a discriminating power of up to 77% for analysis made in the negative mode. The whole mass spectra were then compared using the same methodology, allowing for a better DP in the negative mode of 92% using the Pearson correlation on standardized data. The positive mode results generally yielded a lower differential power (DP) than the negative mode due to a higher intra-variability compared to the inter-variability in the mass spectra of the ink samples.

A mass spectrometric study of some pesticides /|nK. S. Subramanian. -- 260 St. Catharines, Ont. : [s. n.],

The fragmentation processes in the mass spectra of a series of organophosphorus, organochlorine, thio and dithiocarbamate as well as a number of miscellaneous pesticides have been studied i n detail by using the Bendix timeof- flight, MS-12 single-focussing and MS-30 double-focussing mass spectrometers. Interpretation of all the spectra have been presented; their mode s of dissociation elucidated, aided by metastable transitions wherever possible and the structures of the various f ragmentation species postulated wherever f easible. The fragmentation mechanisms are based on the concepts of inductive, resonance and steric ef~ects. Multiple bond cleavages accompanied by simultaneous bond formation and rearrangement reactions involving cycli c t r ansition states have clarified t he formation of various ions . Due emphasis has been placed on the effect of the functional groups or substituents in altering the mass spectral behaviour of the pesticides as they form the basis for the identifi cation of the otherwise identical pesticides. The organophosphorus pesticides which have been studied include i) the phosphates (eg: DDVP and Phosdrin ); ii) phosphorothionates (eg: Parathion, 0-2, 4 dichloro phenyl 0, O-diethyl thionophosphate); iii) phosphorothioites (eg: Tributyl phosphorotrithioite); i V) phosphorothioates (eg: Ethion) and v) phosphorodithioates (eg: Carbophenolthion). Cleavages and rearrangements of the ester moiety dominate the spectrum of phosdrin while that of DDVP is + dominated by t he fragmentation modes of the (OH30)2P=0 + moiety. Fragmentation §f the (CH30)2P=S characterises the spectrum of (OH30)2"P -Cl while cleavages of the + (C2H50 )2P=S species mark the spectra of parathion and 0-2, 4- di chlorophenyl O, O-diethyl thiophosphate. The 0(, cl eavages of the thioether f unction rather than + cleavages of the (C2H50)2P=S signify the spectrum of carbophenolthion. Tributyl phosphorotrithioite behaves more like an aliphatic hydrocarbon than like the corresponding phosphites. The isopropyl and butyl esters of 2, 4 dichlorophenoxy acetic acid show cleavage and rearrangement ions typical of an ester. In spite of its structural similari ty to pp' - DDT and pp' - DDD, Kalthane has a completely different mass spectral behaviour due to the influence of its hydroxyl function. The thiocarbamate pesticides studied include Eptam and Perbulate. Both are structurally similar but having different alkyl substituents on nitrogen and sulphur. This structurQlsimilarity leads to similar types of (N-C), (O-S) and (S-alkyl cleavages). However, perbulate differs from Eptam in showing a rearrangement ion at mle 161 and in forming an isocyanate ion as the base peak. In Eptam the base peak i s the alkyl ion. The dithiocarbamate, Vegadex, resembles the thiocarbamates in undergoing simple cleavages but it differs from them in having a weak parent ion; in the formation of its base peak and in undergoing a series of rearrangement reactions. The miscellaneous pesticides studied include 1-Naphthalene acetic aCid- methyl ester, Fiperonyl butoxide and Allethrin. The ester i s stable to electron impact and shows only fewer ions. Piper onyl butoxide, a polyether, shows characteristics of an et her, alcohol and aldehyde . Allethrin is regarded as an ester of the type R-C-O-R1 with n R being a substituted cyclopr opane moiety and o Rt, a substituted cyclopentenone mOiety. Accordingly it shows cleavage ions typical of an aliphatic ester and undergoes bond ruptures of the cyclic moieties to give unusual ions. Its base peak is an odd electron ion, quite contrary to expectations.

Comparative electron impact and fast atom bombardment mass spectrometric studies of some HMPA adducts of phenyltin and phenyllead halides and studies of strong hydrogen bonding by FAB-MS

The fragmentation patterns and mass spectra of some phenyl tin and -lead halide adducts with hexamethylphosphoramide are compared by subjecting them t~ electron impact and fast atom bombardment ionization in a mass spectrometer. This comparison is restricted to the metal-containing ions. Ligand-exchange mechanisms of some of the metal-containing species are explored by FAB-MS. Several moisturesensitive organo-metallics and H-bonded systems have been examined by FAB for attempted characterization, but without any success. Scavenging and trapping of water molecules by complex aggregates in solutions of quaternary ammonium fluorides and hydroxides are investigated by FAB to complement previous NMR-studies.

Fast atom bombardment mass spectrometry of the coordination complexes of the ligand 5, 5, 7, 12, 12, 14- hexamenthyl-1, 4, 8, 11-tetraazacycloteradecane

A number of metal complexes containing the ligand 5,5,7,12,12,14-hexamethyl-l,4,8,11-tetra-azatetradecane were synthesized and analyzed using electron impact (EI) and fast atom bombardment (FAB). The FAB mass spectra were obtained in positive and negative ion mode. FAB in the positive ion mode proved to be the most successful technique for the identification of these compounds. In the majority of cases the spectra obtained using positive ion FAB were structurally informative, although not all showed molecular (M+) or quasimolecular ([M+H]+) ions. The fragmentations observed were characteristic of the ligands, and were interpreted based on the chemistry of these compounds.

Mass spectral studies of some pentafluorophenyl derivatives of group V /|nby Andrew Tanner Rake. -- 260 St. Catharines, Ont. : [s. n.],

The mass spectra of compounds of t he series (C6F5 )3-n MP~ (n = 1,2,3, M = P and As ), (C6F5>3Sb, Ph) Sb and (C6F5 )2SbPh have been studied in detail and the important modes of fragmentation were e1ucidated, a ided by metastable ions. Various trends attributed to the central atom and or the . substituent groups have been noted and, where applicable, compared to recent studies on related phenyl and pentafluorophenyl compounds of groups IV and V. The mass spectra of fluorine containing organometallic compounds exhibit characteristic migrations of fluorine to t he central atom, giving an increasing abundance of MF+, MF2+' and RMF+ (R = Ph or C6F5) ions on descending the group_ The mass spectra of pentafluorophenyl , antimony, and arsenic compounds show a greater fragmentation of the aromatic ring than those of phosphorus. The mixed phenyl pentafluorophenyl derivatives show a characteristic pattern depending on the number of phenyl grm.lps present but show t he general characteristics of both the tris(phenyl) and tris(pentafluorophenyl) compounds. The diphenyl pentafluorophenyl der ivatives show the loss of biphenyl ion as the most import ant step, the los s of phenyl t o give the i on PhMC6F5 + being of secondary importance. The ,bis(pentafluorophenyl) phenyl derivatives fragment primarily by loss of PhC6F5 to give C6F5M+ ions, the abundance of t hese increasing r apidly from phosphorus to arsenic. This species then, exhibits a characteristic fragmentation observed in the tris(penta- fluorophenyl ) compounds. However, the abundance of (C6F5)2M+ species in these compounds i s small. I ons of the type C6H4MC6F4 + and tetrafluorobiphenylene ions C6H4C6F4 + also are observed on substitution of a phenyl group for a penta- fluorophenyl group. The fully fluorinated species (C6F4)2M+ is not observed, although octafluorobiphenylene ions , (C6F4)2+' are evident in several spectra . The appearance potentials of the major ions were obtatned from the ionisation efficiency curves. Attempts were made to correlate these to the effect of the central atom in substituent groups, but the large errors involved prevented the reaching of quantitative conclusions, although it would appear that the electron is removed from the ligand in the ionisation of t he parent molecule .

The fast atom bombardment mass spectrometric analysis of the carbamate pesticide carbaryl

The carbamate pesticide, carbaryl, was quantitatively studied using fast atom bombardment mass spectrometry (FAB-MS). Mass spectra were obtained in the positive ion-mode using both 2-nitrophenyloctyl ether (NPOE) and 3-nitrobenzyl alcohol (NBA) as matrix liquids. The sample was applied by three different techniques; simple mixing, solvent mixing and surface precipitation. Smaller volumes of matrix liquid were found to produce more favourable ion currents. Detection limits were largely independent of the matrix or application technique used. The relationship between ion current and the mass of analyte was found to be intricately related to the choice of matrix liquid.

Automatic internal calibration in liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry of protein digests

Accurately measured peptide masses can be used for large-scale protein identification from bacterial whole-cell digests as an alternative to tandem mass spectrometry (MS/MS) provided mass measurement errors of a few parts-per-million (ppm) are obtained. Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) routinely achieves such mass accuracy either with internal calibration or by regulating the charge in the analyzer cell. We have developed a novel and automated method for internal calibration of liquid chromatography (LC)/FTICR data from whole-cell digests using peptides in the sample identified by concurrent MS/MS together with ambient polydimethyl-cyclosiloxanes as internal calibrants in the mass spectra. The method reduced mass measurement error from 4.3 +/- 3.7 ppm to 0.3 +/- 2.3 ppm in an E. coli LC/FTICR dataset of 1000 MS and MS/MS spectra and is applicable to all analyses of complex protein digests by FTICRMS. Copyright (c) 2006 John Wiley & Sons, Ltd.

Mass spectral characterization of submicron biogenic organic particles in the Amazon Basin

Submicron atmospheric particles in the Amazon Basin were characterized by a high-resolution aerosol mass spectrometer during the wet season of 2008. Patterns in the mass spectra closely resembled those of secondary-organic-aerosol (SOA) particles formed in environmental chambers from biogenic precursor gases. In contrast, mass spectral indicators of primary biological aerosol particles (PBAPs) were insignificant, suggesting that PBAPs contributed negligibly to the submicron fraction of particles during the period of study. For 40% of the measurement periods, the mass spectra indicate that in-Basin biogenic SOA production was the dominant source of the submicron mass fraction, contrasted to other periods (30%) during which out-of-Basin organic-carbon sources were significant on top of the baseline in-Basin processes. The in-Basin periods had an average organic-particle loading of 0.6 mu g m(-3) and an average elemental oxygen-to-carbon (O:C) ratio of 0.42, compared to 0.9 mu g m(-3) and 0.49, respectively, during periods of out-of-Basin influence. On the basis of the data, we conclude that most of the organic material composing submicron particles over the Basin derived from biogenic SOA production, a finding that is consistent with microscopy observations made in a concurrent study. This source was augmented during some periods by aged organic material delivered by long-range transport. Citation: Chen, Q., et al. (2009), Mass spectral characterization of submicron biogenic organic particles in the Amazon Basin, Geophys. Res. Lett., 36, L20806, doi: 10.1029/2009GL039880.

Atmospheric pressure photoionization mass spectrometry as a tool for the investigation of the hydrolysis reaction mechanisms of phosphite antioxidants

The hydrolysis reaction mechanism of phosphite antioxidants is investigated by liquid chromatography-mass spectrometry (LC/MS). The phosphites were chosen because they differed in chemical structure and phosphorus content. Dopant assisted-atmospheric pressure photoionization (DA-APPI) is chosen as the ion source for (lie ionization of the compounds. [it our previous work, DA-APPI was shown to offer an attractive alternative to atmospheric pressure chemical ionization (APCI) since it provided background-ion free mass spectra and higher sensitivity [M. Papanastasiou, et al., Polymer Degradation and Stability 91 (11) (2006) 2675-2682]. In positive ion mode, the molecules are generally detected in their protonated form. In negative ion mode, the phosphites are unstable and only fragment ions are observed: these however, are characteristic of each phosphite and may be used for the identification of the analytes in complex mixtures. The analytes under investigation are exposed to accelerated humid ageing conditions and their hydrolytic pathway and stability is investigated. Different substituents around the phosphorus atom are shown to have a significant effect on the stability of the phosphites, with phenol substituents producing very hydrolytically stable structures. Alkanox P24 and PEP-36 follow a similar hydrolytic pathway via the scission of the first and then the second P-O-phenol bonds, eventually leading to the formation of phenol, Phosphorous acid and pentaerythritol as end products. HP-10 exhibits a rather different Structure and the products detected suggest scission of either the P-O-hydrocarbon or one of the P-O-phenol bonds. A phenomenon similar to that of autocatalysis is observed for all phosphites and is attributed to the formation of dialkyl phosphites as intermediate products. (C) 2008 Elsevier B.V. All rights reserved.

Description of Heavy Quark Mass by Lippmann-Schwinger Equation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of liver S9 enzymes on somalargine and solasodine cytotoxicity and mass spectrometric fragmentation

The steroidal glycoalkaloid solamargine and its parent aglycone solasodine, isolated from Solanum palinacanthum, were studied in vitro for cytotoxicity and biotransformation by the hepatic S9 fraction as the metabolic activating system. The MTT uptake assay was used to determine viability after 24 h in RAW 264.7 mouse macrophage-like and SiHa cells exposed to various concentrations of the alkaloids in the presence and absence of the hepatic S9 microsomal fraction. The dose-response curves were established for solamargine and solasodine in the presence and absence of external metabolizing system. From these data, the cytotoxic index (CI50) was calculated with mean values of 7.2 and 13.6 μg/mL for Raw cells and 8.6 and 26.0 μg/mL for SiHa cells, respectively. Mass spectrometry was performed to compare the fragmentation patterns of the alkaloids to predict metabolism by the S9 fraction. The mass spectra demonstrated a distinct fragmentation patterns for solamargine and solasodine after the addition of the S9 fraction. In the present study, we demonstrate that the cytotoxic effect of solamargine and solasodine and their metabolites prepared in vitro by biotransformation with the S9 fraction are comparable. These findings suggest that the metabolic activation system S9 fraction may fail to suppress the cytotoxicity of these alkaloids. © 2013 Springer-Verlag Berlin Heidelberg.

Studies of jet mass in dijet and W/Z + jet events

Invariant mass spectra for jets reconstructed using the anti-k T and CambridgeAachen algorithms are studied for different jet grooming techniques in data corresponding to an integrated luminosity of 5 fb-1, recorded with the CMS detector in proton-proton collisions at the LHC at a center-of-mass energy of 7 TeV. Leading-order QCD predictions for inclusive dijet and W/Z+jet production combined with parton-shower Monte Carlo models are found to agree overall with the data, and the agreement improves with the implementation of jet grooming methods used to distinguish merged jets of large transverse momentum from softer QCD gluon radiation. © 2013 CERN for the benefit of the CMS collaboration.