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Sample records for dissociation tandem mass

  1. A tandem mass spectrometer for collision-induced dissociation

    NASA Astrophysics Data System (ADS)

    1982-02-01

    A tandem mass spectrometer is described for studies of collision-induced dissociation. This instrument is especially suited for investigations on organic molecules, e.g., biochemical substances, for m/z values up to 1000. The first stage is formed by a conventional EI source and a sector magnet, and has a mass resolution of about 600. The first stage is provided with a collision gas cell at the site of the detector slit. In the second stage the fragment ions are post-accelerated in order to reduce the relative energy-spread and to increase the resolution and transmission. The fragment spectrum is analyzed by a second magnet (R = 750 mm, deflection angle = 15 deg) and simultaneously recorded. Quadrupoles are added in order to vary the dispersion and to aid focussing. The ratio between the highest and lowest masses in a simultaneously detected spectrum may vary from 4 : 1 to 1.06 : 1. The resolution can be as high as 600, and the transmission from the collision cell to the CEMA ranges from 60 to 100%; the detection sensitivity can be as high as 1 ion per 10 s.

  2. Decoding Split and Pool Combinatorial Libraries with Electron Transfer Dissociation Tandem Mass Spectrometry

    PubMed Central

    Sarkar, Mohosin; Pascal, Bruce D.; Steckler, Caitlin; Aquino, Claudio; Micalizio, Glenn C.; Kodadek, Thomas; Chalmers, Michael J.

    2015-01-01

    Screening of bead-based split and pool combinatorial chemistry libraries is a powerful approach to aid the discovery of new chemical compounds able to interact with, and modulate the activities of, protein targets of interest. Split and pool synthesis provides for large and well diversified chemical libraries, in this case comprised of oligomers generated from a well-defined starting set. At the end of the synthesis, each bead in the library displays many copies of a unique oligomer sequence. Because the sequence of the oligomer is not known at the time of screening, methods for decoding of the sequence of each screening “hit” are essential. Here we describe an electron transfer dissociation (ETD) based tandem mass spectrometry approach for the decoding of mass-encoded split and pool libraries. We demonstrate that the newly described “chiral oligomers of pentenoic amides (COPAs)” yield non-sequence-specific product ions upon collisional activated dissociation; however, complete sequence information can be obtained with ETD. To aid in the decoding of libraries from MS and MS/MS data, we have incorporated 79Br/81Br isotope “tags” to differentiate N- and C-terminal product ions. In addition, we have created “Hit-Find,” a software program that allows users to generate libraries in silico. The user can then search all possible members of the chemical library for those that fall within a user-defined mass error. PMID:23636859

  3. Characterization of the iron-binding properties of pyoverdine using electron-capture dissociation-tandem mass spectrometry.

    PubMed

    Qi, Yulin; Hayen, Heiko; Volmer, Dietrich A

    2016-02-01

    Pyoverdines (PVD) are a group of siderophores produced by fluorescent Pseudomonads. Identification of PVD variants mostly relies on liquid chromatography-tandem mass spectrometry (LC-MS/MS) using collision-induced dissociation (CID). Here, both CID and the novel dissociation technique electron-capture dissociation (ECD) were applied to characterize PVD succinamide and its Fe(III)-chelated complex. The results clearly showed that ECD produced diagnostic side chain fragmentation of the PVD peptide chain and preserved the labile Fe(III) binding to the chromophore in contrast to CID. The ECD technique is therefore expected to support the understanding of strain-specific Fe(III) transport processes of PVDs.

  4. A review of electron-capture and electron-transfer dissociation tandem mass spectrometry in polymer chemistry.

    PubMed

    Hart-Smith, Gene

    2014-01-15

    Mass spectrometry (MS)-based studies of synthetic polymers often characterise detected polymer components using mass data alone. However when mass-based characterisations are ambiguous, tandem MS (MS/MS) offers a means by which additional analytical information may be collected. This review provides a synopsis of two particularly promising methods of dissociating polymer ions during MS/MS: electron-capture and electron-transfer dissociation (ECD and ETD, respectively). The article opens with a summary of the basic characteristics and operating principles of ECD and ETD, and relates these techniques to other methods of dissociating gas-phase ions, such as collision-induced dissociation (CID). Insights into ECD- and ETD-based MS/MS, gained from studies into proteins and peptides, are then discussed in relation to polymer chemistry. Finally, ECD- and ETD-based studies into various classes of polymer are summarised; for each polymer class, ECD- and ETD-derived data are compared to CID-derived data. These discussions identify ECD and ETD as powerful means by which unique and diagnostically useful polymer ion fragmentation data may be generated, and techniques worthy of increased utilisation by the polymer chemistry community.

  5. Identification of cross-linked peptides after click-based enrichment using sequential collision-induced dissociation and electron transfer dissociation tandem mass spectrometry.

    PubMed

    Chowdhury, Saiful M; Du, Xiuxia; Tolić, Nikola; Wu, Si; Moore, Ronald J; Mayer, M Uljana; Smith, Richard D; Adkins, Joshua N

    2009-07-01

    Chemical cross-linking combined with mass spectrometry can be a powerful approach for the identification of protein-protein interactions and for providing constraints on protein structures. However, enrichment of cross-linked peptides is crucial to reduce sample complexity before mass spectrometric analysis. In addition compact cross-linkers are often preferred to provide short spacer lengths, surface accessibility to the protein complexes, and must have reasonable solubility under conditions where the native complex structure is stable. In this study, we present a novel compact cross-linker that contains two distinct features: (1) an alkyne tag and (2) a small molecule detection tag (NO(2)) to maintain reasonable solubility in water. The alkyne tag enables enrichment of the cross-linked peptides after proteolytic cleavage and coupling of an affinity tag using alkyne-azido click chemistry. Neutral loss of the small NO(2) moiety provides a secondary means of detecting cross-linked peptides in MS/MS analyses, providing additional confidence in peptide identifications. We show the labeling efficiency of this cross-linker, which we termed CLIP (click-enabled linker for interacting proteins) using ubiquitin. The enrichment capability of CLIP is demonstrated for cross-linked ubiquitin in highly complex E. coli cell lysates. Sequential collision-induced dissociation tandem mass spectrometry (CID-MS/MS) and electron transfer dissociation (ETD)-MS/MS of intercross-linked peptides (two peptides connected with a cross-linker) are also demonstrated for improved automated identification of cross-linked peptides.

  6. Determining the Binding Sites of β-Cyclodextrin and Peptides by Electron-Capture Dissociation High Resolution Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Qi, Yulin; Geib, Timon; Volmer, Dietrich A.

    2015-07-01

    Cyclodextrins (CDs) are a group of cyclic oligosaccharides, which readily form inclusion complexes with hydrophobic compounds to increase bioavailability, thus making CDs ideal drug excipients. Recent studies have also shown that CDs exhibit a wide range of protective effects, preventing proteins from aggregation, degradation, and folding. These effects strongly depend on the binding sites on the protein surface. CDs only exhibit weak interactions with amino acids, however; conventional analytical techniques therefore usually fail to reveal the exact location of the binding sites. Moreover, some studies even suggest that CD inclusion complexes are merely electrostatic adducts. Here, electron capture dissociation (ECD) was applied in this proof-of-concept study to examine the exact nature of the CD/peptide complexes, and CD binding sites were unambiguously located for the first time via Fourier-transform ion cyclotron resonance (FTICR) tandem mass spectrometry.

  7. Determining the Binding Sites of β-Cyclodextrin and Peptides by Electron-Capture Dissociation High Resolution Tandem Mass Spectrometry.

    PubMed

    Qi, Yulin; Geib, Timon; Volmer, Dietrich A

    2015-07-01

    Cyclodextrins (CDs) are a group of cyclic oligosaccharides, which readily form inclusion complexes with hydrophobic compounds to increase bioavailability, thus making CDs ideal drug excipients. Recent studies have also shown that CDs exhibit a wide range of protective effects, preventing proteins from aggregation, degradation, and folding. These effects strongly depend on the binding sites on the protein surface. CDs only exhibit weak interactions with amino acids, however; conventional analytical techniques therefore usually fail to reveal the exact location of the binding sites. Moreover, some studies even suggest that CD inclusion complexes are merely electrostatic adducts. Here, electron capture dissociation (ECD) was applied in this proof-of-concept study to examine the exact nature of the CD/peptide complexes, and CD binding sites were unambiguously located for the first time via Fourier-transform ion cyclotron resonance (FTICR) tandem mass spectrometry.

  8. Characterization of an Ion Mobility-Multiplexed Collision Induced Dissociation- Tandem Time-of-Flight Mass Spectrometry Approach

    SciTech Connect

    Ibrahim, Yehia M.; Prior, David C.; Baker, Erin Shammel; Smith, Richard D.; Belov, Mikhail E.

    2010-06-01

    The confidence in peptide (and protein) identifications with ion mobility spectrometry time-of-flight mass spectrometry (IMS-TOFMS) is expected to drastically improve with the addition of information from an efficient ion dissociation step prior to MS detection. High throughput IMS-TOFMS analysis imposes a strong need for multiplexed ion dissociation approaches where multiple precursor ions yield complex sets of fragment ions that are often intermingled with each other in both the drift time and m/z domains. We have developed and evaluated a novel approach for collision-induced dissociation (CID) with an IMS-TOFMS instrument. It has been shown that precursor ions activated inside an rf-device with an axial dc-electric field produce abundant fragment ions which are radially confined with the rf-field and collisionally cooled at an elevated pressure, resulting in high CID efficiencies comparable or higher than those measured in triple-quadrupole instruments We have also developed an algorithm for deconvoluting these complex multiplexed tandem MS spectra by clustering both the precursor and fragment ions into the matching drift time profiles and by effectively utilizing high mass measurement accuracy of the TOFMS. In a single IMS separation with a tryptic digest of bovine serum albumin (BSA), we have reliably identified 20 unique peptides using multiplexed CID approach downstream of the IMS separation. Peptides were identified based upon the correlation between the precursor and fragment drift time profiles and by matching the profile representative masses to those of in silico BSA tryptic peptides and their fragments. The false discovery rate (FDR) of peptide identifications from multiplexed MS/MS spectra was less than 1%.

  9. Defining the disulfide bonds of insulin-like growth factor-binding protein-5 by tandem mass spectrometry with electron transfer dissociation and collision-induced dissociation.

    PubMed

    Nili, Mahta; Mukherjee, Aditi; Shinde, Ujwal; David, Larry; Rotwein, Peter

    2012-01-06

    The six high-affinity insulin-like growth factor-binding proteins (IGFBPs) comprise a conserved family of secreted molecules that modulate IGF actions by regulating their half-life and access to signaling receptors, and also exert biological effects that are independent of IGF binding. IGFBPs are composed of cysteine-rich amino- (N-) and carboxyl- (C-) terminal domains, along with a cysteine-poor central linker segment. IGFBP-5 is the most conserved IGFBP, and contains 18 cysteines, but only 2 of 9 putative disulfide bonds have been mapped to date. Using a mass spectrometry (MS)-based strategy combining sequential electron transfer dissociation (ETD) and collision-induced dissociation (CID) steps, in which ETD fragmentation preferentially induces cleavage of disulfide bonds, and CID provides exact disulfide linkage assignments between liberated peptides, we now have definitively mapped 5 disulfide bonds in IGFBP-5. In addition, in conjunction with ab initio molecular modeling we are able to assign the other 4 disulfide linkages to within a GCGCCXXC motif that is conserved in five IGFBPs. Because of the nature of ETD fragmentation MS experiments were performed without chemical reduction of IGFBP-5. Our results not only establish a disulfide bond map of IGFBP-5 but also define a general approach that takes advantage of the specificity of ETD and the scalability of tandem MS, and the predictive power of ab initio molecular modeling to characterize unknown disulfide linkages in proteins.

  10. Precursor charge state prediction for electron transfer dissociation tandem mass spectra.

    PubMed

    Sharma, Vagisha; Eng, Jimmy K; Feldman, Sergey; von Haller, Priska D; MacCoss, Michael J; Noble, William S

    2010-10-01

    Electron-transfer dissociation (ETD) induces fragmentation along the peptide backbone by transferring an electron from a radical anion to a protonated peptide. In contrast with collision-induced dissociation, side chains and modifications such as phosphorylation are left intact through the ETD process. Because the precursor charge state is an important input to MS/MS sequence database search tools, the ability to accurately determine the precursor charge is helpful for the identification process. Furthermore, because ETD can be applied to large, highly charged peptides, the need for accurate precursor charge state determination is magnified. Otherwise, each spectrum must be searched repeatedly using a large range of possible precursor charge states. To address this problem, we have developed an ETD charge state prediction tool based on support vector machine classifiers that is demonstrated to exhibit superior classification accuracy while minimizing the overall number of predicted charge states. The tool is freely available, open source, cross platform compatible, and demonstrated to perform well when compared with an existing charge state prediction tool. The program is available from http://code.google.com/p/etdz/.

  11. Identification of reactive cysteines in a protein using arsenic labeling and collision-induced dissociation tandem mass spectrometry.

    PubMed

    Lu, Meiling; Wang, Hailin; Wang, Zhongwen; Li, Xing-Fang; Le, X Chris

    2008-08-01

    Trivalent arsenicals have high affinity for thiols (such as free cysteines) in proteins. We describe here the use of this property to develop a collision-induced dissociation (CID) tandem mass spectrometry (MS/MS) technique for the identification of reactive cysteines in proteins. A trivalent arsenic species, dimethylarsinous acid (DMA (III)), with a residue mass (103.9607) and mass defect distinct from the normal 20 amino acids, was used to selectively label reactive cysteine residues in proteins. The CID fragment ions of the arsenic-labeled sequences shifted away from the more abundant normal fragments that would otherwise overlap with the ions of interest. Along with the internal and immonium ions, the arsenic-labeled fragment ions served as MS/MS signatures for identification of the binding sites and for assessment of the relative reactivity of individual cysteine residues in a protein. Using this method, we have identified two highly reactive binding sites in rat hemoglobin (Hb): Cys-13alpha and Cys-125beta. Cys-13alpha was bound to DMA (III) in the Hb of rats fed with arsenic, and this binding was responsible for arsenic accumulation in rat blood, while Cys-125beta was found to bind to glutathione in rat blood. This study revealed the relative reactivity of the cysteines in rat Hb in the following decreasing order: Cys-13alpha > Cys-111alpha > Cys-104alpha and Cys-13alpha > Cys-125beta > Cys-93beta. Arsenic-labeling is easy and fast for identification of active binding sites without enzymatic digestion and acid hydrolysis, and useful for characterization and identification of metal binding sites in other proteins.

  12. Toward a suitable structural analysis of gene delivery carrier based on polycationic carbohydrates by electron transfer dissociation tandem mass spectrometry.

    PubMed

    Przybylski, Cédric; Benito, Juan M; Bonnet, Véronique; Mellet, Carmen Ortiz; García Fernández, José M

    2016-12-15

    Polycationic carbohydrates represent an attractive class of biomolecules for several applications and particularly as non viral gene delivery vectors. In this case, the establishment of structure-biological activity relationship requires sensitive and accurate characterization tools to both control and achieve fine structural deciphering. Electrospray-tandem mass spectrometry (ESI-MS/MS) appears as a suitable approach to address these questions. In the study herein, we have investigated the usefulness of electron transfer dissociation (ETD) to get structural data about five polycationic carbohydrates demonstrated as promising gene delivery agents. A particular attention was paid to determine the influence of charge states as well as both fluoranthene reaction time and supplementary activation (SA) on production of charge reduced species, fragmentation yield, varying from 2 to 62%, as well as to obtain the most higher both diversity and intensity of fragments, according to charge states and targeted compounds. ETD fragmentation appeared to be mainly directed toward pending group rather than carbohydrate cyclic scaffold leading to a partial sequencing for building blocks when amino groups are close to carbohydrate core, but allowing to complete structural deciphering of some of them, such as those including dithioureidocysteaminyl group which was not possible with CID only. Such findings clearly highlight the potential to help the rational choice of the suitable analytical conditions, according to the nature of the gene delivery molecules exhibiting polycationic features. Moreover, our ETD-MS/MS approach open the way to a fine sequencing/identification of grafted groups carried on various sets of oligo-/polysaccharides in various fields such as glycobiology or nanomaterials, even with unknown or questionable extraction, synthesis or modification steps.

  13. Elucidating collision induced dissociation products and reaction mechanisms of protonated uracil by coupling chemical dynamics simulations with tandem mass spectrometry experiments.

    PubMed

    Molina, Estefanía Rossich; Ortiz, Daniel; Salpin, Jean-Yves; Spezia, Riccardo

    2015-12-01

    In this study we have coupled mixed quantum-classical (quantum mechanics/molecular mechanics) direct chemical dynamics simulations with electrospray ionization/tandem mass spectrometry experiments in order to achieve a deeper understanding of the fragmentation mechanisms occurring during the collision induced dissociation of gaseous protonated uracil. Using this approach, we were able to successfully characterize the fragmentation pathways corresponding to ammonia loss (m/z 96), water loss (m/z 95) and cyanic or isocyanic acid loss (m/z 70). Furthermore, we also performed experiments with isotopic labeling completing the fragmentation picture. Remarkably, fragmentation mechanisms obtained from chemical dynamics simulations are consistent with those deduced from isotopic labeling.

  14. Formation and reshuffling of disulfide bonds in bovine serum albumin demonstrated using tandem mass spectrometry with collision-induced and electron-transfer dissociation.

    PubMed

    Rombouts, Ine; Lagrain, Bert; Scherf, Katharina A; Lambrecht, Marlies A; Koehler, Peter; Delcour, Jan A

    2015-07-20

    Thermolysin hydrolyzates of freshly isolated, extensively stored (6 years, 6 °C, dry) and heated (60 min, 90 °C, in excess water) bovine serum albumin (BSA) samples were analyzed with liquid chromatography (LC) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using alternating electron-transfer dissociation (ETD) and collision-induced dissociation (CID). The positions of disulfide bonds and free thiol groups in the different samples were compared to those deduced from the crystal structure of native BSA. Results revealed non-enzymatic posttranslational modifications of cysteine during isolation, extensive dry storage, and heating. Heat-induced extractability loss of BSA was linked to the impact of protein unfolding on the involvement of specific cysteine residues in intermolecular and intramolecular thiol-disulfide interchange and thiol oxidation reactions. The here developed approach holds promise for exploring disulfide bond formation and reshuffling in various proteins under conditions relevant for chemical, biochemical, pharmaceutical and food processing.

  15. Fundamentals of tandem mass spectrometry: a dynamics study of simple C-C bond cleavage in collision-activated dissociation of polyatomic ions at low energy.

    PubMed

    Shukla, A K; Qian, K; Anderson, S; Futrell, J H

    1990-02-01

    The loss of methyl radical in collision-activated dissociation (CAD) of acetone and propane molecular ions has been studied at low energy using a tandem hybrid mass spectrometer. Although the two processes are very similar chemically and energetically, very different dynamical features are observed. Acetyl ions from acetone ion are predominantly backward-scattered, with intensity maxima lying inside and outside the elastic scattering circle, confirming our previous observation that electronically excited states are important in low-energy acetone CAD. Ethyl ions from propane ion show a forward-scattered peak maximum at a nonzero scattering angle, which is consistent with generally accepted models for vibrational excitation and redistribution of energy before dissociation. Both processes demonstrate that CAD at low energy proceeds via small-impact-parameter collisions with momentum transfer. Comparison of the present results with higher energy CAD dynamics studies and earlier work leads to some tentative general conclusions about energy transfer in these processes.

  16. High-energy collision induced dissociation fragmentation pathways of peptides, probed using a multiturn tandem time-of-flight mass spectrometer 'MULTUM-TOF/TOF'

    SciTech Connect

    Toyoda, Michisato; Giannakopulos, Anastassios E.; Colburn, Alex W.; Derrick, Peter J.

    2007-07-15

    A new multiturn tandem time-of-flight (TOF) mass spectrometer 'MULTUM-TOF/TOF' has been designed and constructed. It consists of a matrix-assisted laser desorption/ionization ion source, a multiturn TOF mass spectrometer, a collision cell, and a quadratic-field ion mirror. The multiturn TOF mass spectrometer can overcome the problem of precursor ion selection in TOF, due to insufficient time separation between two adjacent TOF peaks, by increasing the number of cycles. As a result, the total TOF increases with the increase in resolving power. The quadratic-field ion mirror allows temporal focusing for fragment ions with different kinetic energies. Product ion spectra from monoisotopically selected precursor ions of angiotensin I, substance P, and bradykinin have been obtained. The fragment ions observed are mainly the result of high-energy collision induced dissociation.

  17. Liquid chromatography electron capture dissociation tandem mass spectrometry (LC-ECD-MS/MS) versus liquid chromatography collision-induced dissociation tandem mass spectrometry (LC-CID-MS/MS) for the identification of proteins.

    PubMed

    Creese, Andrew J; Cooper, Helen J

    2007-05-01

    Electron capture dissociation (ECD) offers many advantages over the more traditional fragmentation techniques for the analysis of peptides and proteins, although the question remains: How suitable is ECD for incorporation within proteomic strategies for the identification of proteins? Here, we compare LC-ECD-MS/MS and LC-CID-MS/MS as techniques for the identification of proteins. Experiments were performed on a hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer. Replicate analyses of a six-protein (bovine serum albumin, apo-transferrin, lysozyme, cytochrome c, alcohol dehydrogenase, and beta-galactosidase) tryptic digest were performed and the results analyzed on the basis of overall protein sequence coverage and sequence tag lengths within individual peptides. The results show that although protein coverage was lower for LC-ECD-MS/MS than for LC-CID-MS/MS, LC-ECD-MS/MS resulted in longer peptide sequence tags, providing greater confidence in protein assignment.

  18. INFRARED SPECTRUM OF POTASSIUM-CATIONIZED TRIETHYLPHOSPHATE GENERATED USING TANDEM MASS SPECTROMETRY AND INFRARED MULTIPLE PHOTON DISSOCIATION

    SciTech Connect

    Gary S. Groenewold; Christopher M. Leavitt; Ryan P. Dain; Jos Oomens; Jeff Steill; van Stipdonk, Michael J.

    2009-09-01

    Tandem mass spectrometry and wavelength selective infrared photodissociation was used to generate an infrared spectrum of gas-phase triethylphosphate cationized by attachment of K+. Prominent absorptions were observed in the region of 900 to 1300 cm-1 that are characteristic of phosphate P=O and P-O-R stretches. The relative positions and intensities of the IR absorptions were reproduced well by density functional theory (DFT) calculations performed using the B3LYP functional and the 6-31+g(d), 6-311+g(d,p) and 6-311++G(3df,2pd) basis sets. Because of good correspondence between experiment and theory for the cation, DFT was then used to generate a theoretical spectrum for neutral triethylphosphate, which in turn accurately reproduces the IR spectrum of the neat liquid when solvent effects are included in the calculations.

  19. Infrared spectrum of potassium-cationized triethylphosphate generated using tandem mass spectrometry and infrared multiple photon dissociation.

    PubMed

    Groenewold, Gary S; Leavitt, Christopher M; Dain, Ryan P; Oomens, Jos; Steill, Jeffrey D; van Stipdonk, Michael J

    2009-09-01

    Tandem mass spectrometry and wavelength-selective infrared photodissociation were used to generate an infrared spectrum of gas-phase triethylphosphate cationized by attachment of K(+). Prominent absorptions were observed in the region of 900 to 1300 cm(-1) that are characteristic of phosphate P=O and P-O-R stretches. The relative positions and intensities of the IR absorptions were reproduced well by density functional theory (DFT) calculations performed using the B3LYP functional and the 6-31+G(d), 6-311+G(d,p) and 6-311++G(3df,2pd) basis sets. Because of good correspondence between experiment and theory for the cation, DFT was then used to generate a theoretical spectrum for neutral triethylphosphate, which in turn accurately reproduces the IR spectrum of the neat liquid when solvent effects are included in the calculations.

  20. Tandem mass spectrometry acquisition approaches to enhance identification of protein-protein interactions using low-energy collision-induced dissociative chemical crosslinking reagents.

    PubMed

    Soderblom, Erik J; Bobay, Benjamin G; Cavanagh, John; Goshe, Michael B

    2007-01-01

    Chemical crosslinking combined with mass spectrometry is a useful tool for studying the topological organization of multiprotein interactions, but it is technically challenging to identify peptides involved in a crosslink using tandem mass spectrometry (MS/MS) due to the presence of product ions originating from both peptides within the same crosslink. We have previously developed a novel set of collision-induced dissociative chemical crosslinking reagents (CID-CXL reagents) that incorporate a labile bond within the linker which readily dissociates at a single site under low-energy collision-induced dissociation (CID) to enable independent isolation and sequencing of the crosslinked peptides by traditional MS/MS and database searching. Alternative low-energy CID events were developed within the in-source region by increasing the multipole DC offset voltage (ISCID) or within the ion trap by increasing the collisional excitation (ITCID). Both dissociation events, each having their unique advantages, occur without significant backbone fragmentation to the peptides, thus permitting subsequent CID to be applied to these distinct peptide ions for generation of suitable product ion spectra for database searching. Each approach was developed and applied to a chemical crosslinking study involving the N-terminal DNA-binding domain of AbrB (AbrBN), a transition-state regulator in Bacillus subtilis. A total of thirteen unique crosslinks were identified using the ITCID approach which represented a significant improvement over the eight unique crosslinks identified using the ISCID approach. The ability to segregate intrapeptide and interpeptide crosslinks using ITCID represents the first step towards high-throughput analysis of protein-protein crosslinks using our CID-CXL reagents.

  1. Identification of lanthionine and lysinoalanine in heat-treated wheat gliadin and bovine serum albumin using tandem mass spectrometry with higher-energy collisional dissociation.

    PubMed

    Rombouts, Ine; Lambrecht, Marlies A; Carpentier, Sebastien C; Delcour, Jan A

    2016-04-01

    The present manuscript reports on the identification of various dehydroamino acid-derived bonds and cross-links resulting from thermal treatment (excess water, 240 min, 130 °C) of two model food proteins, bovine serum albumin, and wheat gliadin. S-Carbamidomethylated tryptic and chymotryptic digests of unheated (control) and heated serum albumin and gliadin, respectively, were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-ESI-MS/MS) with higher-energy collisional dissociation (HCD). Heat-induced β-elimination of cystine, serine and threonine, and subsequent Michael addition of cysteine and lysine to dehydroalanine and 3-methyl-dehydroalanine were demonstrated. Lanthionine, lysinoalanine, 3-methyl-lanthionine, and 3-methyl-lysinoalanine were identified. The detection of inter-chain lanthionine in both bovine serum albumin and wheat gliadin suggests the significance of these cross-links for food texture.

  2. Comparison of high- and low-energy collision-induced dissociation tandem mass spectrometry in the analysis of glycoalkaloids and their aglycons.

    PubMed

    Claeys, M; Van den Heuvel, H; Chen, S; Derrick, P J; Mellon, F A; Price, K R

    1996-02-01

    Four aglycons (tomatidine, demissidine, solanidine, and solasodine) and three glycoalkaloids (α-tomatine, α-chaconine, and α-solanine) have been analyzed by positive ion liquid secondary ion high-energy and low-energy collision-induced dissociation (CID) tandem mass Spectrometry, performed on a four-sector (EBEB) and a hybrid (EBQQ) instrument, respectively. Both high- and low-energy collision-induced dissociation mass spectra of [M+H](+) ions of these compounds provided structural information that aided the characterization of the different aglycons and of the carbohydrate sequence and linkage sites in the glycoalkaloids. Low-energy CID favors charge-driven fragmentation of the aglycon rings, whilst high-energy CID spectra are more complex and contain additional ions that appear to result from charge-remote fragmentations, multiple cleavages, or complex charge-driven rearrangements. With respect to the structural characterization of the carbohydrate part, low-energy CID fragmentations of sugar residues in the glycoalkaloids generate Y n (+) ions and some low intensity Z n (+) ions; the high-energy spectra also exhibit strong (1,5)X n (+) ions, formed by multiple cleavage of the sugar ring, and significant Z n (+) ions.

  3. Formation and reshuffling of disulfide bonds in bovine serum albumin demonstrated using tandem mass spectrometry with collision-induced and electron-transfer dissociation

    PubMed Central

    Rombouts, Ine; Lagrain, Bert; Scherf, Katharina A.; Koehler, Peter; Delcour, Jan A.

    2015-01-01

    Thermolysin hydrolyzates of freshly isolated, extensively stored (6 years, 6 °C, dry) and heated (60 min, 90 °C, in excess water) bovine serum albumin (BSA) samples were analyzed with liquid chromatography (LC) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using alternating electron-transfer dissociation (ETD) and collision-induced dissociation (CID). The positions of disulfide bonds and free thiol groups in the different samples were compared to those deduced from the crystal structure of native BSA. Results revealed non-enzymatic posttranslational modifications of cysteine during isolation, extensive dry storage, and heating. Heat-induced extractability loss of BSA was linked to the impact of protein unfolding on the involvement of specific cysteine residues in intermolecular and intramolecular thiol-disulfide interchange and thiol oxidation reactions. The here developed approach holds promise for exploring disulfide bond formation and reshuffling in various proteins under conditions relevant for chemical, biochemical, pharmaceutical and food processing. PMID:26193081

  4. A Fundamental Tandem Mass Spectrometry Study of the Collision-Activated Dissociation of Small Deprotonated Molecules Related to Lignin.

    PubMed

    Marcum, Christopher L; Jarrell, Tiffany M; Zhu, Hanyu; Owen, Benjamin C; Haupert, Laura J; Easton, Mckay; Hosseinaei, Omid; Bozell, Joseph; Nash, John J; Kenttämaa, Hilkka I

    2016-12-20

    The collision-activated fragmentation pathways and reaction mechanisms of 34 deprotonated model compounds representative of lignin degradation products were explored experimentally and computationally. The compounds were evaporated and ionized by using negative-ion mode electrospray ionization doped with NaOH to produce abundant deprotonated molecules. The ions were isolated and subjected to collision-activated dissociation (CAD). Their fragment ions were then isolated and also subjected to CAD. This was repeated until no further fragmentation was observed (up to MS(6) ). This approach enabled the identification of characteristic reaction pathways and delineation of reasonable fragmentation mechanisms for deprotonated molecules containing various functional groups. The varying fragmentation patterns observed for different types of compounds allow for the identification of the functionalities in these compounds. This information was utilized to identify the presence of specific functionalities and their combinations in molecules in an organosolv lignin sample.

  5. Protonation sites and dissociation mechanisms of t-butylcarbamates in tandem mass spectrometric assays for newborn screening.

    PubMed

    Spáčil, Zdeněk; Hui, Renjie; Gelb, Michael H; Tureček, František

    2011-10-01

    Structures of tert-butylcarbamate ions in the gas-phase and methanol solution were studied for simple secondary and tertiary carbamates as well as for carbamate-containing products and internal standards for lysosomal enzyme assays used in newborn screening of a α-galactosidase A deficiency (Fabry disease), mucopolysaccharidosis I (Hurler disease), and mucopolysaccharidosis II (Hunter disease). The protonation of simple t-butylcarbamates can occur at the carbonyl group, which is the preferred site in the gas phase. Protonation in methanol solution is more favorable if occurring at the carbamate nitrogen atom. The protonation of more complex t-butylcarbamates occurs at amide and carbamate carbonyl groups, and the ions are stabilized by intramolecular hydrogen bonding, which is affected by solvation. Tertiary carbamates containing aminophenol amide groups were calculated to have substantially greater gas-phase basicities than secondary carbamates containing coumarin amide groups. The main diagnostically important ion dissociation by elimination of 2-methylpropene (isobutylene, i-C(4)H(8)) and carbon dioxide is shown by experiment and theory to proceed in two steps. Energy-resolved collision-induced dissociation of the Hurler's disease enzymatic product ion, which is a coumarin-diamine linker-t-butylcarbamate conjugate (3a(+)), indicated separate energy thresholds for the loss of i-C(4)H(8) and CO(2). Computational investigation of the potential energy surface along two presumed reaction pathways indicated kinetic preference for the migration of a t-butyl hydrogen atom to the carbamate carbonyl resulting in the isobutylene loss. The consequent loss of CO(2) required further proton migrations that had to overcome energy barriers.

  6. Identification of Cross-Linked Peptides after Click-Based Enrichment Using Sequential Collision-Induced Dissociation and Electron Transfer Dissociation Tandem Mass Spectrometry

    SciTech Connect

    Chowdhury, Saiful M.; Du, Xiuxia; Tolic, Nikola; Wu, Si; Moore, Ronald J.; Mayer, M. Uljana; Smith, Richard D.; Adkins, Joshua N.

    2009-07-01

    Chemical cross-linking combined with mass spectrometric analysis is emerging as a powerful technique for protein-protein interaction and protein structure elucidation studies.1 Cross-linkers covalently link two interacting proteins, often with chemistries specific to certain amino acid side chains. After enzymatic digestion of the proteins, the resulting cross-linked peptides can be subjected to analysis by LC-MS(/MS) to identify cross-linked species.2,3 For studying protein interactions using chemical cross-linking towards global discovery-based applications, the critical needs are the development of cross-linkers that are highly specific, amenable to LC-MS/MS, and resulting spectra are interpretable by bioinformatics tools to automatically assign cross-linked peptides with high confidence.4-10 As recently mentioned by Aebersold and co-workers, due to the low relative abundances of cross-linking products compared to their unmodified counterparts, enrichment of cross-linked species is also highly desirable to improve the likelihood of unambiguous identification of cross-linked peptides.6 Most of the currently available enrichable cross-linkers are bulky and are not amenable to studying protein-protein interactions in vivo. To discover protein-protein interactions with high confidence, there is a need for chemical cross-linkers that can effectively label protein complexes, utilize mass spectrometry based bottom-up proteomics analysis pipelines and also contains enrichment functionality.

  7. Structure and end-group analysis of complex hexanediol-neopentylglycol-adipic acid copolyesters by matrix-assisted laser desorption/ionization collision-induced dissociation tandem mass spectrometry.

    PubMed

    Weidner, Steffen M; Falkenhagen, Jana; Knop, Karin; Thünemann, Andreas

    2009-09-01

    Sequences and end groups of complex copolyesters were determined by fragmentation analysis by means of matrix-assisted laser desorption/ionization collision-induced dissociation tandem mass spectrometry (MALDI CID MS/MS). The complexity of the crude copolyester mixture was reduced by a chromatographic separation followed by a MALDI time-of-flight (TOF) investigation of fractions. Due to overlapping compositional and end-group information a clear assignment of end groups was very difficult. However, the fragmentation of suitable precursor ions resulted in typical fragment ion patterns and, therefore, enabled a fast and unambiguous determination of the end groups and composition of this important class of polymers.

  8. Improved collision-induced dissociation analysis of peptides by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry through 3-sulfobenzoic acid succinimidyl ester labeling.

    PubMed

    Alley, William R; Mechref, Yehia; Klouckova, Iveta; Novotny, Milos V

    2007-01-01

    The sulfonation reagent, a succinimidyl ester of 3-sulfobenzoic acid, has been synthesized for effective peptide sequencing. It is capable of incorporating an additional mobile proton into the peptide backbone, thus, facilitating efficient collision-induced dissociation. This reagent is easily and inexpensively prepared in short time. Tandem mass spectra of the guanidinated and reagent-sulfonated peptides consist mainly of the y-ion series with higher intensities than those observed for solely guanidinated peptides. These enhanced tandem MS attributes significantly improved MASCOT total-ion scores, thus, allowing more confident peptide sequencing. This derivatization was also very effective for the analysis of tryptic digest of human blood serum proteins separated by two-dimensional gel electrophoresis. When used in LC-MALDI/MS/MS format, this type of derivatization does not adversely affect chromatographic efficiencies.

  9. Characterization of 4-hydroxy-2-nonenal-modified peptides by liquid chromatography-tandem mass spectrometry using data-dependent acquisition: neutral loss-driven MS3 versus neutral loss-driven electron capture dissociation.

    PubMed

    Rauniyar, Navin; Stevens, Stanley M; Prokai-Tatrai, Katalin; Prokai, Laszlo

    2009-01-15

    Reactive oxygen species generated during oxidative stress can lead to unfavorable cellular consequences, predominantly due to formation of 4-hydroxy-2-nonenal (HNE) during lipid peroxidation. Data-dependent and neutral loss (NL)-driven MS(3) acquisition have been reported for the identification of HNE adducts by mass spectrometry-based proteomics. However, the limitation associated with this method is the ambiguity in correct assignment of the HNE modification site when more than one candidate site is present as MS(3) is triggered on the neutral loss ion. We introduce NL-triggered electron capture dissociation tandem mass spectrometry (NL-ECD-MS/MS) for the characterization of HNE-modification sites in peptides. With this method performed using a hybrid linear ion trap-Fourier transform ion cyclotron resonance (FTICR) mass spectrometer, ECD in the FTICR unit of the instrument is initiated on precursor ions of peptides showing the neutral loss of 156 Da corresponding to an HNE molecule in the prescan acquired via collision-induced dissociation tandem mass spectrometry in the linear ion trap. In addition to manifold advantages associated with the ECD method of backbone fragmentation, including extensive sequence fragments, ECD tends to retain the HNE group during MS/MS of the precursor ion, facilitating the correct localization of the modification site. The results also suggest that predisposition of a peptide molecular ion to lose HNE during collision-induced dissociation-based fragmentation is independent of its charge state (2+ or 3+). In addition, we have demonstrated that coupling of solid-phase enrichment of HNE-modified peptides facilitates the detection of this posttranslational modification by NL-driven strategies for low-abundance proteins that are susceptible to substoichiometric carbonylation during oxidative stress.

  10. Simultaneous Transmission Mode Collision-Induced Dissociation and Ion/Ion Reactions for Top-Down Protein Identification/Characterization Using a Quadrupole/Time-of-Flight Tandem Mass Spectrometer

    PubMed Central

    Liu, Jian; Huang, Teng-Yi; McLuckey, Scott A.

    2009-01-01

    Simultaneous transmission mode collision-induced dissociation (CID) and ion/ion proton transfer reactions have been implemented on a quadrupole/time-of-flight (TOF) tandem mass spectrometer. Reagent anions were trapped in a pressurized quadrupole collision cell by applying appropriate DC voltages while multiply protonated protein precursor ions were injected into the collision cell at energies sufficient to give rise to CID. Intact precursor ions as well as fragment ions underwent ion/ion proton transfer reactions during their passage through the collision cell and on to an orthogonal acceleration TOF mass analyzer. The resulting product ion spectrum was then submitted to deconvolution to yield a “zero-charge” spectrum, which was then matched against in silico produced spectra derived from a protein database. Dramatic improvements in the scores associated with correct matches were obtained relative to CID data without benefit of ion/ion reactions for proteins as large as carbonic anhydrase (29 kDa). The parameters that most affect the extent of ion/ion proton transfer during transmission through the instrument include the number of anions stored in the collision cell, the amplitude of the radio-frequency trapping voltage, the voltage of the LINAC potential associated with the collision cell, and the collision gas pressure. This work demonstrates that it is possible to effect whole protein tandem mass spectrometry with simultaneous CID, ion/ion reactions, and mass analysis for high duty cycle top-down protein characterization. PMID:19281259

  11. Identification of epoxide functionalities in protonated monofunctional analytes by using ion/molecule reactions and collision-activated dissociation in different ion trap tandem mass spectrometers.

    PubMed

    Eismin, Ryan J; Fu, Mingkun; Yem, Sonoeun; Widjaja, Fanny; Kenttämaa, Hilkka I

    2012-01-01

    A mass spectrometric method has been delineated for the identification of the epoxide functionalities in unknown monofunctional analytes. This method utilizes gas-phase ion/molecule reactions of protonated analytes with neutral trimethyl borate (TMB) followed by collision-activated dissociation (CAD) in an ion trapping mass spectrometer (tested for a Fourier-transform ion cyclotron resonance and a linear quadrupole ion trap). The ion/molecule reaction involves proton transfer from the protonated analyte to TMB, followed by addition of the analyte to TMB and elimination of methanol. Based on literature, this reaction allows the general identification of oxygen-containing analytes. Vinyl and phenyl epoxides can be differentiated from other oxygen-containing analytes, including other epoxides, based on the loss of a second methanol molecule upon CAD of the addition/methanol elimination product. The only other analytes found to undergo this elimination are some amides but they also lose O = B-R (R = group bound to carbonyl), which allows their identification. On the other hand, other epoxides can be differentiated from vinyl and phenyl epoxides and from other monofunctional analytes based on the loss of (CH(3)O)(2)BOH or formation of protonated (CH(3)O)(2)BOH upon CAD of the addition/methanol elimination product. For propylene oxide and 2,3-dimethyloxirane, the (CH(3)O)(2)BOH fragment is more basic than the hydrocarbon fragment, and the diagnostic ion (CH(3)O)(2)BOH (2) (+) is formed. These reactions involve opening of the epoxide ring. The only other analytes found to undergo (CH(3)O)(2)BOH elimination are carboxylic acids, but they can be differentiated from the rest based on several published ion/molecule reaction methods. Similar results were obtained in the Fourier-transform ion cyclotron resonance and linear quadrupole ion trap mass spectrometer.

  12. Portable Tandem Mass Spectrometer Analyzer

    DTIC Science & Technology

    1991-07-01

    The planned instrument was to be small enough to be portable in small vehicles and was to be able to use either an atmospheric pressure ion source or a...conventional electron impact/chemical ionization ion source. In order to accomplish these developments an atmospheric pressure ionization source was...developed for a compact, commercially available tandem quadrupole mass spectrometer. This ion source could be readily exchanged with the conventional

  13. Dissociation of biomolecules using a ultraviolet matrix-assisted laser desorption/ionisation time-of-flight/curved field reflectron tandem mass spectrometer equipped with a differential-pumped collision cell.

    PubMed

    Belgacem, Omar; Bowdler, Andrew; Brookhouse, Ian; Brancia, Francesco L; Raptakis, Emmanuel

    2006-01-01

    A commercial matrix-assisted laser desorption/ionisation time-of-flight (MALDI-ToF) instrument equipped with a curved field reflectron (CFR) was modified in order to perform collision-induced dissociation (CID) on a variety of biomolecules. The incorporation of a high-resolution ion gate together with a collision cell within the field-free region allowed tandem mass analysis (MS/MS), without the necessity to decelerate the precursor ions prior to activation. The simultaneous detection of all product ions remained possible by using the CFR. To test the MS/MS performances, ACTH (fragment 1-17), a complex high mannose carbohydrate (Man)(8)(GlcNac)(2) and a lysophosphatidylcholine lipid (18:1) were analysed on the modified instrument. Direct comparison with the low-energy product ion spectra, acquired on a MALDI quadrupole ion trap (QIT) two-stage reflectron time-of flight (ReToF) mass spectrometer, showed significant differences in the types of product ions observed. The additional ions detected were a clear indication of the high-energy fragmentation processes occurring in the collision cell.

  14. Online immunoaffinity liquid chromatography/tandem mass spectrometry determination of a type II collagen peptide biomarker in rat urine: Investigation of the impact of collision-induced dissociation fluctuation on peptide quantitation.

    PubMed

    Berna, Michael; Schmalz, Chris; Duffin, Kevin; Mitchell, Peter; Chambers, Mark; Ackermann, Brad

    2006-09-15

    Proteolytic fragments of type II collagen, a major component of joint tissue, have recently been identified as biomarkers for osteoarthritis, a progressive disease associated with cartilage degeneration. A liquid chromatography/tandem mass spectrometry (MS/MS) assay that utilizes online immunoaffinity chromatography and column switching was developed in our laboratory for the neoepitope of type II collagen (NET2C). During method development, peptide collision-induced dissociation (CID) was found to be a significant source of assay variation, which exceeded 10% CV, despite the fact that a stable-isotope-labeled (SIL) internal standard was used to minimize imprecision. This phenomenon was studied in detail using peptides and associated SIL internal standards of varying lengths and amino acid compositions. Variability in peptide CID necessitated the monitoring of multiple MS/MS transitions to obtain acceptable assay precision. The assay was subsequently validated to measure NET2C concentrations in rat urine over the range of 0.1 to 10 ng/mL. The interday accuracy and precision ranged from 3.9 to 13.1 (%CV) and 10.7 to 5.3 (%RE), respectively, across the range of validated concentrations. A specific application of the assay is presented in which the role of estrogen deficiency in the development and progression of osteoarthritis was investigated. In this study, the effect of estrogen on lowering NET2C concentrations in urine in ovariectomized rats was demonstrated.

  15. A simultaneous determination method for 5-fluorouracil and its metabolites in human plasma with linear range adjusted by in-source collision-induced dissociation using hydrophilic interaction liquid chromatography-electrospray ionization-tandem mass spectrometry.

    PubMed

    Ishii, Hideaki; Shimada, Miki; Yamaguchi, Hiroaki; Mano, Nariyasu

    2016-11-01

    We applied a new technique for quantitative linear range shift using in-source collision-induced dissociation (CID) to complex biological fluids to demonstrate its utility. The technique was used in a simultaneous quantitative determination method of 5-fluorouracil (5-FU), an anticancer drug for various solid tumors, and its metabolites in human plasma by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS). To control adverse effects after administration of 5-FU, it is important to monitor the plasma concentration of 5-FU and its metabolites; however, no simultaneous determination method has yet been reported because of vastly different physical and chemical properties of compounds. We developed a new analytical method for simultaneously determining 5-FU and its metabolites in human plasma by LC/ESI-MS/MS coupled with the technique for quantitative linear range shift using in-source CID. Hydrophilic interaction liquid chromatography using a stationary phase with zwitterionic functional groups, phosphorylcholine, was suitable for separation of 5-FU from its nucleoside and interfering endogenous materials. The addition of glycerin into acetonitrile-rich eluent after LC separation improved the ESI-MS response of high polar analytes. Based on the validation results, linear range shifts by in-source CID is the reliable technique even with complex biological samples such as plasma. Copyright © 2016 John Wiley & Sons Ltd.

  16. Mass spectrometry and tandem mass spectrometry of citrus limonoids.

    PubMed

    Tian, Qingguo; Schwartz, Steven J

    2003-10-15

    Methods for atmospheric pressure chemical ionization tandem mass spectrometry (APCI-MS/MS) of citrus limonoid aglycones and electrospray ionization tandem mass spectrometry (ESI-MS/MS) of limonoid glucosides are reported. The fragmentation patterns of four citrus limonoid aglycones (limonin, nomilin, obacunone, and deacetylnomilin) and six limonoid glucosides, that is, limonin 17-beta-D-glucopyranoside (LG), nomilin 17-beta-D-glucopyranoside (NG), nomilinic acid 17-beta-D-glucopyranoside (NAG), deacetyl nomilinic acid 17-beta-D-glucopyranoside (DNAG), obacunone 17-beta-D-glucopyranoside (OG), and obacunoic acid 17-beta-D-glucopyranoside (OAG) were investigated using a quadruple mass spectrometer in low-energy collisionally activated dissociation (CAD). The four limonoid aglycones and four limonoid glucosides (LG, OG, NAG, and DNAG) were purified from citrus seeds; the other two limonoid glucosides (NG and OAG) were tentatively identified in the crude extract of grapefruit seeds by ESI mass spectrometry in both positive and negative ion analysis. Ammonium hydroxide or acetic acid was added to the mobile phase to facilitate ionization. During positive ion APCI analysis of limonoid aglycones, protonated molecular ion, [M + H]+, or adduct ion, [M + NH3 + H]-, was formed as base peaks when ammonium hydroxide was added to the mobile phase. Molecular anions or adduct ions with acetic acid ([M + HOAc - H] and [M + HOAc]-) or a deprotonated molecular ion were produced during negative ion APCI analysis of limonoid aglycones, depending on the mobile-phase modifier used. Positive ion ESI-MS of limonoid glucosides produced adduct ions of [M + H + NH3]+, [M + Na]+, and [M + K]+ when ammonium hydroxide was added to the mobile phase. After collisionally activated dissociation (CAD) of the limonoid aglycone molecular ions in negative ion APCI analysis, fragment ions indicated structural information of the precursor ions, showing the presence of methyl, carboxyl, and oxygenated ring

  17. Structural Characterization of Carbohydrates by Fourier Transform Tandem Mass Spectrometry

    PubMed Central

    Zhou, Wen; Håkansson, Kristina

    2012-01-01

    Fourier transform tandem mass spectrometry (MS/MS) provides high mass accuracy, high sensitivity, and analytical versatility and has therefore emerged as an indispensable tool for structural elucidation of biomolecules. Glycosylation is one of the most common posttranslational modifications, occurring in ~50% of proteins. However, due to the structural diversity of carbohydrates, arising from non-template driven biosynthesis, achievement of detailed structural insight is highly challenging. This review briefly discusses carbohydrate sample preparation and ionization methods, and highlights recent developments in alternative high-resolution MS/MS strategies, including infrared multiphoton dissociation (IRMPD), electron capture dissociation (ECD), and electron detachment dissociation (EDD), for carbohydrates with a focus on glycans and proteoglycans from mammalian glycoproteins. PMID:22389641

  18. ANALYSIS OF POLYCYCLIC AROMATIC HYDROCARBONS BY ION TRAP TANDEM MASS SPECTROMETRY

    EPA Science Inventory

    An ion-trap mass spectrometer with a wave board and tandem mass spectrometry software was used to analyze gas chromatographically separated polycyclic aromatic hydrocarbons (PAHs) by using collision-induced dissociation (CID). The nonresonant (multiple collision) mode was used to...

  19. Collision-induced dissociation with Fourier transform mass spectrometry

    SciTech Connect

    Cody, R.B.; Burnier, R.C.; Freiser, B.S.

    1982-01-01

    Collision-induced dissociations (CID) is demonstrated on a number of primary and secondary ions using a Nicolet prototype Fourier transform mass spectrometer (FT-MS). Like the triple quadrupole technique, CID using FT-MS is a relatively low energy and efficient process. The ability to study a wide range of ion-molecule reaction products is exemplified by results on proton-bound dimers and transition metal containing ionic species. Variation of collision energy by varying the RF irradiation level can provide information about product distributions as a function of energy as well as yield ion structural information. Like the triple quadrupole technique, no slits are employed and virtually all of the fragment ions formed by the CID process may be detected. Unlike all previous mass spectrometric techniques for studying CID, a tandem instrument is not required, and different experiments are performed by making software modifications rather than hardware modifications.

  20. Quality evaluation of tandem mass spectral libraries.

    PubMed

    Oberacher, Herbert; Weinmann, Wolfgang; Dresen, Sebastian

    2011-06-01

    Tandem mass spectral libraries are gaining more and more importance for the identification of unknowns in different fields of research, including metabolomics, forensics, toxicology, and environmental analysis. Particularly, the recent invention of reliable, robust, and transferable libraries has increased the general acceptance of these tools. Herein, we report on results obtained from thorough evaluation of the match reliabilities of two tandem mass spectral libraries: the MSforID library established by the Oberacher group in Innsbruck and the Weinmann library established by the Weinmann group in Freiburg. Three different experiments were performed: (1) Spectra of the libraries were searched against their corresponding library after excluding either this single compound-specific spectrum or all compound-specific spectra prior to searching; (2) the libraries were searched against each other using either library as reference set or sample set; (3) spectra acquired on different mass spectrometric instruments were matched to both libraries. Almost 13,000 tandem mass spectra were included in this study. The MSforID search algorithm was used for spectral matching. Statistical evaluation of the library search results revealed that principally both libraries enable the sensitive and specific identification of compounds. Due to higher mass accuracy of the QqTOF compared with the QTrap instrument, matches to the MSforID library were more reliable when comparing spectra with both libraries. Furthermore, only the MSforID library was shown to be efficiently transferable to different kinds of tandem mass spectrometers, including "tandem-in-time" instruments; this is due to the coverage of a large range of different collision energy settings-including the very low range-which is an outstanding characteristics of the MSforID library.

  1. Electrospray tandem mass spectrometric investigations of morphinans.

    PubMed

    Raith, Klaus; Neubert, Reinhard; Poeaknapo, Chotima; Boettcher, Christian; Zenk, Meinhart H; Schmidt, Jürgen

    2003-11-01

    In this study positive ESI tandem mass spectra of the [M + H]+ ions of morphinan alkaloids obtained using an ion trap MS were compared with those from a triple quadrupole MS. This allows to assess the differences of the tandem-in-time versus the tandem-in-space principle, often hampering the development of ESI MS/MS libraries. Fragmentation pathways and possible fragment ion structures were discussed. In order to obtain elemental composition, accurate mass measurements were performed. According to the MS/MS fragmentation pathway, the investigated compounds can be grouped into 4 subsets: (1) morphine and codeine, (2) morphinone, codeinone, and neopinone, (3) thebaine and oripavine, (4) salutaridine and salutaridinol. Salutaridinol-7-O-acetate shows a different fragmentation behavior because of the favored loss of acetic acid. Although most fragment ions occur in both ion trap and triple quad tandem mass spectra, some are exclusively seen in either type. For triple quad, quadrupole time-of-flight and FT-ICR MS/MS, the base peak of morphine results from an ion at m/z 165 that contains neither nitrogen nor oxygen. This ion is not found in ion trap MS/MS, but in subsequential MS3 and MS4.

  2. Fast atom bombardment tandem mass spectrometry of carotenoids

    SciTech Connect

    van Breeman, R.B.; Schmitz, H.H.; Schwartz, S.J.

    1995-02-01

    Positive ion fast atom bombardment (FAB) tandem mass spectrometry (MS-MS) using a double-focusing mass spectrometer with linked scanning at constant B/E and high-energy collisionally activated dissociation (CAD) was used to differentiate 17 different cartenoids, including {beta}-apo-8{prime}- carotenal, astaxanthin, {alpha}-carotene, {beta}-carotene, {gamma}-carotene, {zeta}-carotene, canthaxanthin, {beta}-cryptoxanthin, isozeaxanthin bis (pelargonate), neoxanthin, neurosporene, nonaprene, lutein, lycopene, phytoene, phytofluene, and zeaxanthin. The carotenoids were either synthetic or isolated from plant tissues. The use of FAB ionization minimized degradation or rearrangement of the carotenoid structures due to the inherent thermal instability generally ascribed to these compounds. Instead of protonated molecules, both polar xanthophylls and nonpolar carotenes formed molecular ions, M{sup {center_dot}+}, during FAB ionization. Following collisionally activated dissociation, fragment ions of selected molecular ion precursors showed structural features indicative of the presence of hydroxyl groups, ring systems, ester groups, and aldehyde groups and the extent of aliphatic polyene conjugation. The fragmentation patterns observed in the mass spectra herein may be used as a reference for the structural determination of carotenoids isolated from plant and animal tissues. 18 refs., 4 figs.

  3. Dissociation techniques in mass spectrometry-based proteomics.

    PubMed

    Jones, Andrew W; Cooper, Helen J

    2011-09-07

    The field of proteomics, the large-scale analysis of proteins, has undergone a huge expansion over the past decade. Mass spectrometry-based proteomics relies on the dissociation of peptide and/or protein ions to provide information on primary sequence and sites of post-translational modifications. Fragmentation techniques include collision-induced dissociation, electron capture dissociation and electron transfer dissociation. Here, we describe each of these techniques and their use in proteomics. The principles, advantages, limitations, and applications are discussed.

  4. Electrospray and tandem mass spectrometry in biochemistry.

    PubMed Central

    Griffiths, W J; Jonsson, A P; Liu, S; Rai, D K; Wang, Y

    2001-01-01

    Over the last 20 years, biological MS has changed out of all recognition. This is primarily due to the development in the 1980s of 'soft ionization' methods that permit the ionization and vaporization of large, polar, and thermally labile biomolecules. These developments in ionization mode have driven the design and manufacture of smaller and cheaper mass analysers, making the mass spectrometer a routine instrument in the biochemistry laboratory today. In the present review the revolutionary 'soft ionization' methods will be discussed with particular reference to electrospray. The mass analysis of ions will be described, and the concept of tandem MS introduced. Where appropriate, examples of the application of MS in biochemistry will be provided. Although the present review will concentrate on the MS of peptides/proteins and lipids, all classes of biomolecules can be analysed, and much excellent work has been done in the fields of carbohydrate and nucleic acid biochemistry. PMID:11311115

  5. Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry

    PubMed Central

    Syka, John E. P.; Coon, Joshua J.; Schroeder, Melanie J.; Shabanowitz, Jeffrey; Hunt, Donald F.

    2004-01-01

    Peptide sequence analysis using a combination of gas-phase ion/ion chemistry and tandem mass spectrometry (MS/MS) is demonstrated. Singly charged anthracene anions transfer an electron to multiply protonated peptides in a radio frequency quadrupole linear ion trap (QLT) and induce fragmentation of the peptide backbone along pathways that are analogous to those observed in electron capture dissociation. Modifications to the QLT that enable this ion/ion chemistry are presented, and automated acquisition of high-quality, single-scan electron transfer dissociation MS/MS spectra of phosphopeptides separated by nanoflow HPLC is described. PMID:15210983

  6. Towards understanding the tandem mass spectra of protonated oligopeptides. 2: The proline effect in collision-induced dissociation of protonated Ala-Ala-Xxx-Pro-Ala (Xxx = Ala, Ser, Leu, Val, Phe, and Trp).

    PubMed

    Bleiholder, Christian; Suhai, Sándor; Harrison, Alex G; Paizs, Béla

    2011-06-01

    The product ion spectra of proline-containing peptides are commonly dominated by y(n) ions generated by cleavage at the N-terminal side of proline residues. This proline effect is investigated in the current work by collision-induced dissociation (CID) of protonated Ala-Ala-Xxx-Pro-Ala (Xxx includes Ala, Ser, Leu, Val, Phe, and Trp) in an electrospray/quadrupole/time-of-flight (QqTOF) mass spectrometer and by quantum chemical calculations on protonated Ala-Ala-Ala-Pro-Ala. The CID spectra of all investigated peptides show a dominant y(2) ion (Pro-Ala sequence). Our computational results show that the proline effect mainly arises from the particularly low threshold energy for the amide bond cleavage N-terminal to the proline residue, and from the high proton affinity of the proline-containing C-terminal fragment produced by this cleavage. These theoretical results are qualitatively supported by the experimentally observed y(2)/b(3) abundance ratios for protonated Ala-Ala-Xxx-Pro-Ala (Xxx = Ala, Ser, Leu, Val, Phe, and Trp). In the post-cleavage phase of fragmentation the N-terminal oxazolone fragment with the Ala-Ala-Xxx sequence and Pro-Ala compete for the ionizing proton for these peptides. As the proton affinity of the oxazolone fragment increases, the y(2)/b(3) abundance ratio decreases.

  7. Electrospray ionization tandem mass spectrometry of ammonium cationized polyethers.

    PubMed

    Nasioudis, Andreas; Heeren, Ron M A; van Doormalen, Irene; de Wijs-Rot, Nicolette; van den Brink, Oscar F

    2011-05-01

    Quaternary ammonium salts (Quats) and amines are known to facilitate the MS analysis of high molar mass polyethers by forming low charge state adduct ions. The formation, stability, and behavior upon collision-induced dissociation (CID) of adduct ions of polyethers with a variety of Quats and amines were studied by electrospray ionization quadrupole time-of-flight, quadrupole ion trap, and linear ion trap tandem mass spectrometry (MS/MS). The linear ion trap instrument was part of an Orbitrap hybrid mass spectrometer that allowed accurate mass MS/MS measurements. The Quats and amines studied were of different degree of substitution, structure, and size. The stability of the adduct ions was related to the structure of the cation, especially the amine's degree of substitution. CID of singly/doubly charged primary and tertiary ammonium cationized polymers resulted in the neutral loss of the amine followed by fragmentation of the protonated product ions. The latter reveals information about the monomer unit, polymer sequence, and endgroup structure. In addition, the detection of product ions retaining the ammonium ion was observed. The predominant process in the CID of singly charged quaternary ammonium cationized polymers was cation detachment, whereas their doubly charged adduct ions provided the same information as the primary and tertiary ammonium cationized adduct ions. This study shows the potential of specific amines as tools for the structural elucidation of high molar mass polyethers.

  8. Electrospray Ionization Tandem Mass Spectrometry of Ammonium Cationized Polyethers

    NASA Astrophysics Data System (ADS)

    Nasioudis, Andreas; Heeren, Ron M. A.; van Doormalen, Irene; de Wijs-Rot, Nicolette; van den Brink, Oscar F.

    2011-05-01

    Quaternary ammonium salts (Quats) and amines are known to facilitate the MS analysis of high molar mass polyethers by forming low charge state adduct ions. The formation, stability, and behavior upon collision-induced dissociation (CID) of adduct ions of polyethers with a variety of Quats and amines were studied by electrospray ionization quadrupole time-of-flight, quadrupole ion trap, and linear ion trap tandem mass spectrometry (MS/MS). The linear ion trap instrument was part of an Orbitrap hybrid mass spectrometer that allowed accurate mass MS/MS measurements. The Quats and amines studied were of different degree of substitution, structure, and size. The stability of the adduct ions was related to the structure of the cation, especially the amine's degree of substitution. CID of singly/doubly charged primary and tertiary ammonium cationized polymers resulted in the neutral loss of the amine followed by fragmentation of the protonated product ions. The latter reveals information about the monomer unit, polymer sequence, and endgroup structure. In addition, the detection of product ions retaining the ammonium ion was observed. The predominant process in the CID of singly charged quaternary ammonium cationized polymers was cation detachment, whereas their doubly charged adduct ions provided the same information as the primary and tertiary ammonium cationized adduct ions. This study shows the potential of specific amines as tools for the structural elucidation of high molar mass polyethers.

  9. Protein Sequencing with Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Ziady, Assem G.; Kinter, Michael

    The recent introduction of electrospray ionization techniques that are suitable for peptides and whole proteins has allowed for the design of mass spectrometric protocols that provide accurate sequence information for proteins. The advantages gained by these approaches over traditional Edman Degradation sequencing include faster analysis and femtomole, sometimes attomole, sensitivity. The ability to efficiently identify proteins has allowed investigators to conduct studies on their differential expression or modification in response to various treatments or disease states. In this chapter, we discuss the use of electrospray tandem mass spectrometry, a technique whereby protein-derived peptides are subjected to fragmentation in the gas phase, revealing sequence information for the protein. This powerful technique has been instrumental for the study of proteins and markers associated with various disorders, including heart disease, cancer, and cystic fibrosis. We use the study of protein expression in cystic fibrosis as an example.

  10. Interpretation of collision-induced fragmentation tandem mass spectra of posttranslationally modified peptides.

    PubMed

    Bunkenborg, Jakob; Matthiesen, Rune

    2007-01-01

    Tandem collision-induced dissociation (CID) mass spectrometry (MS) provides a sensitive means of analyzing the amino acid sequence of peptides. Modern MS instrumentation is capable of rapidly generating many thousands of tandem mass spectra, and protein database search engines have been developed to cope with this avalanche of data. In most studies, there is a schism between discarding perfectly valid data and including nonsensical peptide identifications--this is currently a major bottleneck in data analysis and it calls for manual evaluation of the data. Especially for posttranslationally modified peptides, there is a need for manual validation of the data because search algorithms seldom have been optimized for the identification of modified peptides and because there are many pitfalls for the unwary. This chapter describes some of the issues that should be considered when interpreting and validating low-energy CID tandem mass spectra and gives some useful tables to aid this process.

  11. Structural characterization of poly(amino)ester dendrimers and related impurities by electrospray tandem mass spectrometry.

    PubMed

    Tintaru, Aura; Monnier, Valérie; Bouillon, Camille; Giordanengo, Rémi; Quéléver, Gilles; Peng, Ling; Charles, Laurence

    2010-08-15

    An acid-terminated poly(amino)ester dendrimer was studied by electrospray ionization tandem mass spectrometry to establish its fragmentation pathways, with the aim of using them to investigate the structure of any defective molecules generated during the dendrimer synthesis. This poly(amino)ester dendrimer could be ionized in both polarities but the most structurally relevant dissociation pathways were found from the deprotonated molecule in negative ion mode. The dissociation pattern of this dendrimer is fully described and supported by accurate mass measurements. The main dissociation reactions of the negatively charged polyacidic dendrimer were shown to consist of (i) the release of carbon dioxide and ethene within a branch, which proceeds as many times as intact neutral branches are available; and (ii) the elimination of an entire dendrimer arm. Monitoring the occurrence of these reactions together with any deviation from these two main routes allowed six major dendritic impurities to be structurally characterized.

  12. Infrared multiphoton dissociation of peptide cations in a dual pressure linear ion trap mass spectrometer.

    PubMed

    Gardner, Myles W; Smith, Suncerae I; Ledvina, Aaron R; Madsen, James A; Coon, Joshua J; Schwartz, Jae C; Stafford, George C; Brodbelt, Jennifer S

    2009-10-01

    A dual pressure linear ion trap mass spectrometer was modified to permit infrared multiphoton dissociation (IRMPD) in each of the two cells-the first a high pressure cell operated at nominally 5 x 10(-3) Torr and the second a low pressure cell operated at nominally 3 x 10(-4) Torr. When IRMPD was performed in the high pressure cell, most peptide ions did not undergo significant photodissociation; however, in the low pressure cell peptide cations were efficiently dissociated with less than 25 ms of IR irradiation regardless of charge state. IRMPD of peptide cations allowed the detection of low m/z product ions including the y(1) fragments and immonium ions which are not typically observed by ion trap collision induced dissociation (CID). Photodissociation efficiencies of approximately 100% and MS/MS (tandem mass spectrometry) efficiencies of greater than 60% were observed for both multiply and singly protonated peptides. In general, higher sequence coverage of peptides was obtained using IRMPD over CID. Further, greater than 90% of the product ion current in the IRMPD mass spectra of doubly charged peptide ions was composed of singly charged product ions compared to the CID mass spectra in which the abundances of the multiply and singly charged product ions were equally divided. Highly charged primary product ions also underwent efficient photodissociation to yield singly charged secondary product ions, thus simplifying the IRMPD product ion mass spectra.

  13. Infrared Multiphoton Dissociation of Peptide Cations in a Dual Pressure Linear Ion Trap Mass Spectrometer

    PubMed Central

    Gardner, Myles W.; Smith, Suncerae I.; Ledvina, Aaron R.; Madsen, James A.; Coon, Joshua J.; Schwartz, Jae C.; Stafford, George C.; Brodbelt, Jennifer S.

    2009-01-01

    A dual pressure linear ion trap mass spectrometer was modified to permit infrared multiphoton dissociation (IRMPD) in each of the two cells - the first a high pressure cell operated at nominally 5 × 10-3 Torr and the second a low pressure cell operated at nominally 3 × 10-4 Torr. When IRMPD was performed in the high pressure cell, most peptide ions did not undergo significant photodissociation; however, in the low pressure cell peptide cations were efficiently dissociated with less than 25 ms of IR irradiation regardless of charge state. IRMPD of peptide cations allowed the detection of low m/z product ions including the y1 fragments and immonium ions which are not typically observed by ion trap collision induced dissociation (CID). Photodissociation efficiencies of ~100% and MS/MS (tandem mass spectrometry) efficiencies of greater than 60% were observed for both multiply and singly protonated peptides. In general, higher sequence coverage of peptides was obtained using IRMPD over CID. Further, greater than 90% of the product ion current in the IRMPD mass spectra of doubly charged peptide ions was composed of singly charged product ions compared to the CID mass spectra in which the abundances of the multiply and singly charged product ions were equally divided. Highly charged primary product ions also underwent efficient photodissociation to yield singly charged secondary product ions, thus simplifying the IRMPD product ion mass spectra. PMID:19739654

  14. Effective novel dissociation methods for intact protein: heat-assisted nozzle-skimmer collisionally induced dissociation and infrared multiphoton dissociation using a Fourier transform ion cyclotron resonance mass spectrometer equipped with a micrometal electrospray ionization emitter.

    PubMed

    Yamada, Naoyuki; Suzuki, Ei-Ichiro; Hirayama, Kazuo

    2006-01-01

    Heating of a nano-electrospray ionization (nanoESI) source can improve the dissociation efficiency of collisionally induced dissociation (CID) methods, such as nozzle-skimmer CID (NS-CID) and infrared multiphoton dissociation (IRMPD), for large biomolecule fragmentation. A metal nanoESI emitter was used due to its resistance to heating above 250 degrees C. This novel method for the dissociation of large biomolecular ions is termed "heat-assisted NS-CID" (HANS-CID) or "heat-assisted IRMPD" (HA-IRMPD). Multiple charged nonreduced protein ions (8.6 Da ubiquitin, 14 kDa lysozyme, and 67 kDa bovine serum albumin) were directly dissociated by HANS-CID and HA-IRMPD to effectively yield fragment ions that could be assigned. The fragment ions of ubiquitin by HANS-CID can be analyzed by tandem mass spectrometry (MS/MS) using sustained off-resonance irradiation CID (SORI-CID) and IRMPD. In addition, a native large protein, immunoglobulin G (IgG, 150 kDa), was efficiently dissociated by HA-IRMPD. The product ions that were obtained reflected the domain structure of IgG. However, these product ions of IgG and lysozyme were not dissociated by MS/MS using the same heating energetic methods such as IRMPD and SORI-CID.

  15. Evaluation of pulsed fast-atom bombardment ionization for increased sensitivity of tandem mass spectrometry

    SciTech Connect

    Tecklenburg, R.E. Jr.; Castro, M.E.; Russell, D.H.

    1989-01-15

    The use of pulsed valves for performing fast-atom bombardment (FAB) ionization on a sector ion-beam mass spectrometer is described. The objective of this work is to establish new methods for improving the sensitivity of tandem mass spectrometry. This paper deals with the use of pulsed FAB for improving total ion yields as well as signal-to-noise ratios for collision-induced dissociation and laser-ion beam photodissociation. Pulsing the neutral gas pressure used for FAB ionization results in neutral primary beam densities much greater (by a factor of 10) than those obtainable for continuous FAB. This approach yields enhancements for total ion yields and collision-induced dissociation signals by a factor of 15, while larger gains (> 28) are measured for the comparable photodissociation experiment.

  16. High-Speed Tandem Mass Spectrometric in Situ Imaging by Nanospray Desorption Electrospray Ionization Mass Spectrometry

    SciTech Connect

    Lanekoff, Ingela T.; Burnum-Johnson, Kristin E.; Thomas, Mathew; Short, Joshua TL; Carson, James P.; Cha, Jeeyeon; Dey, Sudhansu K.; Yang, Pengxiang; Prieto Conaway, Maria C.; Laskin, Julia

    2013-10-15

    Nanospray desorption electrospray ionization (nano-DESI) combined with tandem mass spectrometry (MS/MS), high-resolution mass analysis (m/m=17,500 at m/z 200), and rapid spectral acquisition enabled simultaneous imaging and identification of more than 300 molecules from 92 selected m/z windows (± 1 Da) with a spatial resolution of better than 150 um. Uterine sections of implantation sites on day 6 of pregnancy were analyzed in the ambient environment without any sample pre-treatment. MS/MS imaging was performed by scanning the sample under the nano-DESI probe at 10 um/s while acquiring higher-energy collision-induced dissociation (HCD) spectra for a targeted inclusion list of 92 m/z values at a rate of ~6.3 spectra/s. Molecular ions and their corresponding fragments, separated using high-resolution mass analysis, were assigned based on accurate mass measurement. Using this approach, we were able to identify and image both abundant and low-abundance isobaric species within each m/z window. MS/MS analysis enabled efficient separation and identification of isobaric sodium and potassium adducts of phospholipids. Furthermore, we identified several metabolites associated with early pregnancy and obtained the first 2D images of these molecules.

  17. High-speed tandem mass spectrometric in situ imaging by nanospray desorption electrospray ionization mass spectrometry.

    PubMed

    Lanekoff, Ingela; Burnum-Johnson, Kristin; Thomas, Mathew; Short, Joshua; Carson, James P; Cha, Jeeyeon; Dey, Sudhansu K; Yang, Pengxiang; Prieto Conaway, Maria C; Laskin, Julia

    2013-10-15

    Nanospray desorption electrospray ionization (nano-DESI) combined with tandem mass spectrometry (MS/MS), high-resolution mass analysis of the fragment ions (m/Δm = 17 500 at m/z 200), and rapid spectral acquisition enabled simultaneous imaging and identification of a large number of metabolites and lipids from 92 selected m/z windows (±1 Da) with a spatial resolution of better than 150 μm. Mouse uterine sections of implantation sites on day 6 of pregnancy were analyzed in the ambient environment without any sample pretreatment. MS/MS imaging was performed by scanning the sample under the nano-DESI probe at 10 μm/s, while higher-energy collision-induced dissociation (HCD) spectra were acquired for a targeted inclusion list of 92 m/z values at a rate of ∼6.3 spectra/s. Molecular ions and their corresponding fragments, separated by high-resolution mass analysis, were assigned on the basis of accurate mass measurement. Using this approach, we were able to identify and image both abundant and low-abundance isobaric and isomeric species within each m/z window. MS/MS analysis enabled efficient separation and identification of isomeric and isobaric phospholipids that are difficult to separate in full-scan mode. Furthermore, we identified several metabolites associated with early pregnancy and obtained the first 2D images of these molecules.

  18. Multivariate analysis of electron detachment dissociation and infrared multiphoton dissociation mass spectra of heparan sulfate tetrasaccharides differing only in hexuronic acid stereochemistry.

    PubMed

    Oh, Han Bin; Leach, Franklin E; Arungundram, Sailaja; Al-Mafraji, Kanar; Venot, Andre; Boons, Geert-Jan; Amster, I Jonathan

    2011-03-01

    The structural characterization of glycosaminoglycan (GAG) carbohydrates by mass spectrometry has been a long-standing analytical challenge due to the inherent heterogeneity of these biomolecules, specifically polydispersity, variability in sulfation, and hexuronic acid stereochemistry. Recent advances in tandem mass spectrometry methods employing threshold and electron-based ion activation have resulted in the ability to determine the location of the labile sulfate modification as well as assign the stereochemistry of hexuronic acid residues. To facilitate the analysis of complex electron detachment dissociation (EDD) spectra, principal component analysis (PCA) is employed to differentiate the hexuronic acid stereochemistry of four synthetic GAG epimers whose EDD spectra are nearly identical upon visual inspection. For comparison, PCA is also applied to infrared multiphoton dissociation spectra (IRMPD) of the examined epimers. To assess the applicability of multivariate methods in GAG mixture analysis, PCA is utilized to identify the relative content of two epimers in a binary mixture.

  19. Dissociation and Mass Transfer Coefficients for Ammonia Volatilization Models

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Process-based models are being used to predict ammonia emissions from manure sources, but their accuracy has not been fully evaluated for cattle manure. Laboratory trials were conducted to measure the dissociation and mass transfer coefficients for ammonia volatilization from media of buffered ammon...

  20. Ozone-induced dissociation on a modified tandem linear ion-trap: observations of different reactivity for isomeric lipids.

    PubMed

    Poad, Berwyck L J; Pham, Huong T; Thomas, Michael C; Nealon, Jessica R; Campbell, J Larry; Mitchell, Todd W; Blanksby, Stephen J

    2010-12-01

    Ozone-induced dissociation (OzID) exploits the gas-phase reaction between mass-selected lipid ions and ozone vapor to determine the position(s) of unsaturation. In this contribution, we describe the modification of a tandem linear ion-trap mass spectrometer specifically for OzID analyses wherein ozone vapor is supplied to the collision cell. This instrumental configuration provides spatial separation between mass-selection, the ozonolysis reaction, and mass-analysis steps in the OzID process and thus delivers significant enhancements in speed and sensitivity (ca. 30-fold). These improvements allow spectra revealing the double-bond position(s) within unsaturated lipids to be acquired within 1 s: significantly enhancing the utility of OzID in high-throughput lipidomic protocols. The stable ozone concentration afforded by this modified instrument also allows direct comparison of relative reactivity of isomeric lipids and reveals reactivity trends related to (1) double-bond position, (2) substitution position on the glycerol backbone, and (3) stereochemistry. For cis- and trans-isomers, differences were also observed in the branching ratio of product ions arising from the gas-phase ozonolysis reaction, suggesting that relative ion abundances could be exploited as markers for double-bond geometry. Additional activation energy applied to mass-selected lipid ions during injection into the collision cell (with ozone present) was found to yield spectra containing both OzID and classical-CID fragment ions. This combination CID-OzID acquisition on an ostensibly simple monounsaturated phosphatidylcholine within a cow brain lipid extract provided evidence for up to four structurally distinct phospholipids differing in both double-bond position and sn-substitution.

  1. Repeatability and reproducibility of product ion abundances in electron capture dissociation mass spectrometry of peptides.

    PubMed

    Ben Hamidane, Hisham; Vorobyev, Aleksey; Tsybin, Yury O

    2011-01-01

    Site-specific reproducibility and repeatability of electron capture dissociation (ECD) in Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) are of fundamental importance for product ion abundance (PIA)-based peptide and protein structure analysis. However, despite the growing interest in ECD PIA-based applications, these parameters have not yet been investigated in a consistent manner. Here, we first provide a detailed description of the experimental parameters for ECD-based tandem mass spectrometry performed on a hybrid linear ion trap (LTQ) FT-ICR MS. In the following, we describe the evaluation and comparison of ECD and infrared multiphoton dissociation (IRMPD) PIA methodologies upon variation of a number of experimental parameters, for example, cathode potential (electron energy), laser power, electron and photon irradiation periods and pre- irradiation delays, as well as precursor ion number. Ranges of experimental parameters that yielded an average PIA variation below 5% and 15% were determined for ECD and IRMPD, respectively. We report cleavage site-dependent ECD PIA variation below 20% and correlation coefficients between fragmentation patterns superior to 0.95 for experiments performed on three FT-ICR MS instruments. Overall, the encouraging results obtained for ECD PIA reproducibility and repeatability support the use of ECD PIA as a complementary source of information to m/z data in radical-induced dissociation applied for peptide and protein structure analysis.

  2. Protonation Sites, Tandem Mass Spectrometry and Computational Calculations of o-Carbonyl Carbazolequinone Derivatives

    PubMed Central

    Martínez-Cifuentes, Maximiliano; Clavijo-Allancan, Graciela; Zuñiga-Hormazabal, Pamela; Aranda, Braulio; Barriga, Andrés; Weiss-López, Boris; Araya-Maturana, Ramiro

    2016-01-01

    A series of a new type of tetracyclic carbazolequinones incorporating a carbonyl group at the ortho position relative to the quinone moiety was synthesized and analyzed by tandem electrospray ionization mass spectrometry (ESI/MS-MS), using Collision-Induced Dissociation (CID) to dissociate the protonated species. Theoretical parameters such as molecular electrostatic potential (MEP), local Fukui functions and local Parr function for electrophilic attack as well as proton affinity (PA) and gas phase basicity (GB), were used to explain the preferred protonation sites. Transition states of some main fragmentation routes were obtained and the energies calculated at density functional theory (DFT) B3LYP level were compared with the obtained by ab initio quadratic configuration interaction with single and double excitation (QCISD). The results are in accordance with the observed distribution of ions. The nature of the substituents in the aromatic ring has a notable impact on the fragmentation routes of the molecules. PMID:27399676

  3. Noncovalent Shiga-like toxin assemblies: characterization by means of mass spectrometry and tandem mass spectrometry.

    PubMed

    Williams, Jonathan P; Green, Brian N; Smith, Daniel C; Jennings, Keith R; Moore, Katherine A H; Slade, Susan E; Roberts, Lynne M; Scrivens, James H

    2005-06-14

    Shiga-like toxin 1 (SLTx), produced by enterohemorrhagic strains of Escherichia coli (EHEC), belongs to a family of structurally and functionally related AB(5) protein toxins that are associated with human disease. EHEC infection often gives rise to hemolytic colitis, while toxin-induced kidney damage is one of the major causes of hemolytic uremic syndrome (HUS) and acute renal failure in children. As such, an understanding and analysis of the noncovalent interactions that maintain the quaternary structure of this toxin are fundamentally important since such interactions have significant biochemical and medical implications. This paper reports on the analysis of the noncovalent homopentameric complex of Shiga-like toxin B chain (SLTx-B(5)) using electrospray ionization (ESI) triple-quadrupole (QqQ) mass spectrometry (MS) and tandem mass spectrometry (MS/MS) and the analysis of the noncovalent hexameric holotoxin (SLTx-AB(5)) using ESI time-of-flight (TOF) MS. The triple-quadrupole analysis revealed highly charged monomer ions dissociate from the multiprotein complex to form dimer, trimer, and tetramer product ions, which were also seen to further dissociate. The ESI-TOFMS analysis of SLTx-AB(5) revealed the complex remained intact and was observed in the gas phase over a range of pHs. Theses findings demonstrate that the gas-phase structure observed for both the holotoxin and the isoloated B chains correlates well with the structures reported to exist in the solution phase for these proteins. Such analysis provides a rapid screening technique for assessing the noncovalent structure of this family of proteins and other structurally related toxins.

  4. Large-scale identification of endogenous secretory peptides using electron transfer dissociation mass spectrometry.

    PubMed

    Sasaki, Kazuki; Osaki, Tsukasa; Minamino, Naoto

    2013-03-01

    Mass spectrometry-based unbiased analysis of the full complement of secretory peptides is expected to facilitate the identification of unknown biologically active peptides. However, tandem MS sequencing of endogenous peptides in their native form has proven difficult because they show size heterogeneity and contain multiple internal basic residues, the characteristics not found in peptide fragments produced by in vitro digestion. Endogenous peptides remain largely unexplored by electron transfer dissociation (ETD), despite its widespread use in bottom-up proteomics. We used ETD, in comparison to collision induced dissociation (CID), to identify endogenous peptides derived from secretory granules of a human endocrine cell line. For mass accuracy, both MS and tandem MS were analyzed on an Orbitrap. CID and ETD, performed in different LC-MS runs, resulted in the identification of 795 and 569 unique peptides (ranging from 1000 to 15000 Da), respectively, with an overlap of 397. Peptides larger than 3000 Da accounted for 54% in CID and 46% in ETD identifications. Although numerically outperformed by CID, ETD provided more extensive fragmentation, leading to the identification of peptides that are not reached by CID. This advantage was demonstrated in identifying a new antimicrobial peptide from neurosecretory protein VGF (non-acronymic), VGF[554-577]-NH2, or in differentiating nearly isobaric peptides (mass difference less than 2 ppm) that arise from alternatively spliced exons of the gastrin-releasing peptide gene. CID and ETD complemented each other to add to our knowledge of the proteolytic processing sites of proteins implicated in the regulated secretory pathway. An advantage of the use of both fragmentation methods was also noted in localization of phosphorylation sites. These findings point to the utility of ETD mass spectrometry in the global study of endogenous peptides, or peptidomics.

  5. Large-scale Identification of Endogenous Secretory Peptides Using Electron Transfer Dissociation Mass Spectrometry*

    PubMed Central

    Sasaki, Kazuki; Osaki, Tsukasa; Minamino, Naoto

    2013-01-01

    Mass spectrometry-based unbiased analysis of the full complement of secretory peptides is expected to facilitate the identification of unknown biologically active peptides. However, tandem MS sequencing of endogenous peptides in their native form has proven difficult because they show size heterogeneity and contain multiple internal basic residues, the characteristics not found in peptide fragments produced by in vitro digestion. Endogenous peptides remain largely unexplored by electron transfer dissociation (ETD), despite its widespread use in bottom-up proteomics. We used ETD, in comparison to collision induced dissociation (CID), to identify endogenous peptides derived from secretory granules of a human endocrine cell line. For mass accuracy, both MS and tandem MS were analyzed on an Orbitrap. CID and ETD, performed in different LC-MS runs, resulted in the identification of 795 and 569 unique peptides (ranging from 1000 to 15000 Da), respectively, with an overlap of 397. Peptides larger than 3000 Da accounted for 54% in CID and 46% in ETD identifications. Although numerically outperformed by CID, ETD provided more extensive fragmentation, leading to the identification of peptides that are not reached by CID. This advantage was demonstrated in identifying a new antimicrobial peptide from neurosecretory protein VGF (non-acronymic), VGF[554–577]-NH2, or in differentiating nearly isobaric peptides (mass difference less than 2 ppm) that arise from alternatively spliced exons of the gastrin-releasing peptide gene. CID and ETD complemented each other to add to our knowledge of the proteolytic processing sites of proteins implicated in the regulated secretory pathway. An advantage of the use of both fragmentation methods was also noted in localization of phosphorylation sites. These findings point to the utility of ETD mass spectrometry in the global study of endogenous peptides, or peptidomics. PMID:23250050

  6. Sodium Cation Affinities of Commonly Used MALDI Matrices Determined by Guided Ion Beam Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Chinthaka, S. D. M.; Rodgers, M. T.

    2012-04-01

    The sodium cation affinities of six commonly used MALDI matrices are determined here using guided ion beam tandem mass spectrometry techniques. The collision-induced dissociation behavior of six sodium cationized MALDI matrices, Na+(MALDI), with Xe is studied as a function of kinetic energy. The MALDI matrices examined here include: nicotinic acid, quinoline, 3-aminoquinoline, 4-nitroaniline, picolinic acid, and 3-hydroxypicolinic acid. In all cases, the primary dissociation pathway corresponds to endothermic loss of the intact MALDI matrix. The cross section thresholds are interpreted to yield zero and 298 K Na+-MALDI bond dissociation energies (BDEs), or sodium cation affinities, after accounting for the effects of multiple ion-neutral collisions, the kinetic and internal energy distributions of the reactants, and dissociation lifetimes. Density functional theory calculations at the B3LYP/6-311+G(2d,2p)//B3LYP/6-31G* and MP2(full)/6-311+G(2d,2p)//B3LYP/6-31G* levels of theory are used to characterized the structures and energetics for these systems. The calculated BDEs exhibit very good agreement with the measured values for most systems. The experimental and theoretical Na+-MALDI BDEs determined here are compared with those previously measured by cation transfer equilibrium methods.

  7. Challenges and developments in tandem mass spectrometry based clinical metabolomics.

    PubMed

    Ceglarek, Uta; Leichtle, Alexander; Brügel, Mathias; Kortz, Linda; Brauer, Romy; Bresler, Kristin; Thiery, Joachim; Fiedler, Georg Martin

    2009-03-25

    'Clinical metabolomics' aims at evaluating and predicting health and disease risk in an individual by investigating metabolic signatures in body fluids or tissues, which are influenced by genetics, epigenetics, environmental exposures, diet, and behaviour. Powerful analytical techniques like liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) offers a rapid, effective and economical way to analyze metabolic alterations of pre-defined target metabolites in biological samples. Novel hyphenated technical approaches like the combination of tandem mass spectrometry combined with linear ion trap (QTrap mass spectrometry) combines both identification and quantification of known and unknown metabolic targets. We describe new concepts and developments of mass spectrometry based multi-target metabolome profiling in the field of clinical diagnostics and research. Particularly, the experiences from newborn screening provided important insights about the diagnostic potential of metabolite profiling arrays and directs to the clinical aim of predictive, preventive and personalized medicine by metabolomics.

  8. Tandem mass spectrometry of amidated peptides.

    PubMed

    Mouls, Laetitia; Subra, Gilles; Aubagnac, Jean-Louis; Martinez, Jean; Enjalbal, Christine

    2006-11-01

    The behavior of C-terminal amidated and carboxylated peptides upon low-energy collision-induced dissociation (CID) was investigated. Two sets of 76 sequences of variable amino acid compositions and lengths were synthesized as model compounds. In most cases, C-terminal amidated peptides were found to produce, upon CID, an abundant loss of ammonia from the protonated molecules. To validate such MS/MS signatures, the studied peptides contained amino acids that can potentially release ammonia from their side chains, such as asparagine, glutamine, tryptophan, lysine and arginine. Arginine, and to a lesser extent lysine, was shown to induce a competitive fragmentation leading to the loss of ammonia from their side chains, thus interfering with the targeted backbone neutral release. However, when arginine or lysine was located at the C-terminal position mimicking a tryptic digest, losses of ammonia from the arginine side chain and from the peptide backbone were completely suppressed. Such results were discussed in the frame of peptidomic or proteomic studies in an attempt to reveal the presence of C-terminal amidated peptides or proteins.

  9. In Silico Identification Software (ISIS): A Machine Learning Approach to Tandem Mass Spectral Identification of Lipids

    SciTech Connect

    Kangas, Lars J.; Metz, Thomas O.; Isaac, Georgis; Schrom, Brian T.; Ginovska-Pangovska, Bojana; Wang, Luning; Tan, Li; Lewis, Robert R.; Miller, John H.

    2012-05-15

    Liquid chromatography-mass spectrometry-based metabolomics has gained importance in the life sciences, yet it is not supported by software tools for high throughput identification of metabolites based on their fragmentation spectra. An algorithm (ISIS: in silico identification software) and its implementation are presented and show great promise in generating in silico spectra of lipids for the purpose of structural identification. Instead of using chemical reaction rate equations or rules-based fragmentation libraries, the algorithm uses machine learning to find accurate bond cleavage rates in a mass spectrometer employing collision-induced dissocia-tion tandem mass spectrometry. A preliminary test of the algorithm with 45 lipids from a subset of lipid classes shows both high sensitivity and specificity.

  10. In silico identification software (ISIS): a machine learning approach to tandem mass spectral identification of lipids

    PubMed Central

    Kangas, Lars J.; Metz, Thomas O.; Isaac, Giorgis; Schrom, Brian T.; Ginovska-Pangovska, Bojana; Wang, Luning; Tan, Li; Lewis, Robert R.; Miller, John H.

    2012-01-01

    Motivation: Liquid chromatography–mass spectrometry-based metabolomics has gained importance in the life sciences, yet it is not supported by software tools for high throughput identification of metabolites based on their fragmentation spectra. An algorithm (ISIS: in silico identification software) and its implementation are presented and show great promise in generating in silico spectra of lipids for the purpose of structural identification. Instead of using chemical reaction rate equations or rules-based fragmentation libraries, the algorithm uses machine learning to find accurate bond cleavage rates in a mass spectrometer employing collision-induced dissociation tandem mass spectrometry. Results: A preliminary test of the algorithm with 45 lipids from a subset of lipid classes shows both high sensitivity and specificity. Contact: lars.kangas@pnnl.gov Supplementary information: Supplementary data are available at Bioinformatics online. PMID:22592377

  11. Dynamic collision-induced dissociation of peptides in a quadrupole ion trap mass spectrometer.

    PubMed

    Collin, Olivier L; Beier, Matthias; Jackson, Glen P

    2007-07-15

    The fragmentation of natural peptides using dynamic collision-induced dissociation (DCID), a novel fragmentation method for quadrupole ion traps, is demonstrated. Using leucine enkephalin as a diagnostic molecule, the fragmentation efficiencies and energetics of DCID are compared with other methods of collisional activation in ion traps such as conventional on-resonance excitation and high-amplitude short-time excitation (HASTE). A typical fragmentation efficiency of approximately 20% is achieved for DCID, which is significantly lower than conventional CID (maximum near 80%). Tandem mass spectra of two other peptides, substance P and oxidized insulin alpha-chain, demonstrate that product ion spectra for DCID are comparable to conventional or HASTE CID. Because DCID achieves fragmentation during the standard mass acquisition scan, no extra time is necessary for on-resonance excitation or product ion collection, so analysis times are reduced by a minimum of 10-15% depending on the scanning conditions. DCID therefore offers more tandem mass spectra per second than conventional methods of collisional activation, which could be highly advantageous for bottom-up proteomics separations.

  12. Comparative Proteomics of Tandem Mass Spectrometry Analyses for Bacterial Strains Identification and Differentiation

    DTIC Science & Technology

    2012-02-01

    induced dissociation (CID), Electron transfer dissociation , or post-source decay (PSD) of ionized tryptic peptides derived from bacterial proteins , i.e...fingerprints of the bacterial proteins , i.e. MALDI-TOF-MS technique, or amino acid sequences, from tandem MS/MS analysis, of proteins from collision ...William 2002), present in biological threat microorganisms. The vast amount of protein and peptide data generated from a typical LC- tandem MS

  13. Comparison of laser-induced dissociation and high-energy collision-induced dissociation using matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) for peptide and protein identification.

    PubMed

    Macht, Marcus; Asperger, Arndt; Deininger, Sören-Oliver

    2004-01-01

    The fragmentation of peptides under laser-induced dissociation (LID) as well as high-energy collision-induced dissociation (CID) conditions has been investigated. The effect of the different fragmentation mechanisms on the formation of specific fragment ion types and the usability of the resulting spectra, e.g. for high-throughput protein identification, has been evaluated. Also, basic investigations on the influence of the matrix, as well as laser fluence, on the fragment ion formation and the consequences in the spectral appearance are discussed. The preconditions for obtaining 'pure' CID spectra on matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) instruments are evaluated and discussed as well as the differences between LID and CID in the resulting fragment ion types. While containing a wealth of information due to additional fragment ions in comparison with LID, CID spectra are significantly more complex than LID spectra and, due to different fragmentation patterns, the CID spectra are of limited use for protein identification, even under optimized parameter settings, due to significantly lower scores for the individual spectra. Conditions for optimal results regarding protein identification using MALDI-TOF/TOF instruments have been evaluated. For database searches using tandem mass spectrometric data, the use of LID as fragmentation technique in combination with parameter settings supporting the use of internal fragment ions turned out to yield the optimal results.

  14. Classification of Tandem Mass Spectra for Identification of N- and O-linked Glycopeptides

    PubMed Central

    Toghi Eshghi, Shadi; Yang, Weiming; Hu, Yingwei; Shah, Punit; Sun, Shisheng; Li, Xingde; Zhang, Hui

    2016-01-01

    Analysis of intact glycopeptides by mass spectrometry is essential to determining the microheterogeneity of protein glycosylation. Higher-energy collisional dissociation (HCD) fragmentation of glycopeptides generates mono- or disaccharide ions called oxonium ions that carry information about the structure of the fragmented glycans. Here, we investigated the link between glycan structures and the intensity of oxonium ions in the spectra of glycopeptides and utilized this information to improve the identification of glycopeptides in biological samples. Tandem spectra of glycopeptides from fetuin, glycophorin A, ovalbumin and gp120 tryptic digests were used to build a spectral database of N- and O-linked glycopeptides. Logistic regression was applied to this database to develop model to distinguish between the spectra of N- and O-linked glycopeptides. Remarkably, the developed model was found to reliably distinguish between the N- and O-linked glycopeptides using the spectral features of the oxonium ions using verification spectral set. Finally, the performance of the developed predictive model was evaluated in HILIC enriched glycopeptides extracted from human serum. The results showed that pre-classification of tandem spectra based on their glycosylation type improved the identification of N-linked glycopeptides. The developed model facilitates interpretation of tandem mass spectrometry data for assignment of glycopeptides. PMID:27869200

  15. Classification of Tandem Mass Spectra for Identification of N- and O-linked Glycopeptides

    NASA Astrophysics Data System (ADS)

    Toghi Eshghi, Shadi; Yang, Weiming; Hu, Yingwei; Shah, Punit; Sun, Shisheng; Li, Xingde; Zhang, Hui

    2016-11-01

    Analysis of intact glycopeptides by mass spectrometry is essential to determining the microheterogeneity of protein glycosylation. Higher-energy collisional dissociation (HCD) fragmentation of glycopeptides generates mono- or disaccharide ions called oxonium ions that carry information about the structure of the fragmented glycans. Here, we investigated the link between glycan structures and the intensity of oxonium ions in the spectra of glycopeptides and utilized this information to improve the identification of glycopeptides in biological samples. Tandem spectra of glycopeptides from fetuin, glycophorin A, ovalbumin and gp120 tryptic digests were used to build a spectral database of N- and O-linked glycopeptides. Logistic regression was applied to this database to develop model to distinguish between the spectra of N- and O-linked glycopeptides. Remarkably, the developed model was found to reliably distinguish between the N- and O-linked glycopeptides using the spectral features of the oxonium ions using verification spectral set. Finally, the performance of the developed predictive model was evaluated in HILIC enriched glycopeptides extracted from human serum. The results showed that pre-classification of tandem spectra based on their glycosylation type improved the identification of N-linked glycopeptides. The developed model facilitates interpretation of tandem mass spectrometry data for assignment of glycopeptides.

  16. Gas-Phase Fragmentation Behavior of Oxidized Prenyl Peptides by CID and ETD Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Bhawal, Ruchika P.; Shahinuzzaman, A. D. A.; Chowdhury, Saiful M.

    2016-10-01

    Farnesylation and geranylgeranylation are the two types of prenyl modification of proteins. Prenylated peptides are highly hydrophobic and their abundances in biological samples are low. In this report, we studied the oxidized prenylated peptides by electrospray ionization mass spectrometry and identified them by collision-induced dissociation (CID) and electron-transfer dissociation (ETD) tandem mass spectrometry. Modified prenyl peptides were generated utilizing strong and low strength oxidizing agents to selectively oxidize and epoxidize cysteine sulfur and prenyl side chain. We selected three peptides with prenyl motifs and synthesized their prenylated versions. The detailed characteristic fragmentations of oxidized and epoxidized farnesylated and geranylgeranylated peptides were studied side by side with two popular fragmentation techniques. CID and ETD mass spectrometry clearly distinguished the modified version of these peptides. ETD mass spectrometry provided sequence information of the highly labile modified prenyl peptides and showed different characteristic fragmentations compared with CID. A detailed fragmentation of modified geranylgeranylated peptides was compared by CID and ETD mass spectrometry for the first time.

  17. Gas-Phase Fragmentation Behavior of Oxidized Prenyl Peptides by CID and ETD Tandem Mass Spectrometry.

    PubMed

    Bhawal, Ruchika P; Shahinuzzaman, A D A; Chowdhury, Saiful M

    2017-04-01

    Farnesylation and geranylgeranylation are the two types of prenyl modification of proteins. Prenylated peptides are highly hydrophobic and their abundances in biological samples are low. In this report, we studied the oxidized prenylated peptides by electrospray ionization mass spectrometry and identified them by collision-induced dissociation (CID) and electron-transfer dissociation (ETD) tandem mass spectrometry. Modified prenyl peptides were generated utilizing strong and low strength oxidizing agents to selectively oxidize and epoxidize cysteine sulfur and prenyl side chain. We selected three peptides with prenyl motifs and synthesized their prenylated versions. The detailed characteristic fragmentations of oxidized and epoxidized farnesylated and geranylgeranylated peptides were studied side by side with two popular fragmentation techniques. CID and ETD mass spectrometry clearly distinguished the modified version of these peptides. ETD mass spectrometry provided sequence information of the highly labile modified prenyl peptides and showed different characteristic fragmentations compared with CID. A detailed fragmentation of modified geranylgeranylated peptides was compared by CID and ETD mass spectrometry for the first time. Graphical Abstract ᅟ.

  18. Protein identification using nano liquid chromatography-tandem mass spectrometry.

    PubMed

    Negroni, Luc

    2007-01-01

    Tandem mass spectrometry is an efficient technique for the identification of peptides on the basis of their fragmentation pattern (MS/MS scan). It can generate individual spectra for each peptide, thereby creating a powerful tool for protein identification on the basis of peptide characterization. This important advance in automatic data acquisition has allowed an efficient association between liquid chromatography and tandem mass spectrometry, and the use of nanocolumns and nanoelectrospray ionization has dramatically increased the efficiency of this method. Now large sets of peptides can be identified at a femtomole level. At the end of the process, batch processing of the MS/MS spectra produces peptide lists that identify purified proteins or protein mixtures with high confidence.

  19. Verification of automated peptide identifications from proteomic tandem mass spectra

    PubMed Central

    Tabb, David L; Friedman, David B; Ham, Amy-Joan L

    2010-01-01

    Shotgun proteomics yields tandem mass spectra of peptides that can be identified by database search algorithms. When only a few observed peptides suggest the presence of a protein, establishing the accuracy of the peptide identifications is necessary for accepting or rejecting the protein identification. In this protocol, we describe the properties of peptide identifications that can differentiate legitimately identified peptides from spurious ones. The chemistry of fragmentation, as embodied in the ‘mobile proton’ and ‘pathways in competition’ models, informs the process of confirming or rejecting each spectral match. Examples of ion-trap and tandem time-of-flight (TOF/TOF) mass spectra illustrate these principles of fragmentation. PMID:17406459

  20. Development of an advanced spacecraft tandem mass spectrometer

    NASA Technical Reports Server (NTRS)

    Drew, Russell C.

    1992-01-01

    The purpose of this research was to apply current advanced technology in electronics and materials to the development of a miniaturized Tandem Mass Spectrometer that would have the potential for future development into a package suitable for spacecraft use. The mass spectrometer to be used as a basis for the tandem instrument would be a magnetic sector instrument, of Nier-Johnson configuration, as used on the Viking Mars Lander mission. This instrument configuration would then be matched with a suitable second stage MS to provide the benefits of tandem MS operation for rapid identification of unknown organic compounds. This tandem instrument is configured with a newly designed GC system to aid in separation of complex mixtures prior to MS analysis. A number of important results were achieved in the course of this project. Among them were the development of a miniaturized GC subsystem, with a unique desorber-injector, fully temperature feedback controlled oven with powered cooling for rapid reset to ambient conditions, a unique combination inlet system to the MS that provides for both membrane sampling and direct capillary column sample transfer, a compact and ruggedized alignment configuration for the MS, an improved ion source design for increased sensitivity, and a simple, rugged tandem MS configuration that is particularly adaptable to spacecraft use because of its low power and low vacuum pumping requirements. The potential applications of this research include use in manned spacecraft like the space station as a real-time detection and warning device for the presence of potentially harmful trace contaminants of the spacecraft atmosphere, use as an analytical device for evaluating samples collected on the Moon or a planetary surface, or even use in connection with monitoring potentially hazardous conditions that may exist in terrestrial locations such as launch pads, environmental test chambers or other sensitive areas. Commercial development of the technology

  1. Considerations for electron capture dissociation efficiency in FTICR mass spectrometry

    NASA Astrophysics Data System (ADS)

    Gorshkov, Michael V.; Masselon, Christophe D.; Nikolaev, Eugene N.; Udseth, Harold R.; Pasa-Tolic, Ljiljana; Smith, Richard D.

    2004-05-01

    An experimental approach for increasing the efficiency of Electron Capture Dissociation (ECD) with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) is presented. The approach is based on manipulating the spatial distribution of an ion cloud inside an FTICR trap during electron irradiation, which is realized by using both on-resonance pre-excitation of the ions and sustained off-resonance irradiation (SORI). The achieved fragmentation efficiency is compared with the theoretical prediction. This method may be useful in biological applications of FTICR, such as identification of posttranslational modifications in proteins and de novo sequencing, where the ECD technique is most applicable.

  2. Estimating the Efficiency of Phosphopeptide Identification by Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Hsu, Chuan-Chih; Xue, Liang; Arrington, Justine V.; Wang, Pengcheng; Paez Paez, Juan Sebastian; Zhou, Yuan; Zhu, Jian-Kang; Tao, W. Andy

    2017-03-01

    Mass spectrometry has played a significant role in the identification of unknown phosphoproteins and sites of phosphorylation in biological samples. Analyses of protein phosphorylation, particularly large scale phosphoproteomic experiments, have recently been enhanced by efficient enrichment, fast and accurate instrumentation, and better software, but challenges remain because of the low stoichiometry of phosphorylation and poor phosphopeptide ionization efficiency and fragmentation due to neutral loss. Phosphoproteomics has become an important dimension in systems biology studies, and it is essential to have efficient analytical tools to cover a broad range of signaling events. To evaluate current mass spectrometric performance, we present here a novel method to estimate the efficiency of phosphopeptide identification by tandem mass spectrometry. Phosphopeptides were directly isolated from whole plant cell extracts, dephosphorylated, and then incubated with one of three purified kinases—casein kinase II, mitogen-activated protein kinase 6, and SNF-related protein kinase 2.6—along with 16O4- and 18O4-ATP separately for in vitro kinase reactions. Phosphopeptides were enriched and analyzed by LC-MS. The phosphopeptide identification rate was estimated by comparing phosphopeptides identified by tandem mass spectrometry with phosphopeptide pairs generated by stable isotope labeled kinase reactions. Overall, we found that current high speed and high accuracy mass spectrometers can only identify 20%-40% of total phosphopeptides primarily due to relatively poor fragmentation, additional modifications, and low abundance, highlighting the urgent need for continuous efforts to improve phosphopeptide identification efficiency.

  3. Application of Electron Transfer Dissociation Mass Spectrometry in Analyses of Non-enzymatically Glycated Peptides

    SciTech Connect

    Zhang, Qibin; Frolov, Andrej; Tang, Ning; Hoffman, Ralf; van der Goor, Tom; Metz, Thomas O.; Smith, Richard D.

    2007-03-15

    Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in diabetes mellitus research, particularly in the context of development of diabetic complications. The fragmentation behavior of glycated peptides produced from reaction of D-glucose with lysine residues was investigated by electron transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. It was found that high abundance ions corresponding to various degrees of neutral water losses, as well as furylium ion production, dominate the CID spectra, and that the sequence informative b and y ions were rarely observed when Amadori-modified peptides were fragmented. Contrary to what was observed under CID conditions, ions corresponding to neutral losses of water or furylium ion production were not observed in the ETD spectra. Instead, abundant and almost complete series of c and z type ions were observed regardless of whether the modification site was located in the middle of the sequence or close to the N-terminus, greatly facilitating the peptide sequencing. This study strongly suggests that ETD is a better technique for proteomics studies of non-enzymatically glycated peptides and proteins.

  4. Combined electron transfer dissociation-collision-induced dissociation fragmentation in the mass spectrometric distinction of leucine, isoleucine, and hydroxyproline residues in Peptide natural products.

    PubMed

    Gupta, Kallol; Kumar, Mukesh; Chandrashekara, Krishnappa; Krishnan, Kozhalmannom S; Balaram, Padmanabhan

    2012-02-03

    Distinctions between isobaric residues have been a major challenge in mass spectrometric peptide sequencing. Here, we propose a methodology for distinction among isobaric leucine, isoleucine, and hydroxyproline, a commonly found post-translationally modified amino acid with a nominal mass of 113 Da, through a combined electron transfer dissociation-collision-induced dissociation approach. While the absence of c and z(•) ions, corresponding to the Yyy-Xxx (Xxx = Leu, Ile, or Hyp) segment, is indicative of the presence of hydroxyproline, loss of isopropyl (Δm = 43 Da) or ethyl radicals (Δm = 29 Da), through collisional activation of z radical ions, are characteristic of leucine or isoleucine, respectively. Radical migration processes permit distinctions even in cases where the specific z(•) ions, corresponding to the Yyy-Leu or -Ile segments, are absent or of low intensity. This tandem mass spectrometric (MS(n)) method has been successfully implemented in a liquid chromatography-MS(n) platform to determine the identity of 23 different isobaric residues from a mixture of five different peptides. The approach is convenient for distinction of isobaric residues from any crude peptide mixture, typically encountered in natural peptide libraries or proteomic analysis.

  5. Reduction in database search space by utilization of amino acid composition information from electron transfer dissociation and higher-energy collisional dissociation mass spectra.

    PubMed

    Hansen, Thomas A; Kryuchkov, Fedor; Kjeldsen, Frank

    2012-08-07

    With high-mass accuracy and consecutively obtained electron transfer dissociation (ETD) and higher-energy collisional dissociation (HCD) tandem mass spectrometry (MS/MS), reliable (≥97%) and sensitive fragment ions have been extracted for identification of specific amino acid residues in peptide sequences. The analytical benefit of these specific amino acid composition (AAC) ions is to restrict the database search space and provide identification of peptides with higher confidence and reduced false negative rates. The 6706 uniquely identified peptide sequences determined with a conservative Mascot score of >30 were used to characterize the AAC ions. The loss of amino acid side chains (small neutral losses, SNLs) from the charge reduced peptide radical cations was studied using ETD. Complementary AAC information from HCD spectra was provided by immonium ions. From the ETD/HCD mass spectra, 5162 and 6720 reliable SNLs and immonium ions were successfully extracted, respectively. Automated application of the AAC information during database searching resulted in an average 3.5-fold higher confidence level of peptide identification. In addition, 4% and 28% more peptides were identified above the significance level in a standard and extended search space, respectively.

  6. Direct Analysis in Real Time (DART) of an Organothiophosphate at Ultrahigh Resolution by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry and Tandem Mass Spectrometry

    PubMed Central

    Prokai, Laszlo; Stevens, Stanley M.

    2016-01-01

    Direct analysis in real time (DART) is a recently developed ambient ionization technique for mass spectrometry to enable rapid and sensitive analyses with little or no sample preparation. After swab-based field sampling, the organothiophosphate malathion was analyzed using DART-Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) and tandem mass spectrometry (MS/MS). Mass resolution was documented to be over 800,000 in full-scan MS mode and over 1,000,000 for an MS/MS product ion produced by collision-induced dissociation of the protonated analyte. Mass measurement accuracy below 1 ppm was obtained for all DART-generated ions that belonged to the test compound in the mass spectra acquired using only external mass calibration. This high mass measurement accuracy, achievable at present only through FTMS, was required for unequivocal identification of the corresponding molecular formulae. PMID:26784186

  7. Direct Analysis in Real Time (DART) of an Organothiophosphate at Ultrahigh Resolution by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry and Tandem Mass Spectrometry.

    PubMed

    Prokai, Laszlo; Stevens, Stanley M

    2016-01-16

    Direct analysis in real time (DART) is a recently developed ambient ionization technique for mass spectrometry to enable rapid and sensitive analyses with little or no sample preparation. After swab-based field sampling, the organothiophosphate malathion was analyzed using DART-Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) and tandem mass spectrometry (MS/MS). Mass resolution was documented to be over 800,000 in full-scan MS mode and over 1,000,000 for an MS/MS product ion produced by collision-induced dissociation of the protonated analyte. Mass measurement accuracy below 1 ppm was obtained for all DART-generated ions that belonged to the test compound in the mass spectra acquired using only external mass calibration. This high mass measurement accuracy, achievable at present only through FTMS, was required for unequivocal identification of the corresponding molecular formulae.

  8. Isothiocyanates as derivatization reagents for amines in liquid chromatography/electrospray ionization-tandem mass spectrometry.

    PubMed

    Santa, Tomofumi

    2010-09-01

    The applicability of 3-pyridyl isothiocyanate, p-(dimethylamino)phenyl isothiocyanate and m-nitrophenyl isothiocyanate as the derivatization reagents for amines in high-performance liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) was examined. The generated derivatives of amines with these reagents were favorably separated on the reversed-phase column and detected by ESI-MS/MS. The C-N bond of the generated thiourea structure was efficiently cleaved by collision-induced dissociation and gave the single and intense product ion. Among the three reagents, 3-pyridyl isothiocyanate was the most suitable as the derivatization reagent with regard to the reactivity to amines and the detection sensitivity.

  9. Scheme for the direct analysis of organics in the environment by tandem mass spectrometry

    SciTech Connect

    Hunt, D.F.; Shabanowitz, J.; Harvey, T.M.; Coates, M.

    1985-01-01

    Direct analysis of hazardous organic chemicals in the environment by tandem mass spectrometry is described. Liquid and solid chemical wastes and residues from lyophilized aqueous solutions are volatilized directly into the ion source of a triple quadrupole instrument. All or most wet chemical and chromatographic separation steps are eliminated. Analysis of phthalates, aromatic hydrocarbons, chlorocarbons, phenols, amines, and carboxylic acids by functional group and molecular weight is accomplished by using the technique of collision-activated dissociation and a series of 0.5-s neutral loss and parent ion scans under data system control on a triple quadrupole instrument. Both knowns and unknowns are characterized, detection limits are at the 10-100 ppb level, and the total analysis time per sample is typically only 25-30 min.

  10. Accurate and Efficient Resolution of Overlapping Isotopic Envelopes in Protein Tandem Mass Spectra

    PubMed Central

    Xiao, Kaijie; Yu, Fan; Fang, Houqin; Xue, Bingbing; Liu, Yan; Tian, Zhixin

    2015-01-01

    It has long been an analytical challenge to accurately and efficiently resolve extremely dense overlapping isotopic envelopes (OIEs) in protein tandem mass spectra to confidently identify proteins. Here, we report a computationally efficient method, called OIE_CARE, to resolve OIEs by calculating the relative deviation between the ideal and observed experimental abundance. In the OIE_CARE method, the ideal experimental abundance of a particular overlapping isotopic peak (OIP) is first calculated for all the OIEs sharing this OIP. The relative deviation (RD) of the overall observed experimental abundance of this OIP relative to the summed ideal value is then calculated. The final individual abundance of the OIP for each OIE is the individual ideal experimental abundance multiplied by 1 + RD. Initial studies were performed using higher-energy collisional dissociation tandem mass spectra on myoglobin (with direct infusion) and the intact E. coli proteome (with liquid chromatographic separation). Comprehensive data at the protein and proteome levels, high confidence and good reproducibility were achieved. The resolving method reported here can, in principle, be extended to resolve any envelope-type overlapping data for which the corresponding theoretical reference values are available. PMID:26439836

  11. Building and Searching Tandem Mass Spectral Libraries for Peptide Identification*

    PubMed Central

    Lam, Henry

    2011-01-01

    Spectral library searching is an emerging approach in peptide identifications from tandem mass spectra, a critical step in proteomic data analysis. Conceptually, the premise of this approach is that the tandem MS fragmentation pattern of a peptide under some fixed conditions is a reproducible fingerprint of that peptide, such that unknown spectra acquired under the same conditions can be identified by spectral matching. In actual practice, a spectral library is first meticulously compiled from a large collection of previously observed and identified tandem MS spectra, usually obtained from shotgun proteomics experiments of complex mixtures. Then, a query spectrum is then identified by spectral matching using recently developed spectral search engines. This review discusses the basic principles of the two pillars of this approach: spectral library construction, and spectral library searching. An overview of the software tools available for these two tasks, as well as a high-level description of the underlying algorithms, will be given. Finally, several new methods that utilize spectral libraries for peptide identification in ways other than straightforward spectral matching will also be described. PMID:21900153

  12. Characterization of Wax Esters by Electrospray Ionization Tandem Mass Spectrometry: Double Bond Effect and Unusual Product Ions.

    PubMed

    Chen, Jianzhong; Green, Kari B; Nichols, Kelly K

    2015-08-01

    A series of different types of wax esters (represented by RCOOR') were systematically studied by using electrospray ionization (ESI) collision-induced dissociation tandem mass spectrometry (MS/MS) along with pseudo MS(3) (in-source dissociation combined with MS/MS) on a quadrupole time-of-flight (Q-TOF) mass spectrometer. The tandem mass spectra patterns resulting from dissociation of ammonium/proton adducts of these wax esters were influenced by the wax ester type and the collision energy applied. The product ions [RCOOH2](+), [RCO](+) and [RCO-H2O](+) that have been reported previously were detected; however, different primary product ions were demonstrated for the three wax ester types including: (1) [RCOOH2](+) for saturated wax esters, (2) [RCOOH2](+), [RCO](+) and [RCO-H2O](+) for unsaturated wax esters containing only one double bond in the fatty acid moiety or with one additional double bond in the fatty alcohol moiety, and (3) [RCOOH2](+) and [RCO](+) for unsaturated wax esters containing a double bond in the fatty alcohol moiety alone. Other fragments included [R'](+) and several series of product ions for all types of wax esters. Interestingly, unusual product ions were detected, such as neutral molecule (including water, methanol and ammonia) adducts of [RCOOH2](+) ions for all types of wax esters and [R'-2H](+) ions for unsaturated fatty acyl-containing wax esters. The patterns of tandem mass spectra for different types of wax esters will inform future identification and quantification approaches of wax esters in biological samples as supported by a preliminary study of quantification of isomeric wax esters in human meibomian gland secretions.

  13. Characterization of Wax Esters by Electrospray Ionization Tandem Mass Spectrometry: Double Bond Effect and Unusual Product Ions

    PubMed Central

    Chen, Jianzhong; Green, Kari B; Nichols, Kelly K

    2015-01-01

    A series of different types of wax esters (represented by RCOOR′) were systematically studied by using electrospray ionization (ESI) collision-induced dissociation tandem mass spectrometry (MS/MS) along with pseudo MS3 (in-source dissociation combined with MS/MS) on a quadrupole time-of-flight (Q-TOF) mass spectrometer. The tandem mass spectra patterns resulting from dissociation of ammonium/proton adducts of these wax esters were influenced by the wax ester type and the collision energy applied. The product ions [RCOOH2]+, [RCO]+ and [RCO – H2O]+ that have been reported previously were detected; however, different primary product ions were demonstrated for the three wax ester types including: 1) [RCOOH2]+ for saturated wax esters, 2) [RCOOH2]+, [RCO]+ and [RCO – H2O]+ for unsaturated wax esters containing only one double bond in the fatty acid moiety or with one additional double bond in the fatty alcohol moiety, and 3) [RCOOH2]+ and [RCO]+ for unsaturated wax esters containing a double bond in the fatty alcohol moiety alone. Other fragments included [R′]+ and several series of product ions for all types of wax esters. Interestingly, unusual product ions were detected, such as neutral molecule (including water, methanol and ammonia) adducts of [RCOOH2]+ ions for all types of wax esters and [R′ – 2H]+ ions for unsaturated fatty acyl-containing wax esters. The patterns of tandem mass spectra for different types of wax esters will inform future identification and quantification approaches of wax esters in biological samples as supported by a preliminary study of quantification of isomeric wax esters in human meibomian gland secretions. PMID:26178197

  14. Tandem mass spectrometry of poly(methacrylic Acid) oligomers produced by negative mode electrospray ionization.

    PubMed

    Giordanengo, Rémi; Viel, Stéphane; Allard-Breton, Béatrice; Thévand, André; Charles, Laurence

    2009-01-01

    Dissociation of small poly(methyl acrylic acid) (PMAA) anions produced by electrospray was characterized by tandem mass spectrometry. Upon collisional activation, singly, and doubly deprotonated PMAA oligomers were shown to fragment via two major reactions, dehydration and decarboxylation. The elimination of a water molecule would occur between two consecutive acid groups in a charged-remote mechanism, giving rise to cyclic anhydrides, and was shown to proceed as many times as pairs of neutral pendant groups were available. As a result, the number of dehydration steps, together with the abundance of the fragment ions produced after the release of all water molecules, revealed the polymerization degree of the molecule in the particular case of doubly charged oligomers. For singly deprotonated molecules, the exact number of MAA units could be reached from the number of carbon dioxide molecules successively eliminated from the fully dehydrated precursor ions. In contrast to dehydration, decarboxylation reactions would proceed via a charge-induced mechanism. The proposed dissociation mechanisms are consistent with results commonly reported in thermal degradation studies of poly(acrylic acid) resins and were supported by accurate mass measurements. These fragmentation rules were successfully applied to characterize a polymeric impurity detected in the tested PMAA sample.

  15. Does deamidation cause protein unfolding? A top-down tandem mass spectrometry study

    PubMed Central

    Soulby, Andrew J; Heal, Jack W; Barrow, Mark P; Roemer, Rudolf A; O'Connor, Peter B

    2015-01-01

    Deamidation is a nonenzymatic post-translational modification of asparagine to aspartic acid or glutamine to glutamic acid, converting an uncharged amino acid to a negatively charged residue. It is plausible that deamidation of asparagine and glutamine residues would result in disruption of a proteins' hydrogen bonding network and thus lead to protein unfolding. To test this hypothesis Calmodulin and B2M were deamidated and analyzed using tandem mass spectrometry on a Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS). The gas phase hydrogen bonding networks of deamidated and nondeamidated protein isoforms were probed by varying the infra-red multi-photon dissociation laser power in a linear fashion and plotting the resulting electron capture dissociation fragment intensities as a melting curve at each amino acid residue. Analysis of the unfolding maps highlighted increased fragmentation at lower laser powers localized around heavily deamidated regions of the proteins. In addition fragment intensities were decreased across the rest of the proteins which we propose is because of the formation of salt-bridges strengthening the intramolecular interactions of the central regions. These results were supported by a computational flexibility analysis of the mutant and unmodified proteins, which would suggest that deamidation can affect the global structure of a protein via modification of the hydrogen bonding network near the deamidation site and that top down FTICR-MS is an appropriate technique for studying protein folding. PMID:25653127

  16. Polyatomic ion/surface collisions: new methodology in tandem mass spectrometry

    SciTech Connect

    Mabud, M.A.

    1987-01-01

    The excitation of a gas phase ion to induce fragmentation is an important aspect of tandem mass spectrometry. Investigations have been made by using metal surfaces as collision partners to activate polyatomic ions by using ions of 20-150 eV kinetic energy. Among the phenomena investigated are dissociation of the polyatomic ions upon collision with metal surfaces. The extent of dissociation can be controlled by selection of the impact energy. Collision with a surface gives rise to a narrower range of internal energies than do the corresponding ion/gaseous target collisions. Very large amounts of energy can be deposited in polyatomic ions upon collision with a metal target. Even at modest laboratory kinetic energies, the average internal energy deposited in ion/surface collisions exceeds that in gaseous collisions. Charge-exchange of multiply-charged species at the surface also occurs. Although simple charge exchange is observed, dissociative charge exchange is dominant in the cases studied. Dissociation and charge exchange of polyatomic ions in ion/surface collision are also accompanied by reactive collisions between organic ions and gas-covered metal surfaces. Utility of polyatomic ion/surface interaction technique for isomeric ion characterization has also been investigated. The ion/surface interaction technique appears to have excellent ability to distinguish isomeric ions. One advantage of SID is isomeric ion characterization lies in the fact that reactive collisions occur simultaneously and add a new dimension of information to the daughter spectra recorded. The hydrogen and methyl radical abstraction reactions assist in distinguishing closely related isomeric ions.

  17. Cloud parallel processing of tandem mass spectrometry based proteomics data.

    PubMed

    Mohammed, Yassene; Mostovenko, Ekaterina; Henneman, Alex A; Marissen, Rob J; Deelder, André M; Palmblad, Magnus

    2012-10-05

    Data analysis in mass spectrometry based proteomics struggles to keep pace with the advances in instrumentation and the increasing rate of data acquisition. Analyzing this data involves multiple steps requiring diverse software, using different algorithms and data formats. Speed and performance of the mass spectral search engines are continuously improving, although not necessarily as needed to face the challenges of acquired big data. Improving and parallelizing the search algorithms is one possibility; data decomposition presents another, simpler strategy for introducing parallelism. We describe a general method for parallelizing identification of tandem mass spectra using data decomposition that keeps the search engine intact and wraps the parallelization around it. We introduce two algorithms for decomposing mzXML files and recomposing resulting pepXML files. This makes the approach applicable to different search engines, including those relying on sequence databases and those searching spectral libraries. We use cloud computing to deliver the computational power and scientific workflow engines to interface and automate the different processing steps. We show how to leverage these technologies to achieve faster data analysis in proteomics and present three scientific workflows for parallel database as well as spectral library search using our data decomposition programs, X!Tandem and SpectraST.

  18. Resonance activation and collision-induced-dissociation of ions using rectangular wave dipolar potentials in a digital ion trap mass spectrometer.

    PubMed

    Xu, Fuxing; Wang, Liang; Dai, Xinhua; Fang, Xiang; Ding, Chuan-Fan

    2014-04-01

    Collision-induced dissociation (CID) of ions by resonance activation in a quadrupole ion trap is usually accomplished by resonance exciting the ions to higher kinetic energy, whereby the high kinetic energy ions collide with a bath gas, such as helium or argon, inside the trap and dissociate to fragments. A new ion activation method using a well-defined rectangular wave dipolar potential formed by dividing down the trapping rectangular waveform is developed and examined herein. The mass-selected parent ions are resonance excited to high kinetic energies by simply changing the frequency of the rectangular wave dipolar potential and dissociation proceeds. A relationship between the ion mass and the activation waveform frequency is also identified and described. This highly efficient (CID) procedure can be realized by simply changing the waveform frequency of the dipolar potential, which could certainly simplify tandem mass spectrometry analysis methods.

  19. Non-Target Screening of Veterinary Drugs Using Tandem Mass Spectrometry on SmartMass

    NASA Astrophysics Data System (ADS)

    Xia, Bing; Liu, Xin; Gu, Yu-Cheng; Zhang, Zhao-Hui; Wang, Hai-Yan; Ding, Li-Sheng; Zhou, Yan

    2013-05-01

    Non-target screening of veterinary drugs using tandem mass spectrometric data was performed on the SmartMass platform. This newly developed software uses the characteristic fragmentation patterns (CFP) to identify chemicals, especially those containing particular substructures. A mixture of 17 sulfonamides was separated by ultra performance liquid chromatography (UPLC), and SmartMass was used to process the tandem mass spectrometry (MS/MS) data acquired on an Orbitrap mass spectrometer. The data were automatically extracted, and each sulfonamide was recognized and analyzed with a prebuilt analysis rule. By using this software, over 98 % of the false candidate structures were eliminated, and all the correct structures were found within the top 10 of the ranking lists. Furthermore, SmartMass could also be used to identify slightly modified contraband drugs and metabolites with simple prebuilt rules. [Figure not available: see fulltext.

  20. Implementation of Ion/Ion Reactions in a Quadrupole/Time-of-Flight Tandem Mass Spectrometer

    PubMed Central

    Xia, Yu; Chrisman, Paul A.; Erickson, David E.; Liu, Jian; Liang, Xiaorong; Londry, Frank A.; Yang, Min J.; McLuckey, Scott A.

    2008-01-01

    A commercial quadrupole/time-of-flight (QqTOF) tandem mass spectrometer has been adapted for ion/ion reaction studies. To enable mutual storage of oppositely charged ions in a linear ion trap, the oscillating quadrupole field of the second quadrupole of the system (Q2) serves to store ions in the radial dimension while auxiliary RF is superposed on the end lenses of Q2 during the reaction period to create barriers in the axial dimension. A pulsed dual electrospray (ESI) source is directly coupled to the instrument interface for the purpose of proton transfer reactions. Singly and doubly charged protein ions as high in mass as 66 kDa are readily formed and observed after proton transfer reactions. For the modified instrument, the mass resolving power is about 8000 for a wide m/z range and the mass accuracy is ~20 ppm for external calibration and ~5 ppm for internal calibration after ion/ion reactions. Parallel ion parking is demonstrated with a six-component protein mixture, which shows the potential application of reducing spectral complexity and concentrating certain charge states. The current system has high flexibility with respect to defining MSn experiments involving collision-induced dissociation (CID) and ion/ion reactions. Protein precursor and CID product masses can be determined with good accuracy, providing an attractive platform for top-down proteomics. Electron transfer dissociation (ETD) ion/ion reactions are implemented by using a pulsed nano-ESI/atmospheric pressure chemical ionization (APCI) dual source for ionization. The reaction between protonated peptide ions and radical anions of 1,3-dinitrobenzene formed exclusively c- and z- type fragment ions. PMID:16771545

  1. Ion/Neutral, Ion/Electron, Ion/Photon, and Ion/Ion Interactions in Tandem Mass Spectrometry: Do we need them all? Are they enough?

    PubMed Central

    McLuckey, Scott A.; Mentinova, Marija

    2011-01-01

    A range of strategies and tools has been developed to facilitate the determination of primary structures of analyte molecules of interest via tandem mass spectrometry (MS/MS). The two main factors that determine the primary structural information present in an MS/MS spectrum are the type of ion generated from the analyte molecule and the dissociation method. The ion-type subjected to dissociation is determined by the ionization method/conditions and ion transformation processes that might take place after initial gas-phase ion formation. Furthermore, the range of analyte-related ion types can be expanded via derivatization reactions prior to mass spectrometry. Dissociation methods include those that simply alter the population of internal states of the mass-selected ion (i.e., activation methods like collision-induced dissociation) as well as processes that rely on transformation of the ion-type prior to dissociation (e.g., electron capture dissociation). A variety of ionic interactions has been studied for the purpose of ion dissociation and ion transformation that include ion/neutral, ion/photon, ion/electron, and ion/ion interactions. A wide range of phenomena has been observed, many of which have been explored/developed as means for structural analysis. The techniques arising from these phenomena are discussed within the context of the elements of structure determination in tandem mass spectrometry, viz., ion-type definition and dissociation. Unique aspects of the various ion interactions are emphasized along with any barriers to widespread implementation. PMID:21472539

  2. Quantitative Comparison of Tandem Mass Spectra Obtained on Various Instruments

    NASA Astrophysics Data System (ADS)

    Bazsó, Fanni Laura; Ozohanics, Oliver; Schlosser, Gitta; Ludányi, Krisztina; Vékey, Károly; Drahos, László

    2016-08-01

    The similarity between two tandem mass spectra, which were measured on different instruments, was compared quantitatively using the similarity index (SI), defined as the dot product of the square root of peak intensities in the respective spectra. This function was found to be useful for comparing energy-dependent tandem mass spectra obtained on various instruments. Spectral comparisons show the similarity index in a 2D "heat map", indicating which collision energy combinations result in similar spectra, and how good this agreement is. The results and methodology can be used in the pharma industry to design experiments and equipment well suited for good reproducibility. We suggest that to get good long-term reproducibility, it is best to adjust the collision energy to yield a spectrum very similar to a reference spectrum. It is likely to yield better results than using the same tuning file, which, for example, does not take into account that contamination of the ion source due to extended use may influence instrument tuning. The methodology may be used to characterize energy dependence on various instrument types, to optimize instrumentation, and to study the influence or correlation between various experimental parameters.

  3. Structural Characterization of Native Proteins and Protein Complexes by Electron Ionization Dissociation-Mass Spectrometry.

    PubMed

    Li, Huilin; Sheng, Yuewei; McGee, William; Cammarata, Michael; Holden, Dustin; Loo, Joseph A

    2017-03-07

    Mass spectrometry (MS) has played an increasingly important role in the identification and structural and functional characterization of proteins. In particular, the use of tandem mass spectrometry has afforded one of the most versatile methods to acquire structural information for proteins and protein complexes. The unique nature of electron capture dissociation (ECD) for cleaving protein backbone bonds while preserving noncovalent interactions has made it especially suitable for the study of native protein structures. However, the intra- and intermolecular interactions stabilized by hydrogen bonds and salt bridges can hinder the separation of fragments even with preactivation, which has become particularly problematic for the study of large macromolecular proteins and protein complexes. Here, we describe the capabilities of another activation method, 30 eV electron ionization dissociation (EID), for the top-down MS characterization of native protein-ligand and protein-protein complexes. Rich structural information that cannot be delivered by ECD can be generated by EID. EID allowed for the comparison of the gas-phase and the solution-phase structural stability and unfolding process of human carbonic anhydrase I (HCA-I). In addition, the EID fragmentation patterns reflect the structural similarities and differences among apo-, Zn-, and Cu,Zn-superoxide dismutase (SOD1) dimers. In particular, the structural changes due to Cu-binding and a point mutation (G41D) were revealed by EID-MS. The performance of EID was also compared to that of 193 nm ultraviolet photodissociation (UVPD), which allowed us to explore their qualitative similarities and differences as potential valuable tools for the MS study of native proteins and protein complexes.

  4. Sequencing of Oligourea Foldamers by Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Bathany, Katell; Owens, Neil W.; Guichard, Gilles; Schmitter, Jean-Marie

    2013-03-01

    This study is focused on sequence analysis of peptidomimetic helical oligoureas by means of tandem mass spectrometry, to build a basis for de novo sequencing for future high-throughput combinatorial library screening of oligourea foldamers. After the evaluation of MS/MS spectra obtained for model compounds with either MALDI or ESI sources, we found that the MALDI-TOF-TOF instrument gave more satisfactory results. MS/MS spectra of oligoureas generated by decay of singly charged precursor ions show major ion series corresponding to fragmentation across both CO-NH and N'H-CO urea bonds. Oligourea backbones fragment to produce a pattern of a, x, b, and y type fragment ions. De novo decoding of spectral information is facilitated by the occurrence of low mass reporter ions, representative of constitutive monomers, in an analogous manner to the use of immonium ions for peptide sequencing.

  5. Xylose Migration During Tandem Mass Spectrometry of N-Linked Glycans

    NASA Astrophysics Data System (ADS)

    Hecht, Elizabeth S.; Loziuk, Philip L.; Muddiman, David C.

    2017-01-01

    Understanding the rearrangement of gas-phase ions via tandem mass spectrometry is critical to improving manual and automated interpretation of complex datasets. N-glycan analysis may be carried out under collision induced (CID) or higher energy collision dissociation (HCD), which favors cleavage at the glycosidic bond. However, fucose migration has been observed in tandem MS, leading to the formation of new bonds over four saccharide units away. In the following work, we report the second instance of saccharide migration ever to occur for N-glycans. Using horseradish peroxidase as a standard, the beta-1,2 xylose was observed to migrate from a hexose to a glucosamine residue on the (Xyl)Man3GlcNac2 glycan. This investigation was followed up in a complex N-linked glycan mixture derived from stem differentiating xylem tissue, and the rearranged product ion was observed for 75% of the glycans. Rearrangement was not favored in isomeric glycans with a core or antennae fucose and unobserved in glycans predicted to have a permanent core-fucose modification. As the first empirical observation of this rearrangement, this work warrants dissemination so it may be searched in de novo sequencing glycan workflows.

  6. Automated Glycan Sequencing from Tandem Mass Spectra of N-Linked Glycopeptides.

    PubMed

    Yu, Chuan-Yih; Mayampurath, Anoop; Zhu, Rui; Zacharias, Lauren; Song, Ehwang; Wang, Lei; Mechref, Yehia; Tang, Haixu

    2016-06-07

    Mass spectrometry has become a routine experimental tool for proteomic biomarker analysis of human blood samples, partly due to the large availability of informatics tools. As one of the most common protein post-translational modifications (PTMs) in mammals, protein glycosylation has been observed to alter in multiple human diseases and thus may potentially be candidate markers of disease progression. While mass spectrometry instrumentation has seen advancements in capabilities, discovering glycosylation-related markers using existing software is currently not straightforward. Complete characterization of protein glycosylation requires the identification of intact glycopeptides in samples, including identification of the modification site as well as the structure of the attached glycans. In this paper, we present GlycoSeq, an open-source software tool that implements a heuristic iterated glycan sequencing algorithm coupled with prior knowledge for automated elucidation of the glycan structure within a glycopeptide from its collision-induced dissociation tandem mass spectrum. GlycoSeq employs rules of glycosidic linkage as defined by glycan synthetic pathways to eliminate improbable glycan structures and build reasonable glycan trees. We tested the tool on two sets of tandem mass spectra of N-linked glycopeptides cell lines acquired from breast cancer patients. After employing enzymatic specificity within the N-linked glycan synthetic pathway, the sequencing results of GlycoSeq were highly consistent with the manually curated glycan structures. Hence, GlycoSeq is ready to be used for the characterization of glycan structures in glycopeptides from MS/MS analysis. GlycoSeq is released as open source software at https://github.com/chpaul/GlycoSeq/ .

  7. Electron-Induced Dissociation of Peptides in a Triple Quadrupole Mass Spectrometer Retrofitted with an Electromagnetostatic Cell

    NASA Astrophysics Data System (ADS)

    Voinov, Valery G.; Bennett, Samuel E.; Barofsky, Douglas F.

    2015-05-01

    Dissociation of peptides induced by interaction with (free) electrons (electron-induced dissociation, EID) at electron energies ranging from near 0 to >30 eV was carried out using a radio-frequency-free electromagnetostatic (EMS) cell retrofitted into a triple quadrupole mass spectrometer. The product-ion mass spectra exhibited EID originating from electronically excited even-electron precursor ions, reduced radical cations formed by capture of low-energy electrons, and oxidized radical cations produced by interaction with high-energy electrons. The spectra demonstrate, within the limits of the triple quadrupole's resolving power, that high-energy EID product-ion spectra produced with an EMS cell exhibit essentially the same qualitative structural information, i.e., amino acid side-chain (SC) losses and backbone cleavages, as observed in high-energy EID spectra produced with a Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer. The levels of fragmentation efficiency evident in the product-ion spectra recorded in this study, as was the case for those recorded in earlier studies with FT ICR mass spectrometers, is currently at the margin of analytical utility. Given that this shortcoming can be remedied, EMS cells incorporated into QqQ or QqTOF mass spectrometers could make tandem high-energy EID mass spectrometry more widely accessible for analysis of peptides, small singly charged molecules, pharmaceuticals, and clinical samples.

  8. Top-down proteomics with a quadrupole time-of-flight mass spectrometer and collision-induced dissociation.

    PubMed

    Armirotti, Andrea; Benatti, Umberto; Damonte, Gianluca

    2009-03-01

    With slight modifications of the instrumental parameters, we demonstrate that satisfactory top-down data can be obtained with collision-induced dissociation (CID) tandem mass spectrometry on a quadrupole time-of-flight (qTOF) instrument not originally designed for this purpose. Protein identification is achieved with both N- and C-terminal sequence tags and BLAST database searches. The accurate mass measurement of multiply charged fragment ions (mostly y and b-type) supplements the limited set of cleavage sites and provides a high degree of sequence coverage (90-100%). Post-translational modification issues can be addressed too. This approach might help those mass spectrometry (MS) core facilities that are not able to afford very high-resolution instruments, thus expanding the benefits of top-down protein analysis over the worldwide MS community.

  9. Liquid chromatography tandem mass spectrometry in the clinical laboratory.

    PubMed

    Adaway, Joanne E; Keevil, Brian G; Owen, Laura J

    2015-01-01

    Clinical laboratory medicine has seen the introduction and evolution of liquid chromatography tandem mass spectrometry in routine clinical laboratories over the last 10-15 years. There still exists a wide diversity of assays from very esoteric and highly specialist manual assays to more simplified kit-based assays. The technology is not static as manufacturers are continually making improvements. Mass spectrometry is now commonly used in several areas of diagnostics including therapeutic drug monitoring, toxicology, endocrinology, paediatrics and microbiology. Some of the most high throughput analyses or common analytes include vitamin D, immunosuppressant monitoring, androgen measurement and newborn screening. It also offers flexibility for the measurement of analytes in a variety of different matrices which would prove difficult with immunoassays. Unlike immunoassays or high-pressure liquid chromatography assays using ultraviolet or fluorescence detection, mass spectrometry offers better specificity and reduced interferences if attention is paid to potential isobaric compounds. Furthermore, multiplexing, which enables multiple analytes to be measured with the same volume of serum is advantageous, and the requirement for large sample volumes is decreasing as instrument sensitivity increases. There are many emerging applications in the literature. Using mass spectrometry to identify novel isoforms or modified peptides is possible as is quantification of proteins and peptides, with or without protein digests. Future developments by the manufacturers may also include mechanisms to improve the throughput of samples and strategies to decrease the level of skill required by the operators.

  10. A study of resonance electron capture ionization on a quadrupole tandem mass spectrometer.

    PubMed

    Wei, J; Liu, S; Fedoreyev, S A; Voinov, V G

    2000-01-01

    Procedures that allow the realization of resonance electron capture (REC) mode on a commercial triple-quadrupole mass spectrometer, after some simple modifications, are described. REC mass spectrometry (MS) and tandem mass spectrometry (MS/MS) experiments were performed and spectra for some compounds were recorded. In particular, the charge-remote fragmentation (CRF) spectra of [M - H](-) ions of docosanoic and docosenoic acids under low-energy collisionally activated dissociation (CAD) conditions were obtained, and showed that there were no significant differences for [M - H](-) ions produced at different resonances (i.e. for [M - H](-) ions with different structures). This observation was explained on the basis of results obtained from deuterium-labeled fatty acids, which showed that different CRF ions (but with the same m/z value in the absence of labels) could be produced by different mechanisms, and all of them were obviously realized under CAD conditions that made spectra practically indistinguishable. The other example, which compared the REC-MS/MS spectrum of [M - H](-) ions and EI-MS/MS spectrum of M(+.) ions of daidzein, demonstrated the potential of the REC-MS/MS technique for more complex structure elucidation.

  11. Macromolecule Mass Spectrometry: Citation Mining of User Documents

    DTIC Science & Technology

    2006-05-31

    collision - induced dissociation in quadrupole ion trap coupled tandem mass spectrometry. Ion mass spectra analysis of the resulting fragments determines ...Factor 2 ( DISSOCIATION , FRAGMENTS , CID, COLLISIONS , PRECURSOR, TANDEM, MS/MS, ENERGY , CLEAVAGES, IONS , SPECTRA, QUADRAPOLE, TRAP, PATTERNS, PROTONATION...focuses on the use of post- electrospray ionization collision - induced dissociation of macromolecules coupled

  12. Characterisation of poly(alkyl methacrylate)s by means of electrospray ionisation-tandem mass spectrometry (ESI-MS/MS)

    NASA Astrophysics Data System (ADS)

    Jackson, Anthony T.; Slade, Susan E.; Scrivens, James H.

    2004-11-01

    Electrospray ionisation-tandem mass spectrometry (ESI-MS/MS) has been employed for the characterisation of two poly(alkyl methacrylate) polymers, namely poly(methyl methacrylate) (PMMA) and poly(n-butyl methacrylate) (PBMA). Collision-induced dissociation (CID) experiments were performed in a quadrupole orthogonal time-of-flight (ToF) tandem mass spectrometer fitted with a nanospray source. Tandem mass spectra from singly, doubly and triply charged precursor ions (with alkali metals used for cationisation of the oligomers) are shown and the data are compared to those previously generated by means of matrix-assisted laser desorption/ionisation-collision-induced dissociation (MALDI-CID). These data indicate that cations with greater ionic radii may yield the most useful structural information as the mass-to-charge ratio of the precursor ion increases, whereas lithium or sodium ions are proposed to be ideal for obtaining spectra from lower molecular weight oligomers. Fragment ions at low mass-to-charge ratios dominate the spectra. Two series of peaks may be used to calculate the masses of the initiating and terminating end groups of the polymer. Ion peaks of greater mass-to-charge ratios form series that may be used to infer sequence information from the polymers.

  13. Simulation of Two Dimensional Electrophoresis and Tandem Mass Spectrometry for Teaching Proteomics

    ERIC Educational Resources Information Center

    Fisher, Amanda; Sekera, Emily; Payne, Jill; Craig, Paul

    2012-01-01

    In proteomics, complex mixtures of proteins are separated (usually by chromatography or electrophoresis) and identified by mass spectrometry. We have created 2DE Tandem MS, a computer program designed for use in the biochemistry, proteomics, or bioinformatics classroom. It contains two simulations--2D electrophoresis and tandem mass spectrometry.…

  14. An unsupervised machine learning method for assessing quality of tandem mass spectra

    PubMed Central

    2012-01-01

    Background In a single proteomic project, tandem mass spectrometers can produce hundreds of millions of tandem mass spectra. However, majority of tandem mass spectra are of poor quality, it wastes time to search them for peptides. Therefore, the quality assessment (before database search) is very useful in the pipeline of protein identification via tandem mass spectra, especially on the reduction of searching time and the decrease of false identifications. Most existing methods for quality assessment are supervised machine learning methods based on a number of features which describe the quality of tandem mass spectra. These methods need the training datasets with knowing the quality of all spectra, which are usually unavailable for the new datasets. Results This study proposes an unsupervised machine learning method for quality assessment of tandem mass spectra without any training dataset. This proposed method estimates the conditional probabilities of spectra being high quality from the quality assessments based on individual features. The probabilities are estimated through a constraint optimization problem. An efficient algorithm is developed to solve the constraint optimization problem and is proved to be convergent. Experimental results on two datasets illustrate that if we search only tandem spectra with the high quality determined by the proposed method, we can save about 56 % and 62% of database searching time while losing only a small amount of high-quality spectra. Conclusions Results indicate that the proposed method has a good performance for the quality assessment of tandem mass spectra and the way we estimate the conditional probabilities is effective. PMID:22759570

  15. Improved Isobaric Tandem Mass Tag Quantification by Ion Mobility-Mass Spectrometry

    PubMed Central

    Li, Lingjun

    2014-01-01

    Isobaric tandem mass tags are an attractive alternative to mass difference tags and label free approaches for quantitative proteomics due to the high degree of multiplexing that can be performed with their implementation. A drawback of tandem mass tags are that the co-isolation and co-fragmentation of labeled peptide precursors can result in chimeric MS/MS spectra that can underestimate the fold-change expression of each peptide. Two methods (QuantMode and MS3) have addressed this concern for ion trap and orbitrap instruments, but there is still a need to solve this problem for quadrupole time-of-flight (Q-TOF) instruments. Ion mobility (IM) separations coupled to Q-TOF instruments have the potential to mitigate MS/MS spectra chimeracy since IM-MS has the ability to separate ions based on charge, m/z, and collision cross section (CCS). This work presents results that showcase the power of IM-MS to improve tandem mass tag peptide quantitation accuracy by resolving co-isolated differently charged and same charged peptides prior to MS/MS fragmentation. PMID:24677527

  16. Probabilistic consensus scoring improves tandem mass spectrometry peptide identification.

    PubMed

    Nahnsen, Sven; Bertsch, Andreas; Rahnenführer, Jörg; Nordheim, Alfred; Kohlbacher, Oliver

    2011-08-05

    Database search is a standard technique for identifying peptides from their tandem mass spectra. To increase the number of correctly identified peptides, we suggest a probabilistic framework that allows the combination of scores from different search engines into a joint consensus score. Central to the approach is a novel method to estimate scores for peptides not found by an individual search engine. This approach allows the estimation of p-values for each candidate peptide and their combination across all search engines. The consensus approach works better than any single search engine across all different instrument types considered in this study. Improvements vary strongly from platform to platform and from search engine to search engine. Compared to the industry standard MASCOT, our approach can identify up to 60% more peptides. The software for consensus predictions is implemented in C++ as part of OpenMS, a software framework for mass spectrometry. The source code is available in the current development version of OpenMS and can easily be used as a command line application or via a graphical pipeline designer TOPPAS.

  17. Identification of degradation products of indigoids by tandem mass spectrometry.

    PubMed

    Witkoś, Katarzyna; Lech, Katarzyna; Jarosz, Maciej

    2015-11-01

    The study concerns identification of photodegradation products of indigotin, indirubin and isoindigo. Experimental methodology consists of degradation of standard solutions of indigoids in a solar box and analysis of samples taken at different aging time by using capillary high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometric and spectrophotometric detectors. Identification of the formed compounds was based on careful interpretation of the electrospray ionization MS/MS spectra. Apart from the well-known degradation products of indigoids: isatin, isatoic anhydride and anthranilic acid, another seven species were also identified, and their proposed structures were confirmed by high-resolution molecular masses measurements; according to the best knowledge of authors, they have not been reported so far. The obtained results formed the basis for postulating mechanism of the process. Moreover, the MRM (Multiple Reaction Monitoring) method was developed for the identification of natural dyes and their degradation products in textiles of historical value. Apart from such colorants as indigotin and flavonoids, also presence of degradation products of indigoids was confirmed.

  18. Tandem mass spectrometry of peptides using hybrid and four-sector instruments: a comparative study.

    PubMed

    Bean, M F; Carr, S A; Thorne, G C; Reilly, M H; Gaskell, S J

    1991-07-15

    Product-ion spectra produced by high- and low-energy collisionally activated dissociation (CAD) of [M + H]+ ions of a series of peptides (Mr 550-2500) have been compared on four-sector and hybrid tandem mass spectrometers, respectively. The fast atom bombardment product-ion spectra obtained for the smallest peptide analyzed (methionine-enkephalin) were remarkably similar, but substantial differences in fragmentation were observed for the heavier analytes. For peptides with Mr greater than 1000, more complete sequence information was obtained from high-energy CAD on the four-sector instrument. Nevertheless, low-energy CAD on the hybrid mass spectrometer was able to produce partial sequence information even for the largest of the peptides compared. Limits of analysis, defined as the least quantities of analyte for which product-ion spectra of essentially uncompromised quality could be obtained, were similar (ca. 15 pmol) for small peptides, but lower limits were achieved for larger peptides (Mr greater than 1000) with the four-sector instrument. High-energy CAD spectra were found to be highly reproducible, with qualitatively similar spectra obtained over a wide range of operating conditions. In contrast, it was necessary to carefully control collision gas pressures and collision energies in order to obtain good reproducible data for low-energy CAD. Experimental procedures for obtaining reproducible spectra with good sensitivity for peptides on the hybrid instrument are presented.

  19. Electron Capture Dissociation of Sodium-Adducted Peptides on a Modified Quadrupole/Time-of-Flight Mass Spectrometer.

    PubMed

    Voinov, Valery G; Hoffman, Peter D; Bennett, Samuel E; Beckman, Joseph S; Barofsky, Douglas F

    2015-12-01

    Electron capture dissociation (ECD), which generally preserves the position of labile post-translational modifications, can be a powerful method for de novo sequencing of proteins and peptides. In this report, ECD product-ion mass spectra of singly and doubly sodiated, nonphosphorylated, and phosphorylated peptides are presented and compared with the ECD mass spectra of their protonated counterparts. ECD of doubly charged, singly sodiated peptides yielded essentially the same sequence information as was produced by the corresponding doubly protonated peptides. The presence of several sodium binding sites on the polypeptide backbone, however, resulted in more complicated spectra. This situation is aggravated by the zwitterionic equilibrium of the free acid peptide precursors. The product-ion spectra of doubly and triply charged peptides possessing two sodium ions were further complicated by the existence of isomers created by the differential distribution of sodium binding sites. Triply charged, phosphorylated precursors containing one sodium, wherein the sodium is attached exclusively to the PO4 group, were found to be as useful for sequence analysis as the fully protonated species. Although sodium adducts are generally minimized during sample preparation, it appears that they can nonetheless provide useful sequence information. Additionally, they enable straightforward identification of a peptide's charge state, even on low-resolution instruments. The experiments were carried out using a radio frequency-free electromagnetostatic cell retrofitted into the collision-induced dissociation (CID) section of a hybrid quadrupole/time-of-flight tandem mass spectrometer. Graphical Abstract ᅟ.

  20. Electron Capture Dissociation of Sodium-Adducted Peptides on a Modified Quadrupole/Time-of-Flight Mass Spectrometer

    NASA Astrophysics Data System (ADS)

    Voinov, Valery G.; Hoffman, Peter D.; Bennett, Samuel E.; Beckman, Joseph S.; Barofsky, Douglas F.

    2015-12-01

    Electron capture dissociation (ECD), which generally preserves the position of labile post-translational modifications, can be a powerful method for de novo sequencing of proteins and peptides. In this report, ECD product-ion mass spectra of singly and doubly sodiated, nonphosphorylated, and phosphorylated peptides are presented and compared with the ECD mass spectra of their protonated counterparts. ECD of doubly charged, singly sodiated peptides yielded essentially the same sequence information as was produced by the corresponding doubly protonated peptides. The presence of several sodium binding sites on the polypeptide backbone, however, resulted in more complicated spectra. This situation is aggravated by the zwitterionic equilibrium of the free acid peptide precursors. The product-ion spectra of doubly and triply charged peptides possessing two sodium ions were further complicated by the existence of isomers created by the differential distribution of sodium binding sites. Triply charged, phosphorylated precursors containing one sodium, wherein the sodium is attached exclusively to the PO4 group, were found to be as useful for sequence analysis as the fully protonated species. Although sodium adducts are generally minimized during sample preparation, it appears that they can nonetheless provide useful sequence information. Additionally, they enable straightforward identification of a peptide's charge state, even on low-resolution instruments. The experiments were carried out using a radio frequency-free electromagnetostatic cell retrofitted into the collision-induced dissociation (CID) section of a hybrid quadrupole/time-of-flight tandem mass spectrometer.

  1. Identifying drug metallation sites on peptides using electron transfer dissociation (ETD), collision induced dissociation (CID) and ion mobility-mass spectrometry (IM-MS).

    PubMed

    Williams, Jonathan P; Brown, Jeffery M; Campuzano, Iain; Sadler, Peter J

    2010-08-14

    Electron transfer dissociation (ETD) and collision induced dissociation (CID) have been used to locate the precise binding sites for platinum and ruthenium anticancer complexes on the peptide Substance P. We show that ETD combined with ion mobility-mass spectrometry significantly reduces mass spectral complexity and improves the S/N of the product-ions formed.

  2. Differentiation of lisinopril and its RSS diastereomer by liquid chromatography combined with collision-induced dissociation mass spectrometry.

    PubMed

    Sun, Cuirong; Zhu, Peixi; Hu, Nan; Wang, Danhua; Pan, Yuanjiang

    2010-01-01

    A simple and sensitive liquid chromatography tandem multiple-stage mass spectrometry (HPLC/MS/MS) method suitable for bulk lisinopril analysis was developed, by which lisinopril and its RSS isomer were separated and differentiated. In the collision-induced dissociation (CID) mass spectra of the [M + H](+) ions, the abundance of the fragment ion of m/z 246 for lisinopril was about two times higher than the ion of m/z 245; however, the former fragment ion was noted to be a little lower than the latter for RSS isomer at all collision energies. In the CID mass spectra of the [M + Li](+) ion, the abundance of the rearrangement ion of m/z 315 for the RSS isomer was about three times higher than that for lisinopril. Furthermore, the difference was supported by the results of energy-resolved mass spectrometry (ERMS) in the test range of collision energies. Similar differences were also observed between the CID mass spectra of lisinopril and RSS isomer methylester, which indicated that the RSS isomer could be rapidly characterized by the CID mass spectra of both the protonated and lithium adduct ion. Elemental compositions of all the ions were confirmed by Fourier Transform ion cyclotron resonance ESI mass spectrometry (FT-ICR-ESI/MS). In addition, theoretical computations were carried out to support the experimental results.

  3. Dual electrospray ion source for electron-transfer dissociation on a hybrid linear ion trap-orbitrap mass spectrometer.

    PubMed

    Williams, D Keith; McAlister, Graeme C; Good, David M; Coon, Joshua J; Muddiman, David C

    2007-10-15

    A dual electrospray ionization source (ESI) has been modified to simultaneously produce cations and anions, one from each emitter, for performing rapid electron-transfer dissociation (ETD) ion/ion reactions on a hybrid linear ion trap-orbitrap mass spectrometer. Unlike the pulsed dual ESI sources that were used to generate ETD reagent ions, this source separates the emitters in space, rather than time, by physically switching which one is in front of the atmospheric inlet. The new arrangement allows for substantially enhanced spray stability and decreased switching times (tandem-MS spectra per unit time. Herein, we demonstrate the stability of the ETD anion population and the ability to identify several c- and z-type product ions from multiply protonated peptide cations.

  4. 4-HNE adduct stability characterized by collision-induced dissociation and electron transfer dissociation mass spectrometry.

    PubMed

    Fritz, Kristofer S; Kellersberger, Katherine A; Gomez, Jose D; Petersen, Dennis R

    2012-04-16

    4-Hydroxynonenal (4-HNE) alters numerous proteomic and genomic processes. Understanding chemical mechanisms of 4-HNE interactions with biomolecules and their respective stabilities may lead to new discoveries in biomarkers for numerous diseases of oxidative stress. Collision-induced dissociation (CID) and electron transfer dissociation (ETD) MS/MS were utilized to examine the stability of a 4-HNE-Cys Michael adduct. CID conditions resulted in the neutral loss of 4-HNE, also known as a retro-Michael addition reaction (RMA). Consequently, performing ETD fragmentation on this same adduct did not result in RMA. Interestingly, 4-HNE adduct reduction via sodium borohydride (NaBH₄) treatment stabilized against the CID induced RMA. In a direct comparison of three forms of 4-HNE adducts, computational modeling revealed sizable shifts in the shape and orientation of the lowest unoccupied molecular orbital (LUMO) density around the 4-HNE-Cys moiety. These findings demonstrate that ETD MS/MS analysis can be used to improve the detection of 4-HNE-protein modifications by preventing RMA reactions from occurring.

  5. LESSONS IN DE NOVO PEPTIDE SEQUENCING BY TANDEM MASS SPECTROMETRY

    PubMed Central

    Medzihradszky, Katalin F.; Chalkley, Robert J.

    2015-01-01

    Mass spectrometry has become the method of choice for the qualitative and quantitative characterization of protein mixtures isolated from all kinds of living organisms. The raw data in these studies are MS/MS spectra, usually of peptides produced by proteolytic digestion of a protein. These spectra are “translated” into peptide sequences, normally with the help of various search engines. Data acquisition and interpretation have both been automated, and most researchers look only at the summary of the identifications without ever viewing the underlying raw data used for assignments. Automated analysis of data is essential due to the volume produced. However, being familiar with the finer intricacies of peptide fragmentation processes, and experiencing the difficulties of manual data interpretation allow a researcher to be able to more critically evaluate key results, particularly because there are many known rules of peptide fragmentation that are not incorporated into search engine scoring. Since the most commonly used MS/MS activation method is collision-induced dissociation (CID), in this article we present a brief review of the history of peptide CID analysis. Next, we provide a detailed tutorial on how to determine peptide sequences from CID data. Although the focus of the tutorial is de novo sequencing, the lessons learned and resources supplied are useful for data interpretation in general. PMID:25667941

  6. Positive mode electrospray tandem mass spectrometry of poly(methacrylic acid) oligomers.

    PubMed

    Giordanengo, Rémi; Viel, Stéphane; Allard-Breton, Béatrice; Thévand, André; Charles, Laurence

    2009-06-01

    The dissociation of small poly(methacrylic acid) (PMAA) cations produced by electrospray was characterized by tandem mass spectrometry. Similarly to PMAA ions produced in the negative ion mode, the two electrosprayed cationic forms, namely [PMAA+Na](+) and [PMAA-H+2Na](+), were shown to fragment via a major pathway consisting of successive dehydration steps. Elimination of a water molecule would occur between two consecutive acid groups in a charged-remote mechanism and was shown to proceed as many times as pairs of acidic pendant groups were available. As a result, comparing the number of dehydration steps observed in the MS/MS spectrum of two consecutive oligomers from the polymeric distribution reveals the degree of polymerization of the molecule. Secondary less informative reactions were shown to consist of losses of CO and/or CO(2), depending on the nature of the precursor ion. These fragmentation rules could be used to characterize PMAA-based copolymers, as successfully demonstrated for a polymeric impurity in the tested PMAA sample.

  7. Determination of steryl sulphates in invertebrate tissue by liquid chromatography-tandem mass spectrometry.

    PubMed

    Neto, Renato R; Thompson, Anu; Wolff, George A

    2005-11-01

    A method for the identification and quantification of underivatised steryl sulphates in invertebrates by liquid chromatography (LC) coupled with tandem mass spectrometry (MS) involving a single cleanup step has been developed. Negative electrospray ionisation and positive and negative atmospheric-pressure chemical ionisation (APCI) spectra of steryl sulphate showed pseudomolecular ions ([M+H-H2SO4]+ or [M-H]-). Collision-induced dissociation (CID) was efficient only in positive APCI. LC-MS in negative APCI was least susceptible to interference and possible differences in response factors. The detection limits (signal-to-noise ratio of 3) based on cholest-5-enyl-3-sulphate in positive and negative APCI modes are 3.66 and 0.73 pmol microL(-1), respectively. Calibration plots and response factors for cholest-5-enyl-3-sulphate relative to the internal standard, cholecalciferyl-3-sulphate, in both positive and negative polarities, were linear in the concentration range from 1.22 to 16.4 pmol microL(-1) with good coefficients of determination (R2 > 0.98). It is suggested that the structure elucidation of steryl sulphates is best achieved in CID positive APCI mode, whereas their quantification should be carried out using negative APCI.

  8. Tandem mass spectrometric analysis of a mixture of isobars using the survival yield technique.

    PubMed

    Memboeuf, Antony; Jullien, Laure; Lartia, Rémy; Brasme, Bernard; Gimbert, Yves

    2011-10-01

    Collision induced dissociation tandem mass spectrometry experiments were performed to unequivocally separate compounds from an isobaric mixture of two products. The Survival Yield curve was obtained and is shown to consist in a linear combination of the curves corresponding to the two components separately. For such a mixture, a plateau appears on the diagram in lieu of the continuous decrease expected allowing for the structural study of the two components separately. The width of the plateau critically relates to the fragmentation parameters of the two molecular ions, which need to be sufficiently different structurally for the plateau to be observed. However, at constant fragmentation parameters, we have observed the width significantly increases at large m/z. This makes the separation more and more efficient as isobars have larger m/z and the technique complementary to those applicable at low m/z only. We have observed that the vertical position of the plateau relates linearly to the relative concentration of the two compounds that may be useful for quantification. Repeatability was estimated at 2% on a quadrupole ion trap. An advantage of using survival yield curves only, is that a priori knowledge of the respective fragmentation patterns of the two isobars becomes unnecessary. Consequently, similar performances are obtained if fragments are isobaric, which is also demonstrated in our study. The critical case of reverse peptides, at low m/z and similar fragmentation parameters, is also presented as a limitation of the method.

  9. Comparison between Source-induced Dissociation and Collision-induced Dissociation of Ampicillin, Chloramphenicol, Ciprofloxacin, and Oxytetracycline via Mass Spectrometry.

    PubMed

    Lee, Seung Ha; Choi, Dal Woong

    2013-06-01

    Mass spectrometry (MS) is a very powerful instrument that can be used to analyze a wide range of materials such as proteins, peptides, DNA, drugs, and polymers. The process typically involves either chemical or electron (impact) ionization of the analyte. The resulting charged species or fragment is subsequently identified by the detector. Usually, single mass uses source-induced dissociation (SID), whereas mass/mass uses collision-induced dissociation (CID) to analyze the chemical fragmentations Each technique has its own advantages and disadvantages. While CID is most effective for the analysis of pure substances, multiple- step MS is a powerful technique to get structural data. Analysis of veterinary drugs ampicillin, chloramphenicol, ciprofloxacin, and oxytetracycline serves to highlight the slight differences between SID and CID. For example, minor differences were observed between ciprofloxacin and oxytetracycline via SID or CID. However, distinct fragmentation patterns were observed for ampicllin depending on the analysis method. Both SID and CID showed similar fragmentation spectra but different signal intensities for chloramphenicol. There are several factors that can influence the fragmentation spectra, such as the collision energy, major precursor ion, electrospray mode (positive or negative), and sample homogeneity. Therefore, one must select a fragmentation method on an empirical and case-by-case basis.

  10. MassMatrix: A Database Search Program for Rapid Characterization of Proteins and Peptides from Tandem Mass Spectrometry Data

    PubMed Central

    Xu, Hua; Freitas, Michael A.

    2009-01-01

    MassMatrix is a program that matches tandem mass spectra with theoretical peptide sequences derived from a protein database. The program uses a mass accuracy sensitive probabilistic score model to rank peptide matches. The tandem mass spectrometry search software was evaluated by use of a high mass accuracy data set and its results compared with those from Mascot, SEQUEST, X!Tandem, and OMSSA. For the high mass accuracy data, MassMatrix provided better sensitivity than Mascot, SEQUEST, X!Tandem, and OMSSA for a given specificity and the percentage of false positives was 2%. More importantly all manually validated true positives corresponded to a unique peptide/spectrum match. The presence of decoy sequence and additional variable post-translational modifications did not significantly affect the results from the high mass accuracy search. MassMatrix performs well when compared with Mascot, SEQUEST, X!Tandem, and OMSSA with regard to search time. MassMatrix was also run on a distributed memory clusters and achieved search speeds of ~100,000 spectra per hour when searching against a complete human database with 8 variable modifications. The algorithm is available for public searches at http://www.massmatrix.net. PMID:19235167

  11. Quantification of citalopram or escitalopram and their demethylated metabolites in neonatal hair samples by liquid chromatography-tandem mass spectrometry.

    PubMed

    Frison, Giampietro; Favretto, Donata; Vogliardi, Susanna; Terranova, Claudio; Ferrara, Santo Davide

    2008-08-01

    Citalopram and escitalopram are highly selective serotonin reuptake inhibitors widely used in the treatment of depression. They exhibit adverse drug reactions and side effects, however, and the development of specific methods for their determination is of great interest in clinical and forensic toxicology. A liquid chromatography-tandem mass spectrometry method has been developed and validated for the assay of citalopram, escitalopram, and their demethylated metabolites in 10-mg hair samples. The analytes were extracted by incubation in methanol and liquid/liquid extraction with diethyl ether/dichloromethane. Gradient elution on a narrow bore C18 column was realized using clomipramine-d3 as an internal standard. Positive ion electrospray ionization and tandem mass spectrometry determination by collision-induced dissociation were performed in an ion trap mass spectrometer. The method exhibited a linear range of 25 to 2000 pg/mg, a quantification limit of 25 pg/mg for all analytes, relative standard deviations in the range of 12.10 to 9.80 (intraassay), and 13.80 to 11.78 (interassay), and accuracies (as percent recovery of the spiked standards) in the range of 90% to 110%; it was applied to the determination of citalopram and escitalopram and their metabolites in hair samples of two newborns to document their in utero exposure to the drugs. The method proved suitable for neonatal hair analysis of citalopram or escitalopram and was applied to two real cases of gestational exposure.

  12. Highly specific quantification of ergotamine in urine, blood, and hair samples by liquid chromatography-tandem mass spectrometry.

    PubMed

    Favretto, Donata; Frison, Giampietro; Vogliardi, Susanna; Ferrara, Santo Davide

    2007-06-01

    Ergotamine has been used for therapeutic purposes since the 1950s, usually to treat vascular headache. It is highly toxic and in large, repeated doses can produce all the symptoms of ergot poisoning. A selective and sensitive method, based on liquid chromatography-tandem mass spectrometry (LC-MS2), has been developed for quantifying ergotamine in biological fluids with use of a quick and easy sample preparation. Ergotamine and the internal standard, trideuterated lysergic acid diethylamide, were extracted from human urine, blood, and hair by means of liquid-liquid extraction at alkaline pH. Gradient elution on a cyanopropyl column was used for chromatographic separation. Positive ion electrospray ionization and tandem mass spectrometry determination by collision-induced dissociation were performed in an ion trap mass spectrometer. The method was validated and successfully applied to a case of iatrogenic ergotism resulting from the intake of ergotamine tartrate for treating headache. For the first time, ergotamine was identified and quantified in hair. The ergotamine concentrations measured were 320 pg/mL in blood, 100 pg/mL in urine, 24 pg/mg in proximal hair, and 15 pg/mg in distal hair.

  13. Discrimination Between Peptide O-Sulfo- and O-Phosphotyrosine Residues by Negative Ion Mode Electrospray Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Edelson-Averbukh, Marina; Shevchenko, Andrej; Pipkorn, Rüdiger; Lehmann, Wolf D.

    2011-12-01

    Unambiguous differentiation between isobaric sulfated and phosphorylated tyrosine residues (sTyr and pTyr) of proteins by mass spectrometry is challenging, even using high resolution mass spectrometers. Here we show that upon negative ion mode collision-induced dissociation (CID), pTyr- and sTyr-containing peptides exhibit entirely different modification-specific fragmentation patterns leading to a rapid discrimination between the isobaric covalent modifications using the tandem mass spectral data. This study reveals that the ratio between the relative abundances of [M-H-80]- and [M-H-98]- fragment ions in ion-trap CID and higher energy collision dissociation (HCD) spectra of singly deprotonated +80 Da Tyr-peptides can be used as a reliable indication of the Tyr modification group nature. For multiply deprotonated +80 Da Tyr-peptides, CID spectra of sTyr- and pTyr-containing sequences can be readily distinguished based on the presence/absence of the [M-nH-79](n-1)- and [M-nH-79-NL]( n-1)- ( n = 2, 3) fragment ions (NL = neutral loss).

  14. 10 K Ring Electrode Trap - Tandem Mass Spectrometer for Infrared Spectroscopy of Mass Selected Ions

    SciTech Connect

    Goebbert, Daniel J.; Meijer, Gerard; Asmis, Knut R.

    2009-03-17

    A novel instrumental setup for measuring infrared photodissociation spectra of buffer gas cooled, mass-selected ions is described and tested. It combines a cryogenically cooled, linear radio frequency ion trap with a tandem mass spectrometer, optimally coupling continuous ion sources to pulsed laser experiments. The use of six independently adjustable DC potentials superimposed over the trapping radio frequency field provides control over the ion distribution within, as well as the kinetic energy distribution of the ions extracted from the ion trap. The scheme allows focusing the ions in space and time, such that they can be optimally irradiated by a pulsed, widely tunable infrared photodissociation laser. Ion intensities are monitored with a time-of-flight mass spectrometer mounted orthogonally to the ion trap axis.

  15. Low missing mass, single and double diffraction dissociation at the LHC

    SciTech Connect

    Jenkovszky, L. L.; Kuprash, O. E.; Orava, R.; Salii, A.

    2014-12-15

    The cross sections for single and double diffraction dissociation at low missing masses are calculated for the LHC energies on the basis of the dual (Regge) model under the assumption of a dominant contribution of the exchange of the Pomeron Regge pole. The model reproduces the rich resonance structure in the region of low missing masses M{sub x}. Diffractively excited states lie on the nucleon trajectory M{sub x} supplemented with the isolated Roper resonance. Detailed predictions for the squared momentum transfer and missing-mass dependence of the differential and integrated single and double diffraction dissociation in the kinematical range of present and future LHC measurements are given.

  16. Electron capture dissociation mass spectrometry of tyrosine nitrated peptides.

    PubMed

    Jones, Andrew W; Mikhailov, Victor A; Iniesta, Jesus; Cooper, Helen J

    2010-02-01

    In vivo protein nitration is associated with many disease conditions that involve oxidative stress and inflammatory response. The modification involves addition of a nitro group at the position ortho to the phenol group of tyrosine to give 3-nitrotyrosine. To understand the mechanisms and consequences of protein nitration, it is necessary to develop methods for identification of nitrotyrosine-containing proteins and localization of the sites of modification. Here, we have investigated the electron capture dissociation (ECD) and collision-induced dissociation (CID) behavior of 3-nitrotyrosine-containing peptides. The presence of nitration did not affect the CID behavior of the peptides. For the doubly-charged peptides, addition of nitration severely inhibited the production of ECD sequence fragments. However, ECD of the triply-charged nitrated peptides resulted in some singly-charged sequence fragments. ECD of the nitrated peptides is characterized by multiple losses of small neutral species including hydroxyl radicals, water and ammonia. The origin of the neutral losses has been investigated by use of activated ion (AI) ECD. Loss of ammonia appears to be the result of non-covalent interactions between the nitro group and protonated lysine side-chains.

  17. Enrichment and Analysis of Nonenzymatically Glycated Peptides: Boronate Affinity Chromatography Coupled with Electron-Transfer Dissociation Mass Spectrometry

    SciTech Connect

    Zhang, Qibin; Tang, Ning; Brock, Jonathan W.; Mottaz, Heather M.; Ames, Jennifer M.; Baynes, John; Smith, Richard D.; Metz, Thomas O.

    2007-06-01

    Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. However, no effective high-throughput methods exist for identifying proteins containing this low abundance post-translational modification in bottom-up proteomic studies. In this report, phenylboronate affinity chromatography was used in a two-step enrichment scheme to selectively isolate first glycated proteins and then glycated, tryptic peptides from human serum glycated in vitro. Enriched peptides were subsequently analyzed by alternating electron transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. It was observed that ETD fragmentation mode resulted in a significantly higher number of glycated peptide identifications (87.6% of all identified peptides) versus CID mode (17.0% of all identified peptides), when utilizing dual glycation enrichment on both the protein and peptide level. This study illustrates that phenylboronate affinity chromatography coupled with LC-MS/MS with ETD as the fragmentation mode is an efficient approach for analyses of glycated proteins and can have broad applications in studies of diabetes mellitus.

  18. Rapid characterization of urinary metabolites of pibutidine hydrochloride in humans by liquid chromatography/electrospray ionization tandem mass spectrometry.

    PubMed

    Kato, K; Jingu, S; Ogawa, N; Higuchi, S

    1999-01-01

    The metabolic products of pibutidine hydrochloride, a new H(2)-receptor antagonist, in human urine after oral administration of 40 mg/man were characterized by high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) with electrospray ionization (ESI). Two-stage collision-induced dissociation (CID) experiments, with in-source CID by increasing the octapole offset voltage and collision-cell CID, were performed in order to develop a very rapid screening procedure that enhanced selectivity toward pibutidine-related compounds. It was possible to detect metabolites of pibutidine directly from a crude biological matrix without prior extraction, enabling confirmation of the identity of eight metabolites in urine. In addition, the linear range in ESI for pibutidine-related compounds was studied to determine the urinary excretion of pibutidine and its metabolites in humans.

  19. Accurate characterization of carcinogenic DNA adducts using MALDI tandem time-of-flight mass spectrometry

    NASA Astrophysics Data System (ADS)

    Barnes, Charles A.; Chiu, Norman H. L.

    2009-01-01

    Many chemical carcinogens and their in vivo activated metabolites react readily with genomic DNA, and form covalently bound carcinogen-DNA adducts. Clinically, carcinogen-DNA adducts have been linked to various cancer diseases. Among the current methods for DNA adduct analysis, mass spectroscopic method allows the direct measurement of unlabeled DNA adducts. The goal of this study is to explore the use of matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS) to determine the identity of carcinogen-DNA adducts. Two of the known carcinogenic DNA adducts, namely N-(2'-deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenyl-imidazo [4,5-b] pyridine (dG-C8-PhIP) and N-(2'-deoxyguanosin-8yl)-4-aminobiphenyl (dG-C8-ABP), were selected as our models. In MALDI-TOF MS measurements, the small matrix ion and its cluster ions did not interfere with the measurements of both selected dG adducts. To achieve a higher accuracy for the characterization of selected dG adducts, 1 keV collision energy in MALDI-TOF/TOF MS/MS was used to measure the adducts. In comparison to other MS/MS techniques with lower collision energies, more extensive precursor ion dissociations were observed. The detection of the corresponding fragment ions allowed the identities of guanine, PhIP or ABP, and the position of adduction to be confirmed. Some of the fragment ions of dG-C8-PhIP have not been reported by other MS/MS techniques.

  20. Hydrophilic interaction liquid chromatography-electrospray ionization-tandem mass spectrometry of a complex mixture of native and oxidized phospholipids.

    PubMed

    Losito, I; Facchini, L; Diomede, S; Conte, E; Megli, F M; Cataldi, T R I; Palmisano, F

    2015-11-27

    A mixture of native and oxidized phospholipids (PLs), generated by the soybean lipoxygenase type V-catalyzed partial oxidation of a lipid extract obtained from human platelets, was analyzed by Hydrophilic Interaction Liquid Chromatography-ElectroSpray Ionization-Tandem Mass Spectrometry (HILIC-ESI-MS/MS). The complexity of the resulting mixture was remarkable, considering that the starting lipid extract, containing (as demonstrated in a previous study) about 130 native PLs, was enriched with enzymatically generated hydroperoxylated derivatives and chemically generated hydroxylated forms of PLs bearing polyunsaturated side chains. Nonetheless, the described analytical approach proved to be very powerful; indeed, focusing on phosphatidylcolines (PCs), the most abundant PL class in human platelets, about fifty different native/oxidized species could be identified in a single HILIC-ESI-MS/MS run. Low-energy collision induced dissociation tandem MS (CID-MS/MS) experiments on chromatographically separated species showed single neutral losses of H2O2 and H2O to be typical fragmentation pathways of hydroperoxylated PCs, whereas a single H2O loss was observed for hydroxylated ones. Moreover, diagnostic losses of n-hexanal or n-pentanol were exploited to recognize PCs hydroperoxylated on the last but five carbon atom of a ɷ-6 polyunsaturated side chain. Despite the low resolution of the 3D ion trap mass analyzer used, the described HILIC-ESI-MS/MS approach appears very promising for the identification of oxidized lipids in oxidatively stressed complex biological systems.

  1. Electron capture dissociation in a digital ion trap mass spectrometer.

    PubMed

    Ding, Li; Brancia, Francesco L

    2006-03-15

    Electron capture dissociation was implemented in a digital ion trap without using any magnetic field to focus the electrons. Since rectangular waveforms are employed in the DIT for both trapping and dipole excitation, electrons can be injected into the trap when the electric field is constant. Following deceleration, electrons reach the precursor ion cloud. The fragment ions produced by interactions with the electron beam are subsequently analyzed by resonant ejection. [Glu(1)]-Fibrinopeptide B and substance P were used to evaluate the performance of the current design. Fragmentation efficiency of 5.5% was observed for substance P peptide ions. Additionally, analysis of the monophosphorylated peptide FQ[pS]EEQQQTEDELQDK shows that in the resulting c- and z-type ions, the phosphate group is retained on the phophoserine residue, providing information on which amino acid residue the modification is located.

  2. Planetary In Situ Sample Analysis with Tandem Two-Step Laser Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Brinckerhoff, W. B.; Getty, S. A.; Cornish, T. J.; Ecelberger, S. A.; Li, X.; Merrill Floyd, M. A.; Arevalo, R.; Elsila, J.; Callahan, M. P.

    2012-12-01

    -) and organic moieties. Second, by focusing a separate "post-ionization" laser pulse just above the sample surface, we can achieve two-step laser mass spectrometry, or L2MS, in the same highly-miniaturized TOF-MS. L2MS enables selective analysis of aromatic organics even in the presence of a complex mineral matrix. Finally, by introducing an ion optical gate in the flight path, we are able to take advantage of the broad focusing capabilities of the "curved field" reflectron at the core of the TOF-MS to achieve pseudo-tandem structural analysis of selected organics. The high-speed gate is used to admit only the molecular ion/s of interest into the reflectron. Diagnostic fragments of the ion/s obtained through metastable decay or active collision-induced dissociation (CID) remain in focus despite having widely variable velocities and masses. As such even molecular isomers with differing fragmentation pathways may be distinguished through a series of pseudo-tandem mass spectra that could be obtained in an automatic process during a mission. The "real-world" benefits of these enhancements are being fully characterized using a set of synthetic and natural standard samples as well as several planetary analogs and meteorites.

  3. Differential tandem mass spectrometry-based cross-linker: a new approach for high confidence in identifying protein cross-linking

    PubMed Central

    Chakrabarty, Jayanta K.; Naik, Aishwarya G.; Fessler, Michael B.; Munske, Gerhard R.; Chowdhury, Saiful M.

    2017-01-01

    Chemical cross-linking and mass spectrometry are now widely used to analyze large-scale protein-protein interactions. The major challenge in cross-linking approaches is the complexity of the mass spectrometric data. New approaches are required that can identify cross-linked peptides with high-confidence and establish a user-friendly analysis protocol for the biomedical scientific community. Here, we introduce a novel cross-linker that can be selectively cleaved in the gas phase using two differential tandem mass-spectrometric fragmentation methods, such as collision-induced or electron transfer dissociation (CID and ETD). This technique produces two signature mass spectra of the same cross-linked peptide, thereby producing high confidence in identifying the sites of interaction. Further tandem mass spectrometry can also give additional confidence on the peptide sequences. We demonstrate a proof-of-concept for this method using standard peptides and proteins. Peptides and proteins were cross-linked and their fragmentation characteristics were analyzed using CID and ETD tandem mass spectrometry. Two sequential cleavages unambiguously identified cross-linked peptides. In addition, the labeling efficiency of the new cross-linker was evaluated in macrophage immune cells after stimulation with the microbial ligand lipopolysaccharide and subsequent pulldown experiments with biotin-avidin affinity chromatography. We believe this strategy will help advance insights into the structural biology and systems biology of cell signaling. PMID:27649375

  4. Characterization of glycopeptides by combining collision-induced dissociation and electron-transfer dissociation mass spectrometry data.

    PubMed

    Alley, William R; Mechref, Yehia; Novotny, Milos V

    2009-01-01

    Structural characterization of a glycopeptide is not easily attained through collision-induced dissociation (CID), due to the extensive fragmentation of glycan moieties and minimal fragmentation of peptide backbones. In this study, we have exploited the potential of electron-transfer dissociation (ETD) as a complementary approach for peptide fragmentation. Model glycoproteins, including ribonuclease B, fetuin, horseradish peroxidase, and haptoglobin, were used here. In ETD, radical anions transfer an electron to the peptide backbone and induce cleavage of the N-Calpha bond. The glycan moiety is retained on the peptide backbone, being largely unaffected by the ETD process. Accordingly, ETD allows not only the identification of the amino acid sequence of a glycopeptide, but also the unambiguous assignment of its glycosylation site. When data acquired from both fragmentation techniques are combined, it is possible to characterize comprehensively the entire glycopeptide. This is being achieved with a mass spectrometer capable of alternating between CID and ETD on-the-fly during an LC/MS/MS analysis. This is demonstrated here with several tryptic glycopeptides.

  5. A Novel Approach for Untargeted Post-translational Modification Identification Using Integer Linear Optimization and Tandem Mass Spectrometry*

    PubMed Central

    Baliban, Richard C.; DiMaggio, Peter A.; Plazas-Mayorca, Mariana D.; Young, Nicolas L.; Garcia, Benjamin A.; Floudas, Christodoulos A.

    2010-01-01

    A novel algorithm, PILOT_PTM, has been developed for the untargeted identification of post-translational modifications (PTMs) on a template sequence. The algorithm consists of an analysis of an MS/MS spectrum via an integer linear optimization model to output a rank-ordered list of PTMs that best match the experimental data. Each MS/MS spectrum is analyzed by a preprocessing algorithm to reduce spectral noise and label potential complimentary, offset, isotope, and multiply charged peaks. Postprocessing of the rank-ordered list from the integer linear optimization model will resolve fragment mass errors and will reorder the list of PTMs based on the cross-correlation between the experimental and theoretical MS/MS spectrum. PILOT_PTM is instrument-independent, capable of handling multiple fragmentation technologies, and can address the universe of PTMs for every amino acid on the template sequence. The various features of PILOT_PTM are presented, and it is tested on several modified and unmodified data sets including chemically synthesized phosphopeptides, histone H3-(1–50) polypeptides, histone H3-(1–50) tryptic fragments, and peptides generated from proteins extracted from chromatin-enriched fractions. The data sets consist of spectra derived from fragmentation via collision-induced dissociation, electron transfer dissociation, and electron capture dissociation. The capability of PILOT_PTM is then benchmarked using five state-of-the-art methods, InsPecT, Virtual Expert Mass Spectrometrist (VEMS), Modi, Mascot, and X!Tandem. PILOT_PTM demonstrates superior accuracy on both the small and large scale proteome experiments. A protocol is finally developed for the analysis of a complete LC-MS/MS scan using template sequences generated from SEQUEST and is demonstrated on over 270,000 MS/MS spectra collected from a total chromatin digest. PMID:20103568

  6. Alkali metal-cationized serine clusters studied by sonic spray ionization tandem mass spectrometry.

    PubMed

    Nanita, Sergio C; Sokol, Ewa; Cooks, R Graham

    2007-05-01

    Serine solutions containing salts of alkali metals yield magic number clusters of the type (Ser(4)+C)(+), (Ser(8)+C)(+), (Ser(12)+C)(+), and (Ser(17)+2C)(+2) (where C = Li(+), Na(+), K(+), Rb(+), or Cs(+)), in relative abundances which are strongly dependent on the cation size. Strong selectivity for homochirality is involved in the formation of serine tetramers cationized by K(+), Rb(+), and Cs(+). This is also the case for the octamers cationized by the smaller alkalis but there is a strong preference for heterochirality in the octamers cationized by the larger alkali cations. Tandem mass spectrometry shows that the octamers and dodecamers cationized by K(+), Rb(+), and Cs(+) dissociate mainly by the loss of Ser(4) units, suggesting that the neutral tetramers are the stable building blocks of the observed larger aggregates, (Ser(8)+C)(+) and (Ser(12)+C)(+). Remarkably, although the Ser(4) units are formed with a strong preference for homochirality, they aggregate further regardless of their handedness and, therefore, with a preference for the nominally racemic 4D:4L structure and an overall strong heterochiral preference. The octamers cationized by K(+), Rb(+), or Cs(+) therefore represent a new type of cluster ion that is homochiral in its internal subunits, which then assemble in a random fashion to form octamers. We tentatively interpret the homochirality of these tetramers as a consequence of assembly of the serine molecules around a central metal ion. The data provide additional evidence that the neutral serine octamer is homochiral and is readily cationized by smaller ions.

  7. Dissociation and ammonia mass transfer from ammonium solution and dairy cattle manure

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Process-based models are being used to predict ammonia (NH**3) emissions from manure sources, but their accuracy has not been fully evaluated for cattle manure. Laboratory trials were conducted to measure the dissociation and mass transfer coefficient for NH**3 volatilization from media of buffered ...

  8. Reliability of veterinary drug residue confirmation: high resolution mass spectrometry versus tandem mass spectrometry.

    PubMed

    Kaufmann, A; Butcher, P; Maden, K; Walker, S; Widmer, M

    2015-01-26

    Confirmation of suspected residues has been a long time domain of tandem triple quadrupole mass spectrometry (QqQ). The currently most widely used confirmation strategy relies on the use of two selected reaction monitoring signals (SRM). The details of this confirmation procedure are described in detail in the Commission Decision 93/256/EC (CD). On the other hand, high resolution mass spectrometry (HRMS) is nowadays increasingly used for trace analysis. Yet its utility for confirmatory purposes has not been well explored and utilized, since established confirmation strategies like the CD do not yet include rules for modern HRMS technologies. It is the focus of this paper to evaluate the likelihood of false positive and false negative confirmation results, when using a variety of HRMS based measurement modes as compared to conventional QqQ mass spectrometry. The experimental strategy relies on the chromatographic separation of a complex blank sample (bovine liver extract) and the subsequent monitoring of a number of dummy transitions respectively dummy accurate masses. The term "dummy" refers to precursor and derived product ions (based on a realistic neutral loss) whose elemental compositions (CxHyNzOdCle) were produced by a random number generator. Monitoring a large number of such hypothetical SRM's, or accurate masses inevitably produces a number of mass traces containing chromatographic peaks (false detects) which are caused by eluting matrix compounds. The number and intensity of these peaks were recorded and standardized to permit a comparison among the two employed MS technologies. QqQ performance (compounds which happen to produce a response in two SRM traces at identical retention time) was compared with a number of different HRMS(1) and HRMS(2) detection based modes. A HRMS confirmation criterion based on two full scans (an unfragmented and an all ion fragmented) was proposed. Compared to the CD criteria, a significantly lower probability of false

  9. Characterization of nucleic acids by tandem mass spectrometry - The second decade (2004-2013): From DNA to RNA and modified sequences.

    PubMed

    Schürch, Stefan

    2016-07-01

    Nucleic acids play key roles in the storage and processing of genetic information, as well as in the regulation of cellular processes. Consequently, they represent attractive targets for drugs against gene-related diseases. On the other hand, synthetic oligonucleotide analogues have found application as chemotherapeutic agents targeting cellular DNA and RNA. The development of effective nucleic acid-based chemotherapeutic strategies requires adequate analytical techniques capable of providing detailed information about the nucleotide sequences, the presence of structural modifications, the formation of higher-order structures, as well as the interaction of nucleic acids with other cellular components and chemotherapeutic agents. Due to the impressive technical and methodological developments of the past years, tandem mass spectrometry has evolved to one of the most powerful tools supporting research related to nucleic acids. This review covers the literature of the past decade devoted to the tandem mass spectrometric investigation of nucleic acids, with the main focus on the fundamental mechanistic aspects governing the gas-phase dissociation of DNA, RNA, modified oligonucleotide analogues, and their adducts with metal ions. Additionally, recent findings on the elucidation of nucleic acid higher-order structures by tandem mass spectrometry are reviewed. © 2014 Wiley Periodicals, Inc., Mass Spec Rev 35:483-523, 2016.

  10. High explosives vapor detection by atmospheric sampling glow discharge ionization/tandem mass spectrometry

    SciTech Connect

    McLuckey, S.A.; Goeringer, D.E.; Asano, K.G.

    1996-02-01

    The combination of atmospheric sampling glow discharge ionization with tandem mass spectrometry for the detection of traces of high explosives is described. Particular emphasis is placed on use of the quadrupole ion trap as the type of tandem mass spectrometer. Atmospheric sampling glow discharge provides a simple, rugged, and efficient means for anion formation while the quadrupole ion trap provides for efficient tandem mass spectrometry. Mass selective ion accumulation and non-specific ion activation methods can be used to overcome deleterious effects arising from ion/ion interactions. Such interactions constitute the major potential technical barrier to the use of the ion trap for real-time monitoring of targeted compounds in uncontrolled and highly variable matrices. Tailored waveforms can be used to effect both mass selective ion accumulation and ion activation. Concatenated tailored waveforms allow for both functions in a single experiment thereby providing the capability for monitoring several targeted species simultaneously. The combination of atmospheric sampling glow discharge ionization with a state-of-the-art analytical quadrupole ion trap is a highly sensitive and specific detector for traces of high explosives. The combination is also small and inexpensive relative to virtually any other form of tandem mass spectrometry. The science and technology underlying the glow discharge/ion trap combination is sufficiently mature to form the basis for an engineering effort to make the detector portable. 85 refs.

  11. Characterization of γ-carboxylated tryptic peptides by collision-induced dissociation and electron transfer dissociation mass spectrometry.

    PubMed

    Ramström, Margareta; Sandberg, Helena

    2011-01-01

    Vitamin K-dependent carboxylation of glutamic acid (Glu) residues into γ-carboxyglutamic acid (Gla) is a post-translational modification essential for normal protein activity of, for example, proteins involved in the blood coagulation system. These proteins may contain as many as 12 sites for γ-carboxylation within a protein sequence of 45 amino acid residues. In the biopharmaceutical industry, powerful analytical techniques are required for identification and localization of modified sites. We here present comparatively easy and rapid methods for studies of Gla-containing proteins using recent technology. The performances of two mass spectrometric fragmentation techniques, collision-induced dissociation (CID) and electron transfer dissociation (ETD), were evaluated with respect to γ-carboxylated peptides, applying on-line LC-ion trap MS. ETD MS has so far not been reported for Gla-containing peptides and the applicability of CID for heavily γ-carboxylated proteins has not been evaluated. The anticoagulant protein, protein C, containing nine Gla-sites, was chosen as a model protein. After tryptic digestion, three peptides containing Gla-residues were detected by MS; a 1.2 kDa fragment containing two Gla-residues, a 4.5 kDa peptide containing seven residues and also the 5.6 kDa tryptic peptides containing all nine Gla-residues. Regarding the shortest peptide, both CID and ETD provided extensive peptide sequencing. For the larger peptides, fragmentation by CID resulted in loss of the 44 Da CO(2)-group, while little additional fragmentation of the peptide chain was observed. In contrast, ETD resulted in comprehensive fragmentation of the peptide backbone. The study demonstrates that the combination of both techniques would be beneficial and complementary for investigation of γ-carboxylated proteins and peptides.

  12. Dereplication of Flavonoid Glycoconjugates from Adenocalymma imperatoris-maximilianii by Untargeted Tandem Mass Spectrometry-Based Molecular Networking.

    PubMed

    de Oliveira, Gibson Gomes; Carnevale Neto, Fausto; Demarque, Daniel Pecoraro; de Sousa Pereira-Junior, José Antônio; Sampaio Peixoto Filho, Rômulo César; de Melo, Sebastião José; da Silva Almeida, Jackson Roberto Guedes; Lopes, João Luiz Callegari; Lopes, Norberto Peporine

    2016-11-02

    The interpretation of large datasets acquired using high performance liquid chromatography coupled with tandem mass spectrometry represents one of the major challenges in natural products research. Here we propose the use of molecular networking to rapid identify the known secondary metabolites from untargeted MS/MS analysis of Adenocalymma imperatoris-maximilianii plant extracts. The leaves, stems and roots of A. imperatoris-maximilianii were extracted using different solvents according to Snyder selectivity triangle. The samples were analyzed by HPLC coupled with ion trap mass spectrometer in a collision-induced dissociation MS/MS configuration in both positive and negative electrospray ionization modes. Molecular networking simultaneously organized the spectra by cosine similarity. The chemical identification was performed based on the systematic study of the main fragmentation pathways observed for the resulting network. The untargeted tandem mass spectrometry-based molecular networking allowed for the identification of 63 metabolites, mainly mono-, di- and tri-, C- and/or O-glycosyl flavones. Molecular networking was capable not only to dereplicate known flavonoids, but also to point out related prenyl derivatives, described for the first time in Adenocalymma species. The gas-phase reaction route to form the characteristic [M-H2O-(30/60/90)](+) fragments in C-glycosyl flavones was suggested as sequential sugar ring opening followed by retro-aldol elimination involving aldose-ketose isomerization. The use of molecular networking with LC-CID-MS/MS assisted the identification of various isomeric and isobaric flavonoid glycoconjugates by establishing clusters according to the fragmentation similarities. Additionally, the proposed cross-ring sugar cleavages can contribute to the identification of C-glycosides by MS/MS analysis.

  13. Mass fractionation of noble gases in synthetic methane hydrate: Implications for naturally occurring gas hydrate dissociation

    USGS Publications Warehouse

    Hunt, Andrew G.; Stern, Laura; Pohlman, John W.; Ruppel, Carolyn; Moscati, Richard J.; Landis, Gary P.

    2013-01-01

    As a consequence of contemporary or longer term (since 15 ka) climate warming, gas hydrates in some settings may presently be dissociating and releasing methane and other gases to the ocean-atmosphere system. A key challenge in assessing the impact of dissociating gas hydrates on global atmospheric methane is the lack of a technique able to distinguish between methane recently released from gas hydrates and methane emitted from leaky thermogenic reservoirs, shallow sediments (some newly thawed), coal beds, and other sources. Carbon and deuterium stable isotopic fractionation during methane formation provides a first-order constraint on the processes (microbial or thermogenic) of methane generation. However, because gas hydrate formation and dissociation do not cause significant isotopic fractionation, a stable isotope-based hydrate-source determination is not possible. Here, we investigate patterns of mass-dependent noble gas fractionation within the gas hydrate lattice to fingerprint methane released from gas hydrates. Starting with synthetic gas hydrate formed under laboratory conditions, we document complex noble gas fractionation patterns in the gases liberated during dissociation and explore the effects of aging and storage (e.g., in liquid nitrogen), as well as sampling and preservation procedures. The laboratory results confirm a unique noble gas fractionation pattern for gas hydrates, one that shows promise in evaluating modern natural gas seeps for a signature associated with gas hydrate dissociation.

  14. Chemical ionization tandem mass spectrometer for the in situ measurement of methyl hydrogen peroxide

    SciTech Connect

    St Clair, Jason M.; McCabe, David C.; Crounse, John D.; Steiner, Urs; Wennberg, Paul O.

    2010-09-15

    A new approach for measuring gas-phase methyl hydrogen peroxide [(MHP) CH{sub 3}OOH] utilizing chemical ionization mass spectrometry is presented. Tandem mass spectrometry is used to avoid mass interferences that hindered previous attempts to measure atmospheric CH{sub 3}OOH with CF{sub 3}O{sup -} clustering chemistry. CH{sub 3}OOH has been successfully measured in situ using this technique during both airborne and ground-based campaigns. The accuracy and precision for the MHP measurement are a function of water vapor mixing ratio. Typical precision at 500 pptv MHP and 100 ppmv H{sub 2}O is {+-}80 pptv (2 sigma) for a 1 s integration period. The accuracy at 100 ppmv H{sub 2}O is estimated to be better than {+-}40%. Chemical ionization tandem mass spectrometry shows considerable promise for the determination of in situ atmospheric trace gas mixing ratios where isobaric compounds or mass interferences impede accurate measurements.

  15. Activation Energies of Fragmentations of Disaccharides by Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Kuki, Ákos; Nagy, Lajos; Szabó, Katalin E.; Antal, Borbála; Zsuga, Miklós; Kéki, Sándor

    2014-03-01

    A simple multiple collision model for collision induced dissociation (CID) in quadrupole was applied for the estimation of the activation energy (Eo) of the fragmentation processes for lithiated and trifluoroacetated disaccharides, such as maltose, cellobiose, isomaltose, gentiobiose, and trehalose. The internal energy-dependent rate constants k(Eint) were calculated using the Rice-Ramsperger-Kassel-Marcus (RRKM) or the Rice-Ramsperger-Kassel (RRK) theory. The Eo values were estimated by fitting the calculated survival yield (SY) curves to the experimental ones. The calculated Eo values of the fragmentation processes for lithiated disaccharides were in the range of 1.4-1.7 eV, and were found to increase in the order trehalose < maltose < isomaltose < cellobiose < gentiobiose.

  16. Integrated approach for the comprehensive characterization of lipoproteins from human plasma using FPLC and nano-HPLC-tandem mass spectrometry.

    PubMed

    Collins, Lisamarie A; Mirza, Shama P; Kissebah, Ahmed H; Olivier, Michael

    2010-02-04

    The implication of the various lipoprotein classes in the development of atherosclerotic cardiovascular disease has served to focus a great deal of attention on these particles over the past half-century. Using knowledge gained by the sequencing of the human genome, recent research efforts have been directed toward the elucidation of the proteomes of several lipoprotein subclasses. One of the challenges of such proteomic experimentation is the ability to initially isolate plasma lipoproteins subsequent to their analysis by mass spectrometry. Although several methods for the isolation of plasma lipoproteins are available, the most commonly utilized techniques require large sample volumes and may cause destruction and dissociation of lipoprotein particle-associated proteins. Fast protein liquid chromatography (FPLC) is a nondenaturing technique that has been validated for the isolation of plasma lipoproteins from relatively small sample volumes. In this study, we present the use of FPLC in conjunction with nano-HPLC-ESI-tandem mass spectrometry as a new integrated methodology suitable for the proteomic analysis of human lipoprotein fractions. Results from our analysis show that only 200 microl of human plasma suffices for the isolation of whole high density lipoprotein (HDL) and the identification of the majority of all known HDL-associated proteins using mass spectrometry of the resulting fractions.

  17. Simulation of two dimensional electrophoresis and tandem mass spectrometry for teaching proteomics.

    PubMed

    Fisher, Amanda; Sekera, Emily; Payne, Jill; Craig, Paul

    2012-01-01

    In proteomics, complex mixtures of proteins are separated (usually by chromatography or electrophoresis) and identified by mass spectrometry. We have created 2DE Tandem MS, a computer program designed for use in the biochemistry, proteomics, or bioinformatics classroom. It contains two simulations-2D electrophoresis and tandem mass spectrometry. The two simulations are integrated together and are designed to teach the concept of proteome analysis of prokaryotic and eukaryotic organisms. 2DE-Tandem MS can be used as a freestanding simulation, or in conjunction with a wet lab, to introduce proteomics in the undergraduate classroom. 2DE Tandem MS is a free program available on Sourceforge at https://sourceforge.net/projects/jbf/. It was developed using Java Swing and functions in Mac OSX, Windows, and Linux, ensuring that every student sees a consistent and informative graphical user interface no matter the computer platform they choose. Java must be installed on the host computer to run 2DE Tandem MS. Example classroom exercises are provided in the Supporting Information.

  18. MEASUREMENT OF OXIDATIVE STRESS PARAMETERS USING LIQUID CHROMATOGRAPHY - TANDEM MASS SPECTROSCOPY (LC-MS/MS)

    EPA Science Inventory

    What is the study?
    An invited review article. Measurement of oxidative stress parameters using liquid chromatography-tandem mass spectroscopy (LC-MS/MS)
    Why was it done?
    Although oxidative stress is frequently cited as a cause of various adverse biological eff...

  19. Making the Case for Objective Performance Metrics in Newborn Screening by Tandem Mass Spectrometry

    ERIC Educational Resources Information Center

    Rinaldo, Piero; Zafari, Saba; Tortorelli, Silvia; Matern, Dietrich

    2006-01-01

    The expansion of newborn screening programs to include multiplex testing by tandem mass spectrometry requires understanding and close monitoring of performance metrics. This is not done consistently because of lack of defined targets, and interlaboratory comparison is almost nonexistent. Between July 2004 and April 2006 (N = 176,185 cases), the…

  20. Multiresidue analysis of pesticides in straw roughage by liquid chromatography - tandem mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A multiresidue analytical method using a modification of the “quick, easy, cheap, effective, rugged, and safe” (QuEChERS) sample preparation approach combined with liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis was established and validated for the rapid determination of 69 pesti...

  1. Combined Dynamic Arrays for Storing and Searching Semi-Ordered Tandem Mass Spectrometry Data

    Technology Transfer Automated Retrieval System (TEKTRAN)

    When performing bioinformatics analysis on tandem mass spectrometry data, there is a computational need to efficiently store and sort these semi-ordered data sets. To solve this problem, a new data structure based on dynamic arrays was designed and implemented in an algorithm that parses semi-order...

  2. Tandem mass spectrometry newborn screening for inborn errors of intermediary metabolism: abnormal profile interpretation.

    PubMed

    Fernández-Lainez, C; Aguilar-Lemus, J J; Vela-Amieva, M; Ibarra-González, I

    2012-01-01

    Expanded newborn screening for inherited metabolic disorders using tandem mass spectrometry was introduced in 1990's and is widely used around the world. In contrast to conventional screening methods, tandem mass spectrometry does not measure single analytes but identifies and quantifies metabolite profiles; one single blood spot analyzed provides information of about 60 metabolites including amino acids, acylcarnitines and related ratios that enable the diagnosis of approximately 50 different diseases. However, the interpretation of these profiles can become quite complex. The aim of this work is to present in an easy and practical manner a comprehensive compilation of information needed for tandem mass neonatal screening profile interpretation, and basic actions for immediate follow up of abnormal results, including the tests that are required for confirmatory purposes. Other conditions not attributable to metabolic disorders which can lead to an abnormal profile of these markers are also described as well as a series of general recommendations which would be useful for health professionals who are beginning newborn screening for inborn errors of intermediary metabolism using tandem mass spectrometry.

  3. Accurate Mass MS/MS/MS Analysis of Siderophores Ferrioxamine B and E1 by Collision-Induced Dissociation Electrospray Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Sidebottom, Ashley M.; Karty, Jonathan A.; Carlson, Erin E.

    2015-11-01

    Siderophores are bacterially secreted, small molecule iron chelators that facilitate the binding of insoluble iron (III) for reuptake and use in various biological processes. These compounds are classified by their iron (III) binding geometry, as dictated by subunit composition and include groups such as the trihydroxamates (hexadentate ligand) and catecholates (bidentate). Small modifications to the core structure such as acetylation, lipid tail addition, or cyclization, make facile characterization of new siderophores difficult by molecular ion detection alone (MS1). We have expanded upon previous fragmentation-directed studies using electrospray ionization collision-induced dissociation tandem mass spectrometry (ESI-CID-MS/MS/MS) and identified diagnostic MS3 features from the trihydroxamate siderophore class for ferrioxamine B and E1 by accurate mass. Diagnostic features for MS3 include C-C, C-N, amide, and oxime cleavage events with proposed losses of water and -CO from the iron (III) coordination sites. These insights will facilitate the discovery of novel trihydroxamate siderophores from complex sample matrices.

  4. Characterization of noncovalent complexes of antimalarial agents of the artemisinin-type and FE(III)-heme by electrospray mass spectrometry and collisional activation tandem mass spectrometry.

    PubMed

    Pashynska, Vlada A; Van den Heuvel, Hilde; Claeys, Magda; Kosevich, Marina V

    2004-08-01

    In this study, we demonstrate, using electrospray ionization mass spectrometry (ESI-MS) and collision-induced dissociation tandem mass spectrometry (ESI-MS/CID/MS), that stable noncovalent complexes can be formed between Fe(III)-heme and antimalarial agents, i.e., quinine, artemisinin, and the artemisinin derivatives, dihydroartemisinin, alpha- and beta-artemether, and beta-arteether. Differences in the binding behavior of the examined drugs with Fe(III)-heme and the stability of the drug-heme complexes are demonstrated. The results show that all tested antimalarial agents form a drug-heme complex with a 1:1 stoichiometry but that quinine also results in a second complex with the heme dimer. ESI-MS performed on mixtures of pairs of various antimalarial agents with heme indicate that quinine binds preferentially to Fe(III)-heme, while ESI-MS/CID/MS shows that the quinine-heme complex is nearly two times more stable than the complexes formed between heme and artemisinin or its derivatives. Moreover, it is found that dihydroartemisinin, the active metabolite of the artemisinin-type drugs in vivo, results in a Na(+)-containing heme-drug complex, which is as stable as the heme-quinine complex. The efficiency of drug-heme binding of artemisinin derivatives is generally lower and the decomposition under CID higher compared with quinine, but these parameters are within the same order of magnitude. These results suggest that the efficiency of antimalarial agents of the artemisinin-type to form noncovalent complexes with Fe(III)-heme is comparable with that of the traditional antimalarial agent, quinine. Our study illustrates that electrospray ionization mass spectrometry and collision-induced dissociation tandem mass spectrometry are suitable tools to probe noncovalent interactions between heme and antimalarial agents. The results obtained provide insights into the underlying molecular modes of action of the traditional antimalarial agent quinine and of the antimalarials of

  5. Real-time PCR and PCR-tandem Mass Spectrometry for Biodetection

    DTIC Science & Technology

    2005-10-01

    Real - time PCR and PCR- tandem mass spectrometry for biodetection Alvin Fox, University of South Carolina, School of Medicine Report Documentation...TYPE N/A 3. DATES COVERED - 4. TITLE AND SUBTITLE Real - time PCR and PCRtandem mass spectrometry for biodetection 5a. CONTRACT NUMBER 5b...interspace region Bacillus subtilis W23 standard Blank Barn dust House dust Cycle Real - time PCR (16s rRNA) - environmental samples Real - time

  6. Selective Chemoprecipitation and Subsequent Release of Tagged Species for the Analysis of Nitropeptides by Liquid Chromatography–Tandem Mass Spectrometry*

    PubMed Central

    Prokai-Tatrai, Katalin; Guo, Jia; Prokai, Laszlo

    2011-01-01

    Tyrosine nitration is a low-abundance post-translational protein modification that requires appropriate enrichment techniques to enable proteomic analyses. We report a simple yet highly specific method to enrich nitropeptides by chemoprecipitation involving only two straightforward chemical modifications of the nitropeptides before capturing the obtained derivatives with a strategically designed solid-phase active ester reagent. Specifically, capping of the aliphatic amines in the peptides is done first by reductive methylation to preserve the charge state of peptides for electrospray ionization mass spectrometric analysis, followed by reduction of nitrotyrosines to the corresponding aminotyrosines. These peptides are then immobilized on the solid-phase active ester reagent, whereas other peptides carrying no free amino groups are separated from the immobilized species by thoroughly washing the beads from which the tagged peptide derivatives can easily be released by acid-catalyzed hydrolysis at room temperature. The benefits of selective enrichment from a matrix of unmodified peptides for liquid chromatography-tandem mass spectrometry are demonstrated on three synthetic nitropeptides that are nitrated fragments of biologically relevant proteins. Identification of several in vitro nitrated human plasma proteins, also implicated under various pathological processes, by database searches from the enriched and tagged tryptic nitropeptides is presented as a practical application. We also show that converting the nitro-group to the small 4-formylbenzoylamido tag does not significantly alter fragmentation properties upon collision-induced dissociation compared with those of the native nitropeptides, and at the same time this derivatization actually improves electron capture dissociation due to conversion of the electron-predator nitro-group to this novel tag. PMID:21540302

  7. Tandem Mass Spectrometry of Heparan Sulfate Negative Ions: Sulfate Loss Patterns and Chemical Modification Methods for Improvement of Product Ion Profiles

    NASA Astrophysics Data System (ADS)

    Shi, Xiaofeng; Huang, Yu; Mao, Yang; Naimy, Hicham; Zaia, Joseph

    2012-09-01

    Heparan sulfate (HS) is a polysaccharide modified with sulfation, acetylation, and epimerization that enable its binding with protein ligands and regulation of important biological processes. Tandem mass spectrometry has been employed to sequence linear biomolecules e.g., proteins and peptides. However, its application in structural characterization of HS is limited due to the neutral loss of sulfate (SO3) during collisional induced dissociation (CID). In this report, we studied the dissociation patterns of HS disaccharides and demonstrate that the N-sulfate (N-S) bond is especially facile during CID. We identified factors that influence the propensities of such losses from precursor ions and proposed a Free Proton Index (FPI) to help select ions that are able to produce meaningful backbone dissociations. We then investigated the thermodynamics and kinetics of SO3 loss from sulfates that are protonated, deprotonated, and metal-adducted using density functional theory computations. The calculations showed that sulfate loss from a protonated site was much more facile than that from a deprotonated or metal-adducted site. Further, the loss of SO3 from N-sulfate was energetically favored by 3-8 kcal/mol in transition states relative to O-sulfates, making it more prone to this process by a substantial factor. In order to reduce the FPI, representing the number of labile sulfates in HS native chains and oligosaccharides, we developed a series of chemical modifications to selectively replace the N-sulfates of the glucosamine with deuterated acetyl group. These modifications effectively reduced the sulfate density on the HS oligosaccharides and generated considerably more backbone dissociation using on-line LC/tandem MS.

  8. Atmospheric sampling glow discharge ionizataion and triple quadrupole tandem mass spectrometry for explosives vapor detection

    SciTech Connect

    McLuckey, S.A.; Goeringer, D.E.; Asano, K.G.; Hart, K.J.; Glish, G.L.; Grant, B.C.; Chambers, D.M.

    1993-08-01

    The detection and identification of trace vapors of hidden high explosives is an excellent example of a targeted analysis problem. It is desirable to push to ever lower levels the quantity or concentration of explosives material that provides an analytical signal, while at the same time discriminating against all other uninteresting material. The detection system must therefore combine high sensitivity with high specificity. This report describes the philosophy behind the use of atmospheric sampling glow discharge ionization, which is a sensitive, rugged, and convenient means for forming anions from explosives molecules, with tandem mass spectrometry, which provides unparalleled specificity in the identification of explosives-related ions. Forms of tandem mass spectrometry are compared and contrasted to provide a summary of the characteristics to be expected from an explosives detector employing mass spectrometry/mass spectrometry. The instrument developed for the FAA, an atmospheric sampling glow discharge/triple quadrupole mass spectrometer, is described in detail with particular emphasis on the ion source/spectrometer interface and on the capabilities of the spectrometer. Performance characteristics of the system are also described as they pertain to explosives of interest including a description of an automated procedure for the detection and identification of specific explosives. A comparison of various tandem mass spectrometers mated with atmospheric sampling glow discharge is then described and preliminary studies with a vapor preconcentration system provided by the FAA will be described.

  9. Characterization of Tyrosine Nitration and Cysteine Nitrosylation Modifications by Metastable Atom-Activation Dissociation Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Cook, Shannon L.; Jackson, Glen P.

    2011-02-01

    The fragmentation behavior of nitrated and S-nitrosylated peptides were studied using collision induced dissociation (CID) and metastable atom-activated dissociation mass spectrometry (MAD-MS). Various charge states, such as 1+, 2+, 3+, 2-, of modified and unmodified peptides were exposed to a beam of high kinetic energy helium (He) metastable atoms resulting in extensive backbone fragmentation with significant retention of the post-translation modifications (PTMs). Whereas the high electron affinity of the nitrotyrosine moiety quenches radical chemistry and fragmentation in electron capture dissociation (ECD) and electron transfer dissociation (ETD), MAD does produce numerous backbone cleavages in the vicinity of the modification. Fragment ions of nitrosylated cysteine modifications typically exhibit more abundant neutral losses than nitrated tyrosine modifications because of the extremely labile nature of the nitrosylated cysteine residues. However, compared with CID, MAD produced between 66% and 86% more fragment ions, which preserved the labile -NO modification. MAD was also able to differentiate I/L residues in the modified peptides. MAD is able to induce radical ion chemistry even in the presence of strong radical traps and therefore offers unique advantages to ECD, ETD, and CID for determination of PTMs such as nitrated and S-nitrosylated peptides.

  10. Ligand identification of carbohydrate-binding proteins employing a biotinylated glycan binding assay and tandem mass spectrometry.

    PubMed

    Wuhrer, Manfred; van Remoortere, Alexandra; Balog, Crina I A; Deelder, André M; Hokke, Cornelis H

    2010-11-15

    Characterization of protein-carbohydrate interactions at the molecular level is important for understanding many glycan-mediated processes. Here we present a method for the identification of glycan ligands of carbohydrate-binding proteins. The glycans released from natural sources are labeled with biotinamidocaproyl hydrazide (BACH) and subsequently fractionated by high-performance liquid chromatography. Glycan fractions are screened for binding to carbohydrate-binding proteins (CBPs) using a microtitration plate binding assay; CBPs are immobilized, BACH-glycan fractions are added, and bound BACH-glycans are detected using alkaline phosphatase-conjugated streptavidin. The glycan structures in binding fractions are studied by (tandem) mass spectrometry, exoglycosidase treatment, and rechromatography, thereby revealing the glycan motifs recognized by the CBPs. Subsequent surface plasmon resonance experiments using a reverse setup with immobilization of the BACH-glycan ligands on streptavidin-coated surfaces provide more information on glycan-CBP interactions via association and dissociation curves. The presented method is easy and fast, and the required instrumentation is available in many laboratories. The assay is very sensitive given that both the mass spectrometric analysis and the microtitration plate binding assay can be performed on femtomole amounts of BACH-glycans. This approach should be generally applicable to study and structurally identify carbohydrate ligands of anti-glycan antibodies and lectins.

  11. High energy collisions on tandem time-of-flight mass spectrometers.

    PubMed

    Cotter, Robert J

    2013-05-01

    Long before the introduction of matrix-assisted laser desorption/ionization (MALDI), electrospray ionization (ESI), Orbitraps, and any of the other tools that are now used ubiquitously for proteomics and metabolomics, the highest performance mass spectrometers were sector instruments, providing high resolution mass measurements by combining an electrostatic energy analyzer (E) with a high field magnet (B). In its heyday, the four sector mass spectrometer (or EBEB) was the crown jewel, providing the highest performance tandem mass spectrometry using single, high energy collisions to induce fragmentation. During a time in which quadrupole and tandem triple quadrupole instruments were also enjoying increased usage and popularity, there were, nonetheless, some clear advantages for sectors over their low collision energy counterparts. Time-of-flight (TOF) mass spectrometers are high voltage, high vacuum instruments that have much in common with sectors and have inspired the development of tandem instruments exploiting single high energy collisions. In this retrospective, we recount our own journey to produce high performance TOFs and tandem TOFs, describing the basic theory, problems, and the advantages for such instruments. An experiment testing impulse collision theory (ICT) underscores the similarities with sector mass spectrometers where this concept was first developed. Applications provide examples of more extensive fragmentation, side chain cleavages, and charge-remote fragmentation, also characteristic of high energy sector mass spectrometers. Moreover, the so-called curved-field reflectron has enabled the design of instruments that are simpler, collect and focus all of the ions, and may provide the future technology for the clinic, for tissue imaging, and the characterization of microorganisms.

  12. Fast quantitative detection of cocaine in beverages using nanoextractive electrospray ionization tandem mass spectrometry.

    PubMed

    Hu, Bin; Peng, Xuejiao; Yang, Shuiping; Gu, Haiwei; Chen, Huanwen; Huan, Yanfu; Zhang, Tingting; Qiao, Xiaolin

    2010-02-01

    Without any sample pretreatment, effervescent beverage fluids were manually sprayed into the primary ion plume created by using a nanoelectrospray ionization source for direct ionization, and the analyte ions of interest were guided into an ion trap mass spectrometer for tandem mass analysis. Functional ingredients (e.g., vitamins, taurine, and caffeine, etc.) and spiked impurity (e.g., cocaine) in various beverages, such as Red Bull energy drink, Coco-cola, and Pepsi samples were rapidly identified within 1.5 s. The limit of detection was found to be 7-15 fg (S/N = 3) for cocaine in different samples using the characteristic fragment (m/z 150) observed in the MS(3) experiments. Typical relative standard deviation and recovery of this method were 6.9%-8.6% and 104%-108% for direct analysis of three actual samples, showing that nanoextractive electrospray ionization tandem mass spectrometry is a useful technique for fast screening cocaine presence in beverages.

  13. Gas Chromatography/Atmospheric Pressure Chemical Ionization Tandem Mass Spectrometry for Fingerprinting the Macondo Oil Spill.

    PubMed

    Lobodin, Vladislav V; Maksimova, Ekaterina V; Rodgers, Ryan P

    2016-07-05

    We report the first application of a new mass spectrometry technique (gas chromatography combined to atmospheric pressure chemical ionization tandem mass spectrometry, GC/APCI-MS/MS) for fingerprinting a crude oil and environmental samples from the largest accidental marine oil spill in history (the Macondo oil spill, the Gulf of Mexico, 2010). The fingerprinting of the oil spill is based on a trace analysis of petroleum biomarkers (steranes, diasteranes, and pentacyclic triterpanes) naturally occurring in crude oil. GC/APCI enables soft ionization of petroleum compounds that form abundant molecular ions without (or little) fragmentation. The ability to operate the instrument simultaneously in several tandem mass spectrometry (MS/MS) modes (e.g., full scan, product ion scan, reaction monitoring) significantly improves structural information content and sensitivity of analysis. For fingerprinting the oil spill, we constructed diagrams and conducted correlation studies that measure the similarity between environmental samples and enable us to differentiate the Macondo oil spill from other sources.

  14. Linkage Position and Residue Identification of Disaccharides by Tandem Mass Spectrometry and Linear Discriminant Analysis

    SciTech Connect

    Hui Zhang; Steve M. Brokman; Ning Fang; Nicola L. Pohl; Edward S. Yeung

    2008-03-20

    The discrimination of isomeric disaccharides with different linkage types and different monosaccharide residues--glucose (Glc), galactose (Gal), and mannose (Man) at the non-reducing end - was investigated with tandem mass spectrometry (MS/MS) and linear discriminant analysis (LDA). Conventional matrix-assisted laser desorption/ionization (MALDI)-MS has strong interference peaks from matrix ions in the low mass region (<500 Da). This greatly limits the application of MALDI-MS for the analysis of small molecules such as saccharides. We solved this problem by using LDI with acidic fullerene matrix, which gives a very clean background in the low-mass region. Disaccharides with different linkage types give different tandem mass spectral profiles from various cross-ring fragmentation pathways. Disaccharides with the same linkage type but with three different kinds of monosaccharide residues bear the same fragmentation profiles. However, the relative ratios of the fragment ion intensities were found to be distinctly different among the three disaccharide isomers. By employing statistical tools such as LDA to classify the tandem mass spectra, disaccharide isomers with either different linkages or different monosaccharide residues were successfully classified.

  15. Linkage Position and Residue Identification of Disaccharides by Tandem mass Spectrometry and linear Discriminant Analysis

    SciTech Connect

    Hui Zhang; Steve M. Brokman; Ning Fang; Nicola L. Pohl; Edward S. Yeung

    2008-03-20

    The discrimination of isomeric disaccharides with different linkage types and different monosaccharide residues--glucose (Glc), galactose (Gal), and mannose (Man) at the non-reducing end--was investigated with tandem mass spectrometry (MS/MS) and linear discriminant analysis (LDA). Conventional matrix-assisted laser desorption/ionization (MALDI)-MS has strong interference peaks from matrix ions in the low mass region (<500 Da). This greatly limits the application of MALDI-MS for the analysis of small molecules such as saccharides. We solved this problem by using LDI with acidic fullerene matrix, which gives a very clean background in the low-mass region. Disaccharides with different linkage types give different tandem mass spectral profiles from various cross-ring fragmentation pathways. Disaccharides with the same linkage type but with three different kinds of monosaccharide residues bear the same fragmentation profiles. However, the relative ratios of the fragment ion intensities were found to be distinctly different among the three disaccharide isomers. By employing statistical tools such as LDA to classify the tandem mass spectra, disaccharide isomers with either different linkages or different monosaccharide residues were successfully classified.

  16. Shotgun collision-induced dissociation of peptides using a time of flight mass analyzer.

    PubMed

    Purvine, Samuel; Eppel, Jason-Thomas; Yi, Eugene C; Goodlett, David R

    2003-06-01

    Parallel collision-induced dissociation (CID) of peptides rather than serial, as is customary, results in loss of the obvious parent-fragment ion lineage available from CID on a single ion. We report proof-of-principle results suggesting the feasibility of parallel peptide CID, referred to here as shotgun CID, for protein identification when using the measured mass accuracies available from a time of flight mass analyzer and currently available search routines such as SEQUEST. Additionally, we report that parent-fragment ion lineage may be reconstructed from information encoded in the chromatographic single ion current traces of peptides.

  17. A tandem mass spectrometric study of bile acids: interpretation of fragmentation pathways and differentiation of steroid isomers.

    PubMed

    Qiao, Xue; Ye, Min; Liu, Chun-fang; Yang, Wen-zhi; Miao, Wen-juan; Dong, Jing; Guo, De-an

    2012-02-01

    Bile acids are steroids with a pentanoic acid substituent at C-17. They are the terminal products of cholesterol excretion, and play critical physiological roles in human and animals. Bile acids are easy to detect but difficult to identify by using mass spectrometry due to their poly-ring structure and various hydroxylation patterns. In this study, fragmentation pathways of 18 free and conjugated bile acids were interpreted by using tandem mass spectrometry. The analyses were conducted on ion trap and triple quadrupole mass spectrometers. Upon collision-induced dissociation, the conjugated bile acids could cleave into glycine or taurine related fragments, together with the steroid skeleton. Fragmentations of free bile acids were further elucidated, especially by atmospheric pressure chemical ionization mass spectrometry in positive ion mode. Aside from universally observed neutral losses, eliminations occurred on bile acid carbon rings were proposed for the first time. Moreover, four isomeric 5β-cholanic acid hydroxyl derivatives (3α,6α-, 3α,7β-, 3α,7α-, and 3α,12α-) were differentiated using electrospray ionization in negative ion mode: 3α,7β-OH substituent inclined to eliminate H(2)O and CH(2)O(2) groups; 3α,6α-OH substituent preferred neutral loss of two H(2)O molecules; 3α,12α-OH substituent apt to lose the carboxyl in the form of CO(2) molecule; and 3α,7α-OH substituent exhibited no further fragmentation after dehydration. This study provided specific interpretation for mass spectra of bile acids. The results could contribute to bile acid analyses, especially in clinical assays and metabonomic studies.

  18. Tandem mass spectrometry of coprogen and deferoxamine hydroxamic siderophores.

    PubMed

    Simionato, Ana V C; de Souza, Gezimar D; Rodrigues-Filho, Edson; Glick, James; Vouros, Paul; Carrilho, Emanuel

    2006-01-01

    Mechanisms of fragmentation of hydroxamic siderophores are proposed comparing deuterated and nondeuterated samples. Standard siderophores (e.g. deferoxamine and coprogen) were directly injected into both ion trap and linear quadrupole mass spectrometers with electrospray ionization (ESI). Four and two fragmentation steps were carried out for deferoxamine and coprogen (analyzed by positive and negative ESI, respectively). Deferoxamine cleavages occurred in both peptide and hydroxamic bonds while the coprogen fragmentation pattern is more elaborate, since it contains Fe(III) in its structure.

  19. Relative Stability of Peptide Sequence Ions Generated by Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Bythell, Benjamin J.; Hendrickson, Christopher L.; Marshall, Alan G.

    2012-04-01

    We report the use of unimolecular dissociation by infrared radiation for gaseous multiphoton energy transfer to determine relative activation energy (Ea,laser) for dissociation of peptide sequence ions. The sequence ions of interest are mass-isolated; the entire ion cloud is then irradiated with a continuous wave CO2 laser, and the first order rate constant, kd, is determined for each of a series of laser powers. Provided these conditions are met, a plot of the natural logarithm of kd versus the natural logarithm of laser power yields a straight line, whose slope provides a measure of Ea,laser. This method reproduces the Ea values from blackbody radiative dissociation (BIRD) for the comparatively large, singly and doubly protonated bradykinin ions (nominally y 9 and y 9 2+ ). The comparatively small sequence ion systems produce Ea,laser values that are systematic underestimates of theoretical barriers calculated with density functional theory (DFT). However, the relative Ea,laser values are in qualitative agreement with the mobile proton model and available theory. Additionally, novel protonated cyclic-dipeptide (diketopiperazine) fragmentation reactions are analyzed with DFT. FT-ICR MS provides access to sequence ions generated by electron capture dissociation, infrared multiphoton dissociation, and collisional activation methods (i.e., b n , y m , c n , z m • ions).

  20. A comparison of flavonoid glycosides by electrospray tandem mass spectrometry

    NASA Astrophysics Data System (ADS)

    March, Raymond E.; Lewars, Errol G.; Stadey, Christopher J.; Miao, Xiu-Sheng; Zhao, Xiaoming; Metcalfe, Chris D.

    2006-01-01

    A comparison is presented of product ion mass spectra of protonated and deprotonated molecules of kaempferol-3-O-glucoside, quercitin-3-O-glucoside (isoquercitrin), quercitin-3-O-galactoside (hyperoin), apigenin-7-O-glucoside, luteolin-7-O-glucoside, genistein-7-O-glucoside, naringenin-7-O-glucoside (prunin), luteolin-4'-O-glucoside, luteolin-6-C-glucoside (homoorientin, known also as isoorientin), apigenin-8-C-glucoside (vitexin), and luteolin-8-C-glucoside (orientin) together with the product ion mass spectrum of deprotonated kaempferol-7-O-glucoside. All isomeric ions were distinguishable on the basis of their product ion mass spectra. For protonated 3-O-, 7-O-, and 4'-O-glycosides at a collision energy of 46-47 eV, homolytic cleavage of the O-glycosidic bond yielded aglycon Y+ ions, whereas in deprotonated 3-O-, 7-O-, and 4'-O-glycosides, heterolytic and homolytic cleavage of the O-glycosidic bond yielded radical aglycon (Y-H)- and aglycon (Y-) ions. In each case, fragmentation of either the glycan or the aglycon or both was observed. For 6-C- and 8-C-glycosides at a collision energy of 46-47 eV, fragmentation was restricted almost exclusively to the glycan. For luteolin-6-C-glucoside, the integrity of the aglycon structure is preserved at the expense of the glycan for which some 30 fragmentations were observed. Breakdown curves were determined as a function of collision energy for protonated and deprotonated luteolin-6-C-glucoside. An attempt has been made to rationalize the product ion mass spectra derived from C-O- and C-C-luteolin glucosides in terms of computed structures that indicate significant intramolecular hydrogen bonding and rotation of the B-ring to form a coplanar luteolin structure. It is proposed that protonated and deprotonated luteolin-6-C-glucoside may afford examples of cooperative interactive bonding that plays a major role in directing fragmentation.

  1. Tandem Mass Spectrum Sequencing: An Alternative to Database Search Engines in Shotgun Proteomics.

    PubMed

    Muth, Thilo; Rapp, Erdmann; Berven, Frode S; Barsnes, Harald; Vaudel, Marc

    2016-01-01

    Protein identification via database searches has become the gold standard in mass spectrometry based shotgun proteomics. However, as the quality of tandem mass spectra improves, direct mass spectrum sequencing gains interest as a database-independent alternative. In this chapter, the general principle of this so-called de novo sequencing is introduced along with pitfalls and challenges of the technique. The main tools available are presented with a focus on user friendly open source software which can be directly applied in everyday proteomic workflows.

  2. Electrospray ionization collision-induced dissociation mass spectrometry: a tool to characterize synthetic polyaminocarboxylate ferric chelates used as fertilizers.

    PubMed

    Orera, Irene; Orduna, Jesús; Abadía, Javier; Alvarez-Fernández, Ana

    2010-01-01

    Fertilizers based on synthetic polyaminocarboxylate ferric chelates have been known since the 1950s to be successful in supplying Fe to plants. In commercial Fe(III)-chelate fertilizers, a significant part of the water-soluble Fe-fraction consists of still uncharacterized Fe byproducts, whose agronomical value is unknown. Although collision-induced dissociation (CID) tandem mass spectrometry (MS/MS) is a valuable tool for the identification of such compounds, no fragmentation data have been reported for most Fe(III)-chelate fertilizers. The aim of this study was to characterize the CID-MS(2) fragmentation patterns of the major synthetic Fe(III)-chelates used as Fe-fertilizers, and subsequently use this technique for the characterization of commercial fertilizers. Quadrupole-time-of-flight (QTOF) and spherical ion trap mass analyzers equipped with an electrospray ionization (ESI) source were used. ESI-CID-MS(2) spectra obtained were richer when using the QTOF device. Specific differences were found among Fe(III)-chelate fragmentation patterns, even in the case of positional isomers. The analysis of a commercial Fe(III)-chelate fertilizer by high-performance liquid chromatography (HPLC) coupled to ESI-MS(QTOF) revealed two previously unknown, Fe-containing compounds, that were successfully identified by a comprehensive comparison of the ESI-CID-MS(2)(QTOF) spectra with those of pure chelates. This shows that HPLC/ESI-CID-MS(2)(QTOF), along with the Fe(III)-chelate fragmentation patterns, could be a highly valuable tool to directly characterize the water-soluble Fe fraction in Fe(III)-chelate fertilizers. This could be of great importance in issues related to crop Fe-fertilization, both from an agricultural and an environmental point of view.

  3. Multiple losses of neutral C14H14 in the tandem mass spectrometry of several perbenzyl ether intermediates in the synthesis of green tea constituents.

    PubMed

    Lesimple, Alain; Di Falco, Marcos; Richard, Yannick; Lesimple, Souad; Wang, Zhigang; Chan, Tak Hang; Mamer, Orval A

    2005-01-01

    Electrospray and matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS) experiments were used to investigate an unusual fragmentation in collision-induced dissociation (CID) of sodiated and potassiated perbenzyl ether intermediates obtained in the total synthesis of gallate ester constituents of green tea. Prominent fragments correspond to multiple sequential losses of neutral C14H14 that were not observed in the protonated and ammoniated species, that instead present fragment ion series in which members are separated by C7H6. High-resolution MALDI quadrupole time-of-flight (Q-TOF) and electrospray-Fourier transform mass spectrometry (FTMS) were used to confirm elemental compositions of these and related ions.

  4. [Determination of homocysteine by tandem mass spectrometry with chemical ionization].

    PubMed

    Miroshnichenko, I I; Platova, A I; Safarova, T P; Iakovleva, O B

    2014-01-01

    Homocysteine (Hcy) is an intermediate of methionine metabolism. High plasma Hcy concentrations are an independent risk factor for stroke, peripheral vascular disease, deep venous thrombosis, coronary disease, and cognitive deficiency. Apparently, it is a great importance to measure Hcy levels in human blood. A new method for the quantification of Hcy by means of reversed-phase LC/atmospheric pressure chemical ionization mass spectrometry has been developed. The MRM ion transition, m/z 136.0 ® 90.0 was used for Hcy quantification. The limit of detection was 0.4 mM, quantification was performed from 1 mM to 40 mM with coefficient of determination of R2=0,997. The method was applied successfully to Hcy determination in human blood.

  5. Biological Matrix Effects in Quantitative Tandem Mass Spectrometry-Based Analytical Methods: Advancing Biomonitoring

    PubMed Central

    Panuwet, Parinya; Hunter, Ronald E.; D’Souza, Priya E.; Chen, Xianyu; Radford, Samantha A.; Cohen, Jordan R.; Marder, M. Elizabeth; Kartavenka, Kostya; Ryan, P. Barry; Barr, Dana Boyd

    2015-01-01

    The ability to quantify levels of target analytes in biological samples accurately and precisely, in biomonitoring, involves the use of highly sensitive and selective instrumentation such as tandem mass spectrometers and a thorough understanding of highly variable matrix effects. Typically, matrix effects are caused by co-eluting matrix components that alter the ionization of target analytes as well as the chromatographic response of target analytes, leading to reduced or increased sensitivity of the analysis. Thus, before the desired accuracy and precision standards of laboratory data are achieved, these effects must be characterized and controlled. Here we present our review and observations of matrix effects encountered during the validation and implementation of tandem mass spectrometry-based analytical methods. We also provide systematic, comprehensive laboratory strategies needed to control challenges posed by matrix effects in order to ensure delivery of the most accurate data for biomonitoring studies assessing exposure to environmental toxicants. PMID:25562585

  6. The expanding role of tandem mass spectrometry in optimizing diagnosis and treatment of thyroid disease.

    PubMed

    van Deventer, Hendrick E; Soldin, Steven J

    2013-01-01

    This review discusses the state-of-the-art measurement of free and total thyroid hormones in clinical laboratories. We highlight some of the limitations of currently used immunoassays and critically discuss physical separation methods for the measurement of free thyroid hormone. Physical separation methods, such as equilibrium dialysis or ultrafiltration, followed by tandem mass spectrometry for the measurement of free thyroid hormones offer many advantages, which we feel, can deepen our understanding of thyroid hormone metabolism and improve patient diagnosis and care. Problems with direct analogue immunoassay methods for FT4/FT3 as well as immunoassay methods for total T3 at low T3 concentrations and during pregnancy are highlighted. Improved diagnosis and patient management can be achieved utilizing tandem mass spectrometry for these measurements.

  7. Ion Mobility Tandem Mass Spectrometry Enhances Performance of Bottom-up Proteomics

    PubMed Central

    Helm, Dominic; Vissers, Johannes P. C.; Hughes, Christopher J.; Hahne, Hannes; Ruprecht, Benjamin; Pachl, Fiona; Grzyb, Arkadiusz; Richardson, Keith; Wildgoose, Jason; Maier, Stefan K.; Marx, Harald; Wilhelm, Mathias; Becher, Isabelle; Lemeer, Simone; Bantscheff, Marcus; Langridge, James I.; Kuster, Bernhard

    2014-01-01

    One of the limiting factors in determining the sensitivity of tandem mass spectrometry using hybrid quadrupole orthogonal acceleration time-of-flight instruments is the duty cycle of the orthogonal ion injection system. As a consequence, only a fraction of the generated fragment ion beam is collected by the time-of-flight analyzer. Here we describe a method utilizing postfragmentation ion mobility spectrometry of peptide fragment ions in conjunction with mobility time synchronized orthogonal ion injection leading to a substantially improved duty cycle and a concomitant improvement in sensitivity of up to 10-fold for bottom-up proteomic experiments. This enabled the identification of 7500 human proteins within 1 day and 8600 phosphorylation sites within 5 h of LC-MS/MS time. The method also proved powerful for multiplexed quantification experiments using tandem mass tags exemplified by the chemoproteomic interaction analysis of histone deacetylases with Trichostatin A. PMID:25106551

  8. Large Scale Discovery and De Novo-Assisted Sequencing of Cationic Antimicrobial Peptides (CAMPs) by Microparticle Capture and Electron-Transfer Dissociation (ETD) Mass Spectrometry.

    PubMed

    Juba, Melanie L; Russo, Paul S; Devine, Megan; Barksdale, Stephanie; Rodriguez, Carlos; Vliet, Kent A; Schnur, Joel M; van Hoek, Monique L; Bishop, Barney M

    2015-10-02

    The identification and sequencing of novel cationic antimicrobial peptides (CAMPs) have proven challenging due to the limitations associated with traditional proteomics methods and difficulties sequencing peptides present in complex biomolecular mixtures. We present here a process for large-scale identification and de novo-assisted sequencing of newly discovered CAMPs using microparticle capture followed by tandem mass spectrometry equipped with electron-transfer dissociation (ETD). This process was initially evaluated and verified using known CAMPs with varying physicochemical properties. The effective parameters were then applied in the analysis of a complex mixture of peptides harvested from American alligator plasma using custom-made (Bioprospector) functionalized hydrogel particles. Here, we report the successful sequencing process for CAMPs that has led to the identification of 340 unique peptides and the discovery of five novel CAMPs from American alligator plasma.

  9. Liquid Chromatography-Tandem Mass Spectrometry to Define Sortase Cleavage Products.

    PubMed

    Duong, Andrew; Koteva, Kalinka; Sexton, Danielle L; Elliot, Marie A

    2016-01-01

    Sortase enzymes have specific endopeptidase activity, cleaving within a defined pentapeptide sequence at the C-terminal end of their protein substrates. Here, we describe how monitoring sortase cleavage activity can be achieved using peptide substrates. Peptide cleavage can be readily analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS), which allows for the precise definition of cleavage sites. This technique could be used to analyze the peptidase activity of any enzyme, and identify sites of cleavage within any peptide.

  10. Half-life of Si-32 from tandem-accelerator mass spectrometry

    NASA Technical Reports Server (NTRS)

    Elmore, D.; Anantaraman, N.; Fulbright, H. W.; Gove, H. E.; Nishiizumi, K.; Murrell, M. T.; Honda, M.; Hans, H. S.

    1980-01-01

    A newly developed mass-spectrometry technique employing a tandem Van de Graaff accelerator together with a special beam-transport system and heavy-ion detector has been used to determine the half-life of Si-32. The result obtained, 108 plus or minus 18 yr, disagrees with the accepted value of 330 plus or minus 40 yr. The implications of the new half-life of Si-32, which is used for dating studies, are discussed.

  11. Fast multi-blind modification search through tandem mass spectrometry.

    PubMed

    Na, Seungjin; Bandeira, Nuno; Paek, Eunok

    2012-04-01

    With great biological interest in post-translational modifications (PTMs), various approaches have been introduced to identify PTMs using MS/MS. Recent developments for PTM identification have focused on an unrestrictive approach that searches MS/MS spectra for all known and possibly even unknown types of PTMs at once. However, the resulting expanded search space requires much longer search time and also increases the number of false positives (incorrect identifications) and false negatives (missed true identifications), thus creating a bottleneck in high throughput analysis. Here we introduce MODa, a novel "multi-blind" spectral alignment algorithm that allows for fast unrestrictive PTM searches with no limitation on the number of modifications per peptide while featuring over an order of magnitude speedup in relation to existing approaches. We demonstrate the sensitivity of MODa on human shotgun proteomics data where it reveals multiple mutations, a wide range of modifications (including glycosylation), and evidence for several putative novel modifications. Based on the reported findings, we argue that the efficiency and sensitivity of MODa make it the first unrestrictive search tool with the potential to fully replace conventional restrictive identification of proteomics mass spectrometry data.

  12. Fast Multi-blind Modification Search through Tandem Mass Spectrometry*

    PubMed Central

    Na, Seungjin; Bandeira, Nuno; Paek, Eunok

    2012-01-01

    With great biological interest in post-translational modifications (PTMs), various approaches have been introduced to identify PTMs using MS/MS. Recent developments for PTM identification have focused on an unrestrictive approach that searches MS/MS spectra for all known and possibly even unknown types of PTMs at once. However, the resulting expanded search space requires much longer search time and also increases the number of false positives (incorrect identifications) and false negatives (missed true identifications), thus creating a bottleneck in high throughput analysis. Here we introduce MODa, a novel “multi-blind” spectral alignment algorithm that allows for fast unrestrictive PTM searches with no limitation on the number of modifications per peptide while featuring over an order of magnitude speedup in relation to existing approaches. We demonstrate the sensitivity of MODa on human shotgun proteomics data where it reveals multiple mutations, a wide range of modifications (including glycosylation), and evidence for several putative novel modifications. Based on the reported findings, we argue that the efficiency and sensitivity of MODa make it the first unrestrictive search tool with the potential to fully replace conventional restrictive identification of proteomics mass spectrometry data. PMID:22186716

  13. Assigning Peptide Disulfide Linkage Pattern Among Regio-Isomers via Methoxy Addition to Disulfide and Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Durand, Kirt L.; Tan, Lei; Stinson, Craig A.; Love-Nkansah, Chasity B.; Ma, Xiaoxiao; Xia, Yu

    2017-02-01

    Pinpointing disulfide linkage pattern is critical in the characterization of proteins and peptides consisting of multiple disulfide bonds. Herein, we report a method based on coupling online disulfide modification and tandem mass spectrometry (MS/MS) to distinguish peptide disulfide regio-isomers. Such a method relies on a new disulfide bond cleavage reaction in solution, involving methanol as a reactant and 254 nm ultraviolet (UV) irradiation. This reaction leads to selective cleavage of a disulfide bond and formation of sulfenic methyl ester (-SOCH3) at one cysteine residue and a thiol (-SH) at the other. Under low energy collision-induced dissociation (CID), cysteine sulfenic methyl ester motif produces a signature methanol loss (-32 Da), allowing its identification from other possible isomeric structures such as S-hydroxylmethyl (-SCH2OH) and methyl sulfoxide (-S(O)-CH3). Since disulfide bond can be selectively cleaved and modified upon methoxy addition, subsequent MS2 CID of the methoxy addition product provides enhanced sequence coverage as demonstrated by the analysis of bovine insulin. More importantly, this reaction does not induce disulfide scrambling, likely due to the fact that radical intermediates are not involved in the process. An approach based on methoxy addition followed by MS3 CID has been developed for assigning disulfide linkage patterns in peptide disulfide regio-isomers. This methodology was successfully applied to characterizing peptide systems having two disulfide bonds and three disulfide linkage isomers: side-by-side, overlapped, and looped-within-a-loop configurations.

  14. Typing of blood-group antigens on neutral oligosaccharides by negative-ion electrospray ionization tandem mass spectrometry.

    PubMed

    Zhang, Hongtao; Zhang, Shuang; Tao, Guanjun; Zhang, Yibing; Mulloy, Barbara; Zhan, Xiaobei; Chai, Wengang

    2013-06-18

    Blood-group antigens, such as those containing fucose and bearing the ABO(H)- and Lewis-type determinants expressed on the carbohydrate chains of glycoproteins and glycolipids, and also on unconjugated free oligosaccharides in human milk and other secretions, are associated with various biological functions. We have previously shown the utility of negative-ion electrospay ionization tandem mass spectrometry with collision-induced dissociation (ESI-CID-MS/MS) for typing of Lewis (Le) determinants, for example, Le(a), Le(x), Le(b), and Le(y) on neutral and sialylated oligosaccharide chains. In the present report, we extended the strategy to characterization of blood-group A-, B-, and H-determinants on type 1 and type 2 and also on type 4 globoside chains to provide a high sensitivity method for typing of all the major blood-group antigens, including the A, B, H, Le(a), Le(x), Le(b), and Le(y) determinants, present in oligosaccharides. Using the principles established, we identified two minor unknown oligosaccharide components present in the products of enzymatic synthesis by bacterial fermentation. We also demonstrated that the unique fragmentations derived from the D- and (0,2)A-type cleavages observed in ESI-CID-MS/MS, which are important for assigning blood-group and chain types, only occur under the negative-ion conditions for reducing sugars but not for reduced alditols or under positive-ion conditions.

  15. Evaluation and optimization of electron capture dissociation efficiency in fourier transform ion cyclotron resonance mass spectrometry.

    PubMed

    McFarland, Melinda A; Chalmers, Michael J; Quinn, John P; Hendrickson, Christopher L; Marshall, Alan G

    2005-07-01

    Electron capture dissociation (ECD) efficiency has typically been lower than for other dissociation techniques. Here we characterize experimental factors that limit ECD and seek to improve its efficiency. Efficiency of precursor to product ion conversion was measured for a range of peptide (approximately 15% efficiency) and protein (approximately 33% efficiency) ions of differing sizes and charge states. Conversion of precursor ions to products depends on electron irradiation period and maximizes at approximately 5-30 ms. The optimal irradiation period scales inversely with charge state. We demonstrate that reflection of electrons through the ICR cell is more efficient and robust than a single pass, because electrons can cool to the optimal energy for capture, which allows for a wide range of initial electron energy. Further, efficient ECD with reflected electrons requires only a short (approximately 500 micros) irradiation period followed by an appropriate delay for cooling and interaction. Reflection of the electron beam results in electrons trapped in or near the ICR cell and thus requires a brief (approximately 50 micros) purge for successful mass spectral acquisition. Further electron irradiation of refractory precursor ions did not result in further dissociation. Possibly the ion cloud and electron beam are misaligned radially, or the electron beam diameter may be smaller than that of the ion cloud such that remaining precursor ions do not overlap with the electron beam. Several ion manipulation techniques and use of a large, movable dispenser cathode reduce the possibility that misalignment of the ion and electron beams limits ECD efficiency.

  16. An integrated microfluidics-tandem mass spectrometry system for automated protein analysis.

    PubMed

    Figeys, D; Gygi, S P; McKinnon, G; Aebersold, R

    1998-09-15

    We describe an integrated analytical system consisting of a microfluidics device micromachined using photolithography/etching technology, a panel of computer-controlled high-voltage relays, and an electrospray ionization tandem mass spectrometer. Movement of solvents and samples on the device and off the device to the mass spectrometer was achieved by directed electroosmotic pumping induced by the activation of a suitable constellation of high-voltage relays. The system was used for the sequential automated analysis of protein digests. We demonstrate low femtomole per microliter sensitivity of detection and compatibility of the system with the automated analysis of proteins separated by two-dimensional gel electrophoresis.

  17. Derivatization reagents in liquid chromatography/electrospray ionization tandem mass spectrometry.

    PubMed

    Santa, Tomofumi

    2011-01-01

    Liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) is one of the most prominent analytical techniques owing to its inherent selectivity and sensitivity. In LC/ESI-MS/MS, chemical derivatization is often used to enhance the detection sensitivity. Derivatization improves the chromatographic separation, and enhances the mass spectrometric ionization efficiency and MS/MS detectability. In this review, an overview of the derivatization reagents which have been applied to LC/ESI-MS/MS is presented, focusing on the applications to low molecular weight compounds.

  18. Electrospray Ionization Tandem Mass Spectrometry (ESI-MS/MS)-Based Shotgun Lipidomics

    SciTech Connect

    Mezengie, Giorgis I.

    2011-01-11

    In the past decade, many new strategies for mass spectrometry (MS)-based analyses of lipids have been developed. Lipidomics is one of the most promising research fields to emerge as a result of these advances in MS. Currently, mass spectrometric analysis of lipids involves two complementary approaches: direct infusion (shotgun lipidomics) and liquid chromatography coupled to MS. In this chapter, I will demonstrate the approach of shotgun lipidomics using electrospray ionization tandem MS for the analysis of lipid molecular species directly from crude biological extracts of tissue or fluids.

  19. Methylation of acidic moieties in poly(methyl methacrylate-co-methacrylic acid) copolymers for end-group characterization by tandem mass spectrometry.

    PubMed

    Giordanengo, Rémi; Viel, Stéphane; Hidalgo, Manuel; Allard-Breton, Béatrice; Thévand, André; Charles, Laurence

    2010-07-30

    The complete structural characterization of a copolymer composed of methacrylic acid (MAA) and methyl methacrylate (MMA) units was achieved using tandem mass spectrometry. In a first step, collision-induced dissociation (CID) of sodiated MAA-MMA co-oligomers allowed us to determine the co-monomeric composition, the random nature of the copolymer and the sum of the end-group masses. However, dissociation reactions of MAA-based molecules mainly involve the acidic pendant groups, precluding individual characterization of the end groups. Therefore, methylation of all the acrylic acid moieties was performed to transform the MAA-MMA copolymer into a PMMA homopolymer, for which CID mainly proceeds via backbone cleavages. Using trimethylsilyldiazomethane as a derivatization agent, this methylation reaction was shown to be complete without affecting the end groups. Using fragmentation rules established for PMMA polymers together with accurate mass measurements of the product ions and knowledge of reagents used for the studied copolymer synthesis, a structure could be proposed for both end groups and it was found to be consistent with signals obtained in nuclear magnetic resonance spectra.

  20. Analysis of amprolium by hydrophilic interaction liquid chromatography-tandem mass spectrometry.

    PubMed

    Martínez-Villalba, Anna; Moyano, Encarnación; Galceran, M Teresa

    2010-09-10

    We present a fast liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of the coccidiostat amprolium in food samples. Tandem mass spectrometry in a triple quadrupole was used for quantitative purposes, and the information from multiple-stage mass spectrometry in an ion-trap mass analyzer contributed to fragmentation studies. Hydrophilic interaction liquid chromatography (HILIC) in a Fused-Core column using isocratic elution (acetonitrile:formic acid/ammonium formate buffer pH 4, 50 mM (60:40)) successfully analyzed this compound in less than 3 min. The HILIC system was coupled to heated electrospray-MS/MS using highly selective-selected reaction monitoring (H-SRM) to improve sensitivity and selectivity for the analysis of amprolium, after a simple sample treatment based on an "extract and shoot" strategy. Accurate mass measurements were performed to identify the interfering compound responsible for causing matrix ion enhancement in the signal of amprolium. The addition of l-carnitine (the interfering compound) (1 microg L(-1)) to standards and sample extracts allowed the use of the external calibration method for quantitative purposes. The LC-MS/MS (H-SRM) method showed good precision (relative standard deviation, RSD, lower than 13%), accuracy and linearity and allowed the determination of amprolium down to the ppb level (LODs between 0.1 and 0.6 microg kg(-1)).

  1. Quantitative Caffeine Analysis Using a Surface Sampling Probe Electrospray Ionization Tandem Mass Spectrometry System

    SciTech Connect

    Ford, Michael J; Deibel, Michael A.; Tomkins, Bruce A; Van Berkel, Gary J

    2005-01-01

    Quantitative determination of caffeine on reversed-phase C8 thin-layer chromatography plates using a surface sampling electrospray ionization system with tandem mass spectrometry detection is reported. The thin-layer chromatography/electrospray tandem mass spectrometry method employed a deuterium-labeled caffeine internal standard and selected reaction monitoring detection. Up to nine parallel caffeine bands on a single plate were sampled in a single surface scanning experiment requiring 35 min at a surface scan rate of 44 {mu}m/s. A reversed-phase HPLC/UV caffeine assay was developed in parallel to assess the mass spectrometry method performance. Limits of detection for the HPLC/UV and thin-layer chromatography/electrospray tandem mass spectrometry methods determined from the calibration curve statistics were 0.20 ng injected (0.50 {mu}L) and 1.0 ng spotted on the plate, respectively. Spike recoveries with standards and real samples ranged between 97 and 106% for both methods. The caffeine content of three diet soft drinks (Diet Coke, Diet Cherry Coke, Diet Pepsi) and three diet sport drinks (Diet Turbo Tea, Speed Stack Grape, Speed Stack Fruit Punch) was measured. The HPLC/UV and mass spectrometry determinations were in general agreement, and these values were consistent with the quoted values for two of the three diet colas. In the case of Diet Cherry Coke and the diet sports drinks, the determined caffeine amounts using both methods were consistently higher (by 8% or more) than the literature values.

  2. Analysis of phytochelatin-cadmium complexes from plant tissue culture using nano-electrospray ionization tandem mass spectrometry and capillary liquid chromatography/electrospray ionization tandem mass spectrometry.

    PubMed

    Yen, T Y; Villa, J A; DeWitt, J G

    1999-09-01

    Phytochelatins (PCs, also known as class III metallothioneins), a family of sulfhydryl-rich peptides with the formula (gamma-GluCys)(n)Gly(Pc(n), n = 2-11), are induced in plants, yeast and fungi exposed to heavy metals, and are thought to detoxify metals by forming PC- metal complexes. Although PCs have been detected, PC- metal complexes have not been well characterized. In this work, nano-electrospray ionization tandem mass spectrometry (nano-ESI-MS/MS) and capillary liquid chromatography/electrospray ionization tandem mass spectrometry (capillary LC/ESI-MS/MS) methods were used to analyze PC - Cd complexes isolated from Datura innoxia, also known as Jimsonweed, cell culture exposed to Cd. With nano-ESI-MS/MS and capillary LC/ESI-MS/MS we could simultaneously detect the presence of PCs and PC - Cd complexes from plant cell extracts, unambiguously identify these species and elucidate the nature of individual PC - Cd complexes. Phytochelatins with n = 3-6 were detected, as were PC - Cd complexes with PC(3), PC(4) and PC(5). This is the first study to report the size and nature of native PC - Cd complexes from plant tissue samples. These results demonstrate that the direct analysis of plant extracts using nano-ESI-MS/MS and capillary LC/ESI-MS/MS methods is simple and sensitive to the range of PCs and PC - Cd complexes in plants. Hence these methods open up new opportunities for further quantitative analysis of PCs and PC - metal complexes in cell culture and plant systems to understand the relationship between the biosynthesis of these compounds and metal tolerance.

  3. Gas-phase dissociation of ionic liquid aggregates studied by electrospray ionisation mass spectrometry and energy-variable collision induced dissociation.

    PubMed

    Fernandes, Ana M; Coutinho, João A P; Marrucho, Isabel M

    2009-01-01

    Positive singly charged ionic liquid aggregates [(C(n)mim)(m+1)(BF(4))(m)](+) (mim = 3-methylimidazolium; n = 2, 4, 8 and 10) and [(C(4)mim)(m+1)(A)(m)](+) (A = Cl(-), BF(4) (-), PF(6) (-), CF(3)SO(3) (-) and (CF(3)SO(2))(2)N(-)) were investigated by electrospray ionisation mass spectrometry and energy-variable collision induced dissociation. The electrospray ionisation mass spectra (ESI-MS) showed the formation of an aggregate with extra stability for m = 4 for all the ionic liquids with the exception of [C(4)mim][CF(3)SO(3)]. ESI-MS-MS and breakdown curves of aggregate ions showed that their dissociation occurred by loss of neutral species ([C(n)mim][A])(a) with a >or= 1. Variable-energy collision induced dissociation of each aggregate from m = 1 to m = 8 for all the ionic liquids studied enabled the determination of E(cm, 1/2) values, whose variation with m showed that the monomers were always kinetically much more stable than the larger aggregates, independently of the nature of cation and anion. The centre-of-mass energy values correlate well with literature data on ionic volumes and interaction and hydrogen bond energies.

  4. A novel aircraft-based tandem mass spectrometer for atmospheric ion and trace gas measurements

    NASA Astrophysics Data System (ADS)

    Moehler, O.; Reiner, Th.; Arnold, F.

    1993-05-01

    The general design and operation of a novel aircraft-based triple-quadrupole mass spectrometer (TQMS) developed for the improved detection and collisional analysis of atmospheric ions and trace gases are described. The instrument is also suitable for laboratory collision-induced dissociation measurements, studies of ion-molecule reactions, and analytical applications. Highly sensitive and selective trace gas detection by chemical ionization mass spectrometry is also possible using a novel ion injection technique. Result of aircraft-based measurements made with the TQMS are summarized.

  5. A novel approach to collision-induced dissociation (CID) for ion mobility-mass spectrometry experiments.

    PubMed

    Becker, Christopher; Fernandez-Lima, Francisco A; Gillig, Kent J; Russell, William K; Cologna, Stephanie M; Russell, David H

    2009-06-01

    Collision induced dissociation (CID) combined with matrix assisted laser desorption ionization-ion mobility-mass spectrometry (MALDI-IM-MS) is described. In this approach, peptide ions are separated on the basis of mobility in a 15 cm drift cell. Following mobility separation, the ions exit the drift cell and enter a 5 cm vacuum interface with a high field region (up to 1000 V/cm) to undergo collisional activation. Ion transmission and ion kinetic energies in the interface are theoretically evaluated accounting for the pressure gradient, interface dimensions, and electric fields. Using this CID technique, we have successfully fragmented and sequenced a number of model peptide ions as well as peptide ions obtained by a tryptic digest. This instrument configuration allows for the simultaneous determination of peptide mass, peptide-ion sequence, and collision-cross section of MALDI-generated ions, providing information critical to the identification of unknown components in complex proteomic samples.

  6. Improving collision induced dissociation (CID), high energy collision dissociation (HCD), and electron transfer dissociation (ETD) fourier transform MS/MS degradome-peptidome identifications using high accuracy mass information.

    PubMed

    Shen, Yufeng; Tolić, Nikola; Purvine, Samuel O; Smith, Richard D

    2012-02-03

    MS dissociation methods, including collision induced dissociation (CID), high energy collision dissociation (HCD), and electron transfer dissociation (ETD), can each contribute distinct peptidome identifications using conventional peptide identification methods (Shen et al. J. Proteome Res. 2011), but such samples still pose significant informatics challenges. In this work, we explored utilization of high accuracy fragment ion mass measurements, in this case provided by Fourier transform MS/MS, to improve peptidome peptide data set size and consistency relative to conventional descriptive and probabilistic scoring methods. For example, we identified 20-40% more peptides than SEQUEST, Mascot, and MS_GF scoring methods using high accuracy fragment ion information and the same false discovery rate (FDR) from CID, HCD, and ETD spectra. Identified species covered >90% of the collective identifications obtained using various conventional peptide identification methods, which significantly addresses the common issue of different data analysis methods generating different peptide data sets. Choice of peptide dissociation and high-precision measurement-based identification methods presently available for degradomic-peptidomic analyses needs to be based on the coverage and confidence (or specificity) afforded by the method, as well as practical issues (e.g., throughput). By using accurate fragment information, >1000 peptidome components can be identified from a single human blood plasma analysis with low peptide-level FDRs (e.g., 0.6%), providing an improved basis for investigating potential disease-related peptidome components.

  7. High efficiency tandem mass spectrometry analysis using dual linear ion traps.

    PubMed

    Li, Linfan; Zhou, Xiaoyu; Hager, James W; Ouyang, Zheng

    2014-10-07

    Tandem mass spectrometry (MS/MS) plays an essential role in modern chemical analysis. It is used for differentiating isomers and isobars and suppressing chemical noise, which allows high precision quantitation. The MS/MS analysis has been typically applied by isolating the target precursor ions, while disregarding other ions, followed by a fragmentation process that produces the product ions. In this study, configurations of dual linear ion traps were explored to develop high efficiency MS/MS analysis. The ions trapped in the first linear ion trap were axially, mass-selectively transferred to the second linear ion trap for MS/MS analysis. Ions from multiple compounds simultaneously introduced into the mass spectrometer could be sequentially analyzed. This development enables highly efficient use of the sample. For miniature ion trap mass spectrometers with discontinuous atmospheric pressure interfaces, the analysis speed and the quantitation precision can be significantly improved.

  8. Doping control analysis of anabolic steroids in equine urine by gas chromatography-tandem mass spectrometry.

    PubMed

    Wong, April S Y; Leung, Gary N W; Leung, David K K; Wan, Terence S M

    2016-09-08

    Anabolic steroids are banned substances in equine sports. Gas chromatography-mass spectrometry (GC-MS) has been the traditional technique for doping control analysis of anabolic steroids in biological samples. Although liquid chromatography-mass spectrometry (LC/MS) has become an important technique in doping control, the detection of saturated hydroxysteroids by LC-MS remains a problem due to their low ionization efficiency under electrospray. The recent development in fast-scanning gas-chromatography-triple-quadrupole mass spectrometry (GC-MS/MS) has provided a better alternative with a significant reduction in chemical noise by means of selective reaction monitoring. Herein, we present a sensitive and selective method for the screening of over 50 anabolic steroids in equine urine using gas chromatography-tandem mass spectrometry (GC-MS/MS). Copyright © 2016 John Wiley & Sons, Ltd.

  9. Detailed Study of Cyanobacterial Microcystins Using High Performance Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Qi, Yulin; Bortoli, Stella; Volmer, Dietrich A.

    2014-07-01

    Microcystins (MC) are a large group of toxic cyclic peptides, produced by cyanobacteria in eutrophic water systems. Identification of MC variants mostly relies on liquid chromatography (LC) combined with collision-induced dissociation (CID) mass spectrometry. Deviations from the essential amino acid complement are a common feature of these natural products, which makes the CID analysis more difficult and not always successful. Here, both CID and electron capture dissociation (ECD) were applied in combination with ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry to study a cyanobacteria strain isolated from the Salto Grande Reservoir in Sao Paulo State, Brazil, without prior LC separation. CID was shown to be an effective dissociation technique for quickly identifying the MC variants, even those that have previously been difficult to characterize by CID. Moreover, ECD provided even more detailed and complementary information, which enabled us to precisely locate metal binding sites of MCs for the first time. This additional information will be important for environmental chemists to study MC accumulation and production in ecosystems.

  10. Higher energy collision dissociation (HCD) product ion-triggered electron transfer dissociation (ETD) mass spectrometry for the analysis of N-linked glycoproteins.

    PubMed

    Singh, Charandeep; Zampronio, Cleidiane G; Creese, Andrew J; Cooper, Helen J

    2012-09-07

    Large scale mass spectrometry analysis of N-linked glycopeptides is complicated by the inherent complexity of the glycan structures. Here, we evaluate a mass spectrometry approach for the targeted analysis of N-linked glycopeptides in complex mixtures that does not require prior knowledge of the glycan structures or pre-enrichment of the glycopeptides. Despite the complexity of N-glycans, the core of the glycan remains constant, comprising two N-acetylglucosamine and three mannose units. Collision-induced dissociation (CID) mass spectrometry of N-glycopeptides results in the formation of the N-acetylglucosamine (GlcNAc) oxonium ion and a [mannose+GlcNAc] fragment (in addition to other fragments resulting from cleavage within the glycan). In ion-trap CID, those ions are not detected due to the low m/z cutoff; however, they are detected following the beam-type CID known as higher energy collision dissociation (HCD) on the orbitrap mass spectrometer. The presence of these product ions following HCD can be used as triggers for subsequent electron transfer dissociation (ETD) mass spectrometry analysis of the precursor ion. The ETD mass spectrum provides peptide sequence information, which is unobtainable from HCD. A Lys-C digest of ribonuclease B and trypsin digest of immunoglobulin G were separated by ZIC-HILIC liquid chromatography and analyzed by HCD product ion-triggered ETD. The data were analyzed both manually and by search against protein databases by commonly used algorithms. The results show that the product ion-triggered approach shows promise for the field of glycoproteomics and highlight the requirement for more sophisticated data mining tools.

  11. Efficient Polyatomic Interference Reduction in Plasma-Source Mass Spectrometry Via Collision Induced Dissociation

    SciTech Connect

    Jackson, Glen P.; King, Fred L; Duckworth, Douglas {Doug} C

    2003-02-01

    Evidence is provided that illustrates quadrupole ion traps can be used to selectively attenuate strongly bound diatomic ions occurring at the same nominal mass as an analyte ion of interest. Dissociation rates for TaO{sup +} (D{sub 0} {approx} 750 kJ mol{sup -1}) are found to be at least an order of magnitude larger than the loss rate of Au{sup +} due to scattering under 'slow heating' resonance excitation conditions at q{sub z} {approx} 0.67 and using neon as the bath gas. This rate difference is sufficient for the selective removal of this strongly-bound diatomic ion over the loss of the Au{sup +} at the same mass-to-charge ratio. Other examples of quadrupole ion trap CID for the selective reduction of common plasma-generated species are also evaluated by examining the dissociation of GdO{sup +} in the presence of Yb{sup +}, and Cu{sub 2}{sup +} in the presence of Te{sup +}. In each case, a different method of applying the excitation signals is presented, and the attenuation rates for the diatomic species due to CID are substantially larger than scattering losses for the bare metal ions. Evidence is also presented that demonstrates CID can be accomplished in concert with a slow mass analysis scan, thereby providing a means of (1) eliminating polyatomic ions (formed in the plasma or reaction cell) over an extended mass range, (2) recovering metal ion signal from the metal-containing polyatomic ions, and (3) minimizing deleterious secondary reactions of product ions.

  12. Tandem mass spectrometry approach for the investigation of the steroidal metabolism: structure-fragmentation relationship (SFR) in anabolic steroids and their metabolites by ESI-MS/MS analysis.

    PubMed

    Musharraf, Syed Ghulam; Ali, Arslan; Khan, Naik Tameem; Yousuf, Maria; Choudhary, Muhammad Iqbal; Atta-ur-Rahman

    2013-02-01

    Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used to investigate the effect of different substitutions introduced during metabolism on fragmentation patterns of four anabolic steroids including methyltestosterone, methandrostenolone, cis-androsterone and adrenosterone, along with their metabolites. Collision-induced dissociation (CID) analysis was performed to correlate the major product ions of 19 steroids with structural features. The analysis is done to portray metabolic alteration, such as incorporation or reduction of double bonds, hydroxylations, and/or oxidation of hydroxyl moieties to keto functional group on steroidal skeleton which leads to drastically changed product ion spectra from the respective classes of steroids, therefore, making them difficult to identify. The comparative ESI-MS/MS study also revealed some characteristic peaks to differentiate different steroidal metabolites and can be useful for the unambiguous identification of anabolic steroids in biological fluid. Moreover, LC-ESI-MS/MS analysis of fermented extract of methyltestosterone, obtained by Macrophomina phaseolina was also investigated.

  13. Characterization of the triphenylphosphonium derivative of peptides by fast atom bombardment-tandem mass spectrometry, and investigations of the mechanisms of fragmentation of peptides

    SciTech Connect

    Wagner, D.S.

    1992-01-01

    Fast atom bombardment collisionally activated dissociation tandem mass spectrometry is a powerful technique for the determination of the primary structure of peptides. However, there are factors that frequently prevent successful sequence analysis by mass spectrometry. Two such factors are the poor ionization efficiency of some hydrophilic peptides and, for many peptides, ambiguities in interpretation of the spectra when key sequence ions are weak or absent. Novel and simple procedures for preparing ethyl-triphenylphosphonium derivatives of peptides are described. These procedures allow an ethyl-triphenylphosphonium moiety to be selectively attached to either the N- or C-terminus. Modification of peptides by these chemical methods significantly enhances the efficiency of fast atom bombardment ionization. Moreover, upon collisionally activated dissociation, the derivatized peptides generate a predictable series of sequence ions from either the C-terminus or the N-terminus, depending on the location of the ethyl-triphenylphosphonium moiety. The potential utility of the ethyl-triphenylphosphonium derivative in structure elucidation is illustrated by a comparison of the mass spectra of underivatized and derivatized peptides containing up to 20 amino acid residues, or contain an N-terminal blocking group, or contain a phosphate group, or contain a disulfide bond, or contain a backbone modification. When protonated peptide molecules and cationized peptide molecules are subjected to high-energy collisionally activated dissociation, skeletal bonds cleave generating sequence-specific fragment ions. These bond cleavages usually involve H-shifts. The utility of selective deuterium labeling was applied here to elucidate fragmentation mechanisms. Skeletal bond cleavages in the ionized peptide H-VGVAPG-OH were investigated, in which the molecule was analyzed in the protonated form, cationized form, or as the charge-localized ethyl-triphenylphosphonium derivative.

  14. Quantitative profiling of bacteriocins present in dairy-free probiotic preparations of Lactobacillus acidophilus by nanoliquid chromatography-tandem mass spectrometry.

    PubMed

    Nandakumar, Renu; Talapatra, Kesh

    2014-01-01

    Bacteriocins are a heterogeneous group of ribosomally synthesized peptides or proteins with antimicrobial activity, produced predominantly by lactic acid bacteria, with potential applications as biopreservatives and probiotics. We describe here a novel strategy based on a bottom-up, shotgun proteomic approach using nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) with multiple fragmentation techniques for the quantitative profiling of bacteriocins present in the probiotic preparations of Lactobacillus acidophilus. A direct LC-MS/MS analysis with alternate collision-induced dissociation, high-energy collision dissociation, and electron-transfer dissociation fragmentation following a filter-assisted size-exclusion sample prefractionation has resulted in the identification of peptides belonging to 37 bacteriocins or related proteins. Peptides from lactacin F, helveticin J, lysin, avicin A, acidocin M, curvaticin FS47, and carocin D were predominant. The process of freeze drying under vacuum was observed to affect both the diversity and abundance of bacteriocins. Data acquisition using alternating complementary peptide fragmentation modes, especially electron-transfer dissociation, has significantly enhanced the peptide sequence coverage and number of bacteriocin peptides identified. Multi-enzyme proteolytic digestion was observed to increase the sample complexity and dynamic range, lowering the chances of detection of low-abundant bacteriocin peptides by LC-MS/MS. An analytical platform integrating size exclusion prefractionation, nanoLC-MS/MS analysis with multiple fragmentation techniques, and data-dependent decision tree-driven bioinformatic data analysis is novel in bacteriocin research and suitable for the comprehensive bioanalysis of diverse, low-abundant bacteriocins in complex samples.

  15. Ozone-induced dissociation: elucidation of double bond position within mass-selected lipid ions.

    PubMed

    Thomas, Michael C; Mitchell, Todd W; Harman, David G; Deeley, Jane M; Nealon, Jessica R; Blanksby, Stephen J

    2008-01-01

    Ions formed from lipids during electrospray ionization of crude lipid extracts have been mass-selected within a quadrupole linear ion trap mass spectrometer and allowed to react with ozone vapor. Gas-phase ion-molecule reactions between unsaturated lipid ions and ozone are found to yield two primary product ions for each carbon-carbon double bond within the molecule. The mass-to-charge ratios of these chemically induced fragments are diagnostic of the position of unsaturation within the precursor ion. This novel analytical technique, dubbed ozone-induced dissociation (OzID), can be applied both in series and in parallel with conventional collision-induced dissociation (CID) to provide near-complete structural assignment of unknown lipids within complex mixtures without prior fractionation or derivatization. In this study, OzID is applied to a suite of complex lipid extracts from sources including human lens, bovine kidney, and commercial olive oil, thus demonstrating the technique to be applicable to a broad range of lipid classes including both neutral and acidic glycerophospholipids, sphingomyelins, and triacylglycerols. Gas-phase ozonolysis reactions are also observed with different types of precursor ions including [M+H]+, [M+Li]+, [M+Na]+, and [M-H]-: in each case yielding fragmentation data that allow double bond position to be unambiguously assigned. Within the human lens lipid extract, three sphingomyelin regioisomers, namely SM(d18:0/15Z-24:1), SM(d18:0/17Z-24:1), and SM(d18:0/19Z-24:1), and a novel phosphatidylethanolamine alkyl ether, GPEtn(11Z-18:1e/9Z-18:1), are identified using a combination of CID and OzID. These discoveries demonstrate that lipid identification based on CID alone belies the natural structural diversity in lipid biochemistry and illustrate the potential of OzID as a complementary approach within automated, high-throughput lipid analysis protocols.

  16. Exploiting the multiplexing capabilities of tandem mass tags for high-throughput estimation of cellular protein abundances by mass spectrometry.

    PubMed

    Ahrné, Erik; Martinez-Segura, Amalia; Syed, Afzal Pasha; Vina-Vilaseca, Arnau; Gruber, Andreas J; Marguerat, Samuel; Schmidt, Alexander

    2015-09-01

    The generation of dynamic models of biological processes critically depends on the determination of precise cellular concentrations of biomolecules. Measurements of system-wide absolute protein levels are particularly valuable information in systems biology. Recently, mass spectrometry based proteomics approaches have been developed to estimate protein concentrations on a proteome-wide scale. However, for very complex proteomes, fractionation steps are required, increasing samples number and instrument analysis time. As a result, the number of full proteomes that can be routinely analyzed is limited. Here we combined absolute quantification strategies with the multiplexing capabilities of isobaric tandem mass tags to determine cellular protein abundances in a high throughput and proteome-wide scale even for highly complex biological systems, such as a whole human cell line. We generated two independent data sets to demonstrate the power of the approach regarding sample throughput, dynamic range, quantitative precision and accuracy as well as proteome coverage in comparison to existing mass spectrometry based strategies.

  17. Proteomic characterization of integral membrane proteins using thermostatted liquid chromatography coupled with tandem mass spectrometry.

    PubMed

    Moore, Sarah M; Wu, Christine C

    2012-01-01

    Due to the hydrophobicity and localization of integral membrane proteins, they are difficult to study using conventional biochemical methods that are compatible with proteomic analyses. This chapter describes the coupling of multiple crucial steps that lead to the optimized shotgun proteomic analysis of integral membrane proteins while maintaining empirical topology information. Namely, a membrane shaving method is utilized to separate protease accessible peptides from membrane embedded peptides and elevated temperatures during chromatographic separation is utilized to augment the recovery of hydrophobic peptides for in-line analysis using tandem mass spectrometry. This combination of steps facilitates increased identification of membrane proteins while also maintaining information regarding protein topology.

  18. Determination of amphetamine and methamphetamine in umbilical cord using liquid chromatography-tandem mass spectrometry.

    PubMed

    Jones, Joseph; Rios, Rosemarie; Jones, Mary; Lewis, Douglas; Plate, Charles

    2009-11-01

    The use of meconium as a drug-screening matrix for newborns has been the gold standard of care for the past two decades. A recent study using matched pairs of meconium and umbilical cord demonstrated a high degree of agreement. The use of liquid chromatography-tandem mass spectrometry as a means to confirm amphetamines presumptive positive umbilical cord specimens for amphetamine and methamphetamine is described here for the first time. The limit of detection for both compounds was 0.2 ng/g. The limit of quantitation for both compounds was 0.6 ng/g. The assay was linear for both compounds up to 100 ng/g.

  19. Determination of histamine in seafood by hydrophilic interaction chromatography/tandem mass spectrometry.

    PubMed

    Yoshida, Tatsuo; Hamada, Hirotoshi; Murakawa, Hiroshi; Yoshimoto, Hidekazu; Tobino, Toshiaki; Toda, Kei

    2012-01-01

    A simple method was developed to determine histamine, an important compound in chemical food poisoning, by extraction followed by hydrophilic interaction chromatography-tandem mass spectrometry using a hydrophilic column with sulfobetaine-type zwitterion groups. The quantitation range in seafood products was from 0.4 to 200 mg kg(-1) for 5 g food samples. Quantitative recoveries were obtained with four types of seafood product. These results agreed well with those from the more complex, conventional HPLC method, which requires sample derivatization with dansyl chloride.

  20. Tandem mass spectrometric fragmentation patterns of known and new steviol glycosides with structure proposals.

    PubMed

    Zimmermann, Benno F

    2011-06-15

    Stevia rebaudiana contains several steviol glycosides that have a sweet flavor. They are up to 450 times sweeter than sucrose, but some have an undesirable aftertaste. Up to 2010, ten different steviol glycosides have been described from the leaves or purified extracts of S. rebaudiana. In this paper, the tandem mass spectrometric fragmentation patterns of these ten compounds are compiled, along with a scheme for structural elucidation. This scheme is then applied to 12 steviol glycosides that have not yet been described. The proposed structures of five steviol glycosides have been confirmed by other authors.

  1. High-throughput multiclass method for antibiotic residue analysis by liquid chromatography-tandem mass spectrometry.

    PubMed

    Chico, J; Rúbies, A; Centrich, F; Companyó, R; Prat, M D; Granados, M

    2008-12-12

    A simple and rapid method has been developed for the residue analysis of 39 antibiotics (tetracyclines, quinolones, penicillins, sulfonamides and macrolides) in foodstuffs of animal origin. The method combines an effective extraction technique, which uses water-methanol as extracting solvent, with ultra-high-pressure liquid chromatography-tandem mass spectrometry, allowing both confirmation and quantification in a single chromatographic run. The multiresidue method has been validated in chicken muscle matrix according to European Union Decision 2002/657/EC. It has been implemented as a routine method in a Public Health Laboratory, instead of the five plates test and LC methods previously used.

  2. Automated Lipid A Structure Assignment from Hierarchical Tandem Mass Spectrometry Data

    NASA Astrophysics Data System (ADS)

    Ting, Ying S.; Shaffer, Scott A.; Jones, Jace W.; Ng, Wailap V.; Ernst, Robert K.; Goodlett, David R.

    2011-05-01

    Infusion-based electrospray ionization (ESI) coupled to multiple-stage tandem mass spectrometry (MS n ) is a standard methodology for investigating lipid A structural diversity (Shaffer et al. J. Am. Soc. Mass. Spectrom. 18(6), 1080-1092, 2007). Annotation of these MS n spectra, however, has remained a manual, expert-driven process. In order to keep up with the data acquisition rates of modern instruments, we devised a computational method to annotate lipid A MS n spectra rapidly and automatically, which we refer to as hierarchical tandem mass spectrometry (HiTMS) algorithm. As a first-pass tool, HiTMS aids expert interpretation of lipid A MS n data by providing the analyst with a set of candidate structures that may then be confirmed or rejected. HiTMS deciphers the signature ions (e.g., A-, Y-, and Z-type ions) and neutral losses of MS n spectra using a species-specific library based on general prior structural knowledge of the given lipid A species under investigation. Candidates are selected by calculating the correlation between theoretical and acquired MS n spectra. At a false discovery rate of less than 0.01, HiTMS correctly assigned 85% of the structures in a library of 133 manually annotated Francisella tularensis subspecies novicida lipid A structures. Additionally, HiTMS correctly assigned 85% of the structures in a smaller library of lipid A species from Yersinia pestis demonstrating that it may be used across species.

  3. Quantitative analysis of phytosterols in edible oils using APCI liquid chromatography-tandem mass spectrometry.

    PubMed

    Mo, Shunyan; Dong, Linlin; Hurst, W Jeffrey; van Breemen, Richard B

    2013-09-01

    Previous methods for the quantitative analysis of phytosterols have usually used GC-MS and require elaborate sample preparation including chemical derivatization. Other common methods such as HPLC with absorbance detection do not provide information regarding the identity of the analytes. To address the need for an assay that utilizes mass selectivity while avoiding derivatization, a quantitative method based on LC-tandem mass spectrometry (LC-MS-MS) was developed and validated for the measurement of six abundant dietary phytosterols and structurally related triterpene alcohols including brassicasterol, campesterol, cycloartenol, β-sitosterol, stigmasterol, and lupeol in edible oils. Samples were saponified, extracted with hexane and then analyzed using reversed phase HPLC with positive ion atmospheric pressure chemical ionization tandem mass spectrometry and selected reaction monitoring. The utility of the LC-MS-MS method was demonstrated by analyzing 14 edible oils. All six compounds were present in at least some of the edible oils. The most abundant phytosterol in all samples was β-sitosterol, which was highest in corn oil at 4.35 ± 0.03 mg/g, followed by campesterol in canola oil at 1.84 ± 0.01 mg/g. The new LC-MS-MS method for the quantitative analysis of phytosterols provides a combination of speed, selectivity and sensitivity that exceed those of previous assays.

  4. Determination of Imazaquin and its metabolite by liquid chromatography-quadrupole-time of flight tandem mass.

    PubMed

    Yao, H B; Han, G J; Liu, G X; Xie, Y; Wang, C H

    2010-08-01

    A method consisting of solvent extraction followed by liquid chromatography-quadrupole-time of flight- tandem mass spectrometry analysis was developed for the identification of Imazaquin and its metabolite. The relationships between detector response and sample concentrations showed a high degree of linearity (r > 0.998) over the range 0.03-10 microg/g. The recoveries obtained were in the acceptable range of 86%-104% between spiked. The relative standard deviation of this method was 6.4%-17.1%. A 35-day study of Imazaquin degradation was taken in agricultural soil from Binzhou, China. The degradation followed first order kinetics (C = 0.7672e(-0.0774t)), with half-life of less than 8.5 days. Investigation of the by-products from liquid chromatography-quadrupole-time of flight- tandem mass spectrometry has shown that there were four important metabolites 4-methylene-2-(quinolin-2-yl)-1H-imidazol-5(4H)-one, quinoline-3-carbaldehyde, 1-amino-2,3-dimethyl-1-oxobutan-2-ylium and 1H-[1,2]oxazino[4,5-b]quinolin-1-one in the degradation process. The accurate mass measurements error was 5 ppm in this study. The method was successfully applied to the analysis of imazaquin and its metabolite residues in soil.

  5. Quantitative analysis of phytosterols in edible oils using APCI liquid chromatography-tandem mass spectrometry

    PubMed Central

    Mo, Shunyan; Dong, Linlin; Hurst, W. Jeffrey; van Breemen, Richard B.

    2014-01-01

    Previous methods for the quantitative analysis of phytosterols have usually used GC-MS and require elaborate sample preparation including chemical derivatization. Other common methods such as HPLC with absorbance detection do not provide information regarding the identity of the analytes. To address the need for an assay that utilizes mass selectivity while avoiding derivatization, a quantitative method based on LC-tandem mass spectrometry (LC-MS-MS) was developed and validated for the measurement of six abundant dietary phytosterols and structurally related triterpene alcohols including brassicasterol, campesterol, cycloartenol, β-sitosterol, stigmasterol, and lupeol in edible oils. Samples were saponified, extracted with hexane and then analyzed using reversed phase HPLC with positive ion atmospheric pressure chemical ionization tandem mass spectrometry and selected reaction monitoring. The utility of the LC-MS-MS method was demonstrated by analyzing 14 edible oils. All six compounds were present in at least some of the edible oils. The most abundant phytosterol in all samples was β-sitosterol, which was highest in corn oil at 4.35 ± 0.03 mg/g, followed by campesterol in canola oil at 1.84 ± 0.01 mg/g. The new LC-MS-MS method for the quantitative analysis of phytosterols provides a combination of speed, selectivity and sensitivity that exceed those of previous assays. PMID:23884629

  6. Scandium analysis in silicon-containing minerals by inductively coupled plasma tandem mass spectrometry

    NASA Astrophysics Data System (ADS)

    Whitty-Léveillé, Laurence; Drouin, Elisabeth; Constantin, Marc; Bazin, Claude; Larivière, Dominic

    2016-04-01

    This article reports on the development of a new method for the accurate and precise determination of the amount of scandium, Sc, in silicon-containing minerals, based on the use of tandem quadrupole inductively coupled plasma mass spectrometry (ICP-MS/MS). The tandem quadrupole instrument enables new mass filtering configurations, which can reduce polyatomic interferences during the determination of Sc in mineral matrices. He and O2 were used and compared as collision and reaction gases for the removal of interferences at m/z 45 and 61. Using helium gas was ineffective to overcome all of the spectral interferences observed at m/z 45 and particularly for Si-based interferences. However, conversion of Sc+ ions into ScO+ ions (after bombardment with O2 in the octopole reaction system coupled with the use of the instrument in MS/MS mass-shift mode) provided interference-free conditions and sufficiently low limits of detection, down to 3 ng L- 1, to accurately detect Sc. The accuracy of the proposed methodology was assessed by analyzing five different reference materials (BX-N, OKA-2, NIM-L, SY-3 and GH).

  7. Differentiation of hydroxyproline isomers and isobars in peptides by tandem mass spectrometry.

    PubMed

    Kassel, D B; Biemann, K

    1990-08-01

    The isomeric 3- and 4-hydroxyprolines are isobaric with the isomers leucine and isoleucine, and all four have, therefore, the same "residue mass" of 113. Secondary fragmentation processes were found that differentiate the hydroxyproline isomers from each other and from the leucines. Variants of synthetic bradykinin containing one or two hydroxyproline moieties were prepared by using manual Edman degradation and/or enzymatic methods. The tandem mass spectra of these peptides were recorded. The C-terminal wn fragment ions allow the differentiation of 4-hydroxyproline from the 3-isomer and isoleucine, while the N-terminal an ions containing 4-hydroxyproline undergo H2O elimination to differentiate this amino acid from the 3-isomer and leucine. Lys-C digestion of a mussel adhesive protein produced a set of decapeptides varying in the degree of hydroxylation of proline and tyrosine. Heterogeneity with respect to 3-hydroxyproline and 4-hydroxyproline at a certain position in these peptides was assessed by tandem mass spectrometry based on the wn ion series in the CID spectra of these Lys-C peptides. Some N-terminal ions further allow for the differentiation of these two isomeric species.

  8. Determination of Candesartan in Human Plasma with Liquid Chromatography - Tandem Mass Spectrometry.

    PubMed

    Forjan, Vanja; Cvitkovič Maričič, Lea; Prosen, Helena; Brodnjak Vončina, Darinka

    2016-01-01

    A sensitive, specific and rapid liquid chromatography - tandem mass spectrometry method was developed and validated for the determination of candesartan in human plasma. Analyte was separated from endogenous components present in plasma by solid phase extraction. Chromatographic separation was performed on Gemini C18 analytical column using mobile phase acetonitrile - 5 mM ammonium formate pH 2 (90:10, v/v) at flow rate of 0.3 mL/min. For detection, tandem mass spectrometry in SRM mode with positive electrospray ionization was used. The mass transitions m/z 441.1 > 263.1 and 445.1 > 267.1 were used to determine candesartan by using candesartan-d4 as an internal standard. After development, the method was validated according to the requirements of EMA regulatory guidelines in the concentration range 1 - 400 ng/ml in human plasma. Limit of quantification (LLOQ) was 1 ng/ml. The developed and validated method proved to be very fast and reproducible and was therefore successfully implemented in pharmacokinetic and bioequivalence studies with large number of study samples.

  9. Identification of Glycopeptides with Multiple Hydroxylysine O-Glycosylation Sites by Tandem Mass Spectrometry.

    PubMed

    Zhang, Yanlin; Yu, Chuan-Yih; Song, Ehwang; Li, Shuai Cheng; Mechref, Yehia; Tang, Haixu; Liu, Xiaowen

    2015-12-04

    Glycosylation is one of the most common post-translational modifications in proteins, existing in ~50% of mammalian proteins. Several research groups have demonstrated that mass spectrometry is an efficient technique for glycopeptide identification; however, this problem is still challenging because of the enormous diversity of glycan structures and the microheterogeneity of glycans. In addition, a glycopeptide may contain multiple glycosylation sites, making the problem complex. Current software tools often fail to identify glycopeptides with multiple glycosylation sites, and hence we present GlycoMID, a graph-based spectral alignment algorithm that can identify glycopeptides with multiple hydroxylysine O-glycosylation sites by tandem mass spectra. GlycoMID was tested on mass spectrometry data sets of the bovine collagen α-(II) chain protein, and experimental results showed that it identified more glycopeptide-spectrum matches than other existing tools, including many glycopeptides with two glycosylation sites.

  10. Application of dual tree complex wavelet transform in tandem mass spectrometry.

    PubMed

    Murugesan, Selvaraaju; Tay, David B H; Cooke, Ira; Faou, Pierre

    2015-08-01

    Mass Spectrometry (MS) is a widely used technique in molecular biology for high throughput identification and sequencing of peptides (and proteins). Tandem mass spectrometry (MS/MS) is a specialised mass spectrometry technique whereby the sequence of peptides can be determined. Preprocessing of the MS/MS data is indispensable before performing any statistical analysis on the data. In this work, preprocessing of MS/MS data is proposed based on the Dual Tree Complex Wavelet Transform (DTCWT) using almost symmetric Hilbert pair of wavelets. After the preprocessing step, the identification of peptides is done using the database search approach. The performance of the proposed preprocessing technique is evaluated by comparing its performance against Discrete Wavelet Transform (DWT) and Stationary Wavelet Transform (SWT). The preprocessing performed using DTCWT identified more peptides compared to DWT and SWT.

  11. Structural Characterization of Anticancer Drug Paclitaxel and Its Metabolites Using Ion Mobility Mass Spectrometry and Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Lee, Hong Hee; Hong, Areum; Cho, Yunju; Kim, Sunghwan; Kim, Won Jong; Kim, Hugh I.

    2016-02-01

    Paclitaxel (PTX) is a popular anticancer drug used in the treatment of various types of cancers. PTX is metabolized in the human liver by cytochrome P450 to two structural isomers, 3'- p-hydroxypaclitaxel (3 p-OHP) and 6α-hydroxypaclitaxel (6α-OHP). Analyzing PTX and its two metabolites, 3 p-OHP and 6α-OHP, is crucial for understanding general pharmacokinetics, drug activity, and drug resistance. In this study, electrospray ionization ion mobility mass spectrometry (ESI-IM-MS) and collision induced dissociation (CID) are utilized for the identification and characterization of PTX and its metabolites. Ion mobility distributions of 3 p-OHP and 6α-OHP indicate that hydroxylation of PTX at different sites yields distinct gas phase structures. Addition of monovalent alkali metal and silver metal cations enhances the distinct dissociation patterns of these structural isomers. The differences observed in the CID patterns of metalated PTX and its two metabolites are investigated further by evaluating their gas-phase structures. Density functional theory calculations suggest that the observed structural changes and dissociation pathways are the result of the interactions between the metal cation and the hydroxyl substituents in PTX metabolites.

  12. Atmospheric pressure ionization-tandem mass spectrometry of the phenicol drug family.

    PubMed

    Alechaga, Élida; Moyano, Encarnación; Galceran, M Teresa

    2013-11-01

    In this work, the mass spectrometry behaviour of the veterinary drug family of phenicols, including chloramphenicol (CAP) and its related compounds thiamphenicol (TAP), florfenicol (FF) and FF amine (FFA), was studied. Several atmospheric pressure ionization sources, electrospray (ESI), atmospheric pressure chemical ionization and atmospheric pressure photoionization were compared. In all atmospheric pressure ionization sources, CAP, TAP and FF were ionized in both positive and negative modes; while for the metabolite FFA, only positive ionization was possible. In general, in positive mode, [M + H](+) dominated the mass spectrum for FFA, while the other compounds, CAP, TAP and FF, with lower proton affinity showed intense adducts with species present in the mobile phase. In negative mode, ESI and atmospheric pressure photoionization showed the deprotonated molecule [M-H](-), while atmospheric pressure chemical ionization provided the radical molecular ion by electron capture. All these ions were characterized by tandem mass spectrometry using the combined information obtained by multistage mass spectrometry and high-resolution mass spectrometry in a quadrupole-Orbitrap instrument. In general, the fragmentation occurred via cyclization and losses or fragmentation of the N-(alkyl)acetamide group, and common fragmentation pathways were established for this family of compounds. A new chemical structure for the product ion at m/z 257 for CAP, on the basis of the MS(3) and MS(4) spectra is proposed. Thermally assisted ESI and selected reaction monitoring are proposed for the determination of these compounds by ultra high-performance liquid chromatography coupled to tandem mass spectrometry, achieving instrumental detection limits down to 0.1 pg.

  13. A novel “correlated ion and neutral time of flight” method: Event-by-event detection of neutral and charged fragments in collision induced dissociation of mass selected ions

    SciTech Connect

    Teyssier, C.; Fillol, R.; Abdoul-Carime, H.; Farizon, B.; Farizon, M.

    2014-01-15

    A new tandem mass spectrometry (MS/MS) method based on time of flight measurements performed on an event-by-event detection technique is presented. This “correlated ion and neutral time of flight” method allows to explore Collision Induced Dissociation (CID) fragmentation processes by directly identifying not only all ions and neutral fragments produced but also their arrival time correlations within each single fragmentation event from a dissociating molecular ion. This constitutes a new step in the characterization of molecular ions. The method will be illustrated here for a prototypical case involving CID of protonated water clusters H{sup +}(H{sub 2}O){sub n=1–5} upon collisions with argon atoms.

  14. iPE-MMR: An integrated approach to accurately assign monoisotopic precursor masses to tandem mass spectrometric data

    PubMed Central

    Jung, Hee-Jung; Purvine, Samuel O.; Kim, Hokeun; Petyuk, Vladislav A.; Hyung, Seok-Won; Monroe, Matthew E.; Mun, Dong-Gi; Kim, Kyong-Chul; Park, Jong-Moon; Kim, Su-Jin; Tolic, Nikola; Slysz, Gordon W.; Moore, Ronald J.; Zhao, Rui; Adkins, Joshua N.; Anderson, Gordon A.; Lee, Hookeun; Camp, David G.; Yu, Myeong-Hee; Smith, Richard D.; Lee, Sang-Won

    2010-01-01

    Accurate assignment of monoisotopic precursor masses to tandem mass spectrometric (MS/MS) data is a fundamental and critically important step for successful peptide identifications in mass spectrometry based proteomics. Here we describe an integrated approach that combines three previously reported methods of treating MS/MS data for precursor mass refinement. This combined method, “integrated Post-Experiment Monoisotopic Mass Refinement” (iPE-MMR), integrates steps: 1) generation of refined MS/MS data by DeconMSn; 2) additional refinement of the resultant MS/MS data by a modified version of PE-MMR; 3) elimination of systematic errors of precursor masses using DtaRefinery. iPE-MMR is the first method that utilizes all MS information from multiple MS scans of a precursor ion including multiple charge states, in an MS scan, to determine precursor mass. By combining these methods, iPE-MMR increases sensitivity in peptide identification and provides increased accuracy when applied to complex high-throughput proteomics data. PMID:20863060

  15. Mechanism of Formation of the Major Estradiol Product Ions Following Collisional Activation of the Molecular Anion in a Tandem Quadrupole Mass Spectrometer

    NASA Astrophysics Data System (ADS)

    Wooding, Kerry M.; Barkley, Robert M.; Hankin, Joseph A.; Johnson, Christopher A.; Bradford, Andrew P.; Santoro, Nanette; Murphy, Robert C.

    2013-10-01

    The importance of the mass spectral product ion structure is highlighted in quantitative assays, which typically use multiple reaction monitoring (MRM), and in the discovery of novel metabolites. Estradiol is an important sex steroid whose quantitation and metabolite identification using tandem mass spectrometry has been widely employed in numerous clinical studies. Negative electrospray ionization tandem mass spectrometry of estradiol (E2) results in several product ions, including the abundant m/z 183 and 169. Although m/z 183 is one of the most abundant product ions used in many quantitative assays, the structure of m/z 183 has not been rigorously examined. We suggest a structure for m/z 183 and a mechanism of formation consistent with collision induced dissociation (CID) of E2 and several stable isotopes ([D4]-E2, [13C6]-E2, and [D1]-E2). An additional product ion from E2, namely m/z 169, has also been examined. MS3 experiments indicated that both m/z 183 and m/z 169 originate from only E2 [M - H]- m/z 271. These ions, m/z 183 and m/z 169, were also present in the collision induced decomposition mass spectra of other prominent estrogens, estrone (E1) and estriol (E3), indicating that these two product ions could be used to elucidate the estrogenic origin of novel metabolites. We propose two fragmentation schemes to explain the CID data and suggest a structure of m/z 183 and m/z 169 consistent with several isotopic variants and high resolution mass spectrometric measurements.

  16. Identification of anatoxins in blue-green algae food supplements using liquid chromatography-tandem mass spectrometry.

    PubMed

    Draisci, R; Ferretti, E; Palleschi, L; Marchiafava, C

    2001-06-01

    Blue-green algae (cyanobacteria) in tablets and capsules, which are marketed as health food supplements, were investigated for the presence of neurotoxins related to anatoxin-a. These neurotoxins, which are nicotinic agonists, were investigated using isocratic micro-liquid chromatograph-tandem mass spectrometry (micro-LC-MS-MS). The investigated compounds were anatoxin-a and homoanatoxin-a, together with their degradation products, dihydroanatoxin-a, epoxyanatoxin-a, dihydrohomoanatoxin-a and epoxyhomoanatoxin-a which were synthesized from the parent toxins. The analytes were extracted with methanol followed by isocratic chromatography on a micro C18 reversed-phase column using acetonitrile-water, 50:50 (v/v), containing 20 mm acetic acid at 30 microl min(-1). The toxins were ionized in an ionspray (IS) interface operating in the positive ion mode, where the intact protonated molecules, [M + H]+, were generated at m/z 166, m/z 168, m/z 182, m/z 180, m/z 182 and m/z 196, for anatoxin-a, dihydroanatoxin-a, epoxyanatoxin-a, homoanatoxin-a, dihydrohomoanatoxin-a and epoxyhomoanatoxin-a, respectively. These served as precursor ions for collision-induced-dissociation (CID) and diagnostic product ions for these anatoxins were identified to carry out toxin confirmation by selected reaction monitoring (SRM) LC-MS-MS analysis. Dihydrohomoanatoxin-a and a novel isomer of epoxyanatoxin-a were identified in blue-green algae tablets. This finding suggests that a potential human health hazard could be associated with the consumption of these food supplements.

  17. Analysis of epoxyeicosatrienoic and monohydroxyeicosatetraenoic acids esterified to phospholipids in human red blood cells by electrospray tandem mass spectrometry.

    PubMed

    Nakamura, T; Bratton, D L; Murphy, R C

    1997-08-01

    Electrospray ionization (ESI) and tandem mass spectrometry (MS/MS) were used to analyze epoxyeicosatrienoic acids (EETs) and monohydroxyeicosatetraenoic acids (HETEs) isolated from human red blood cell membranes following base hydrolysis. ESI results in the formation of an abundant isobaric carboxylate anion at m/z 319 for both of these oxidized metabolites of arachidonic acid. The product ion spectra from the collision-induced dissociation of this carboxylate anion could be used to identify each of the isomeric eicosanoids from the unique fragment ions of each eicosanoid. The observed product ion spectra were identical with those previously obtained by fast atom bombardment ionization; however, ESI required less EET and HETE for analysis. Both EET and HETE phospholipids were present in human red blood cells (RBCs) and their abundance could be substantially increased by treatment under conditions that would induce free radical oxidation of membrane phospholipids. Following incubation of human RBCs with tert-butyl hydroperoxide (tBuOOH), phospholipids were extracted and purified by normal-phase high-performance liquid chromatography (HPLC) as to glycerophospholipid class containing ethanolamine (GPE), serine (GPS) and choline (GPC) as the polar head group. Each class of phospholipid was hydrolyzed to yield the free carboxylic acid prior to on-line HPLC/ESI-MS/MS analysis. The formation of oxidized arachidonic acid esterified to phospholipids in treated RBCs was found to increase significantly for both esterified EETs in GPE, GPS and GPC which increased 49-, 34- and 59-fold, respectively, and also for esterified HETEs in GPE, GPS and GPC which increased 3-, 4- and 11-fold, respectively, compared with untreated RBCs. These results provide the first characterization of EETs formed non-enzymatically as intact phospholipids in a lipid peroxidation model system.

  18. Assigning Peptide Disulfide Linkage Pattern Among Regio-Isomers via Methoxy Addition to Disulfide and Tandem Mass Spectrometry.

    PubMed

    Durand, Kirt L; Tan, Lei; Stinson, Craig A; Love-Nkansah, Chasity B; Ma, Xiaoxiao; Xia, Yu

    2017-02-13

    Pinpointing disulfide linkage pattern is critical in the characterization of proteins and peptides consisting of multiple disulfide bonds. Herein, we report a method based on coupling online disulfide modification and tandem mass spectrometry (MS/MS) to distinguish peptide disulfide regio-isomers. Such a method relies on a new disulfide bond cleavage reaction in solution, involving methanol as a reactant and 254 nm ultraviolet (UV) irradiation. This reaction leads to selective cleavage of a disulfide bond and formation of sulfenic methyl ester (-SOCH3) at one cysteine residue and a thiol (-SH) at the other. Under low energy collision-induced dissociation (CID), cysteine sulfenic methyl ester motif produces a signature methanol loss (-32 Da), allowing its identification from other possible isomeric structures such as S-hydroxylmethyl (-SCH2OH) and methyl sulfoxide (-S(O)-CH3). Since disulfide bond can be selectively cleaved and modified upon methoxy addition, subsequent MS(2) CID of the methoxy addition product provides enhanced sequence coverage as demonstrated by the analysis of bovine insulin. More importantly, this reaction does not induce disulfide scrambling, likely due to the fact that radical intermediates are not involved in the process. An approach based on methoxy addition followed by MS(3) CID has been developed for assigning disulfide linkage patterns in peptide disulfide regio-isomers. This methodology was successfully applied to characterizing peptide systems having two disulfide bonds and three disulfide linkage isomers: side-by-side, overlapped, and looped-within-a-loop configurations. Graphical Abstract ᅟ.

  19. Effect of D-allose on prostate cancer cell lines: phospholipid profiling by nanoflow liquid chromatography-tandem mass spectrometry.

    PubMed

    Jeong, Rae Ung; Lim, Sangsoo; Kim, Myoung Ok; Moon, Myeong Hee

    2011-08-01

    D-Allose, a rare, naturally occurring monosaccharide, is known to exert anti-proliferative effects on cancer cells. The effects of D-allose on the cellular membranes of hormone-refractory prostate cancer cell line (DU145), hormone-sensitive prostate cancer cell line (LNCaP), and normal prostate epithelial cells (PrEC) were studied at the molecular level by phospholipid (PL) profiling using a shotgun lipidomic method. The molecular structures of 85 PL species including 23 phosphatidylcholines, 12 phosphatidylethanolamines (PEs), 11 phosphatidylserines (PSs), 16 phosphatidylinositols, 9 phosphatidic acids (PAs), and 14 phosphatidylglycerols (PGs) were identified by data-dependent collision-induced dissociation of nanoflow liquid chromatography-tandem mass spectrometry, and the PL amounts were quantified. The addition of D-allose to prostate cancer cell lines during their growth phases had negligible or decreased effects on the relative regulation of PL species, but several new PS molecules (two for DU145 and three for LNCaP) emerged. In contrast, experiments on the PrEC cell line revealed that some high abundant species (14:0/14:0-PE, 16:2/16:0-PG, and 20:6/18:1-PA) showed significant increases in concentration. These findings support a mechanism for the anti-proliferative effect of D-allose on prostate cancer cell lines that involves the induction of programmed cell death since PS molecules are known to induce apoptosis. Principal component analysis was carried out to examine differences in PL distributions among the three cell lines promoted by D-allose.

  20. Triacylglycerol profile in cocoa liquors using MALDI-TOF and LC-ESI tandem mass spectrometry.

    PubMed

    Bono, Luca; Seraglia, Roberta; Roverso, Marco; Di Carro, Marina; Magi, Emanuele

    2014-09-01

    Triacylglycerols are responsible for chocolate's peculiar melting behavior: the type and position of fatty acids on the glycerol molecule strongly affect the melting range of cocoa butter. For this reason, the characterization of triglyceride composition in cocoa products is particularly important. In this work, triacylglycerols extracted from cocoa liquor samples were analyzed by matrix-assisted laser desorption/ionization time-of-flight (TOF) and electrospray ionization tandem mass spectrometry (MS/MS) coupled to liquid chromatography. Extracted samples were initially analyzed by direct injection in MS to obtain information on triglyceride molecular weights; relevant MS parameters were optimized, and the possible formation of the adducts [M + Na](+) and [M + NH(4)](+) was studied. Tandem mass experiments (both with triple quadrupole and TOF/TOF) were performed to study the fragmentation pathways (in particular, the loss of palmitic, stearic and oleic acid) and identify the triacylglycerols in cocoa liquors. Some signals of the spectra obtained with both MS techniques could indicate the presence of diacylglycerols in the cocoa extract, but different experimental evidences demonstrated that they were generated by the in-source fragmentation of triglycerides. A nonaqueous reversed-phase chromatographic separation was also developed and used to support the identification of the analytes; nine triacylglycerols were recognized in the cocoa liquor extracts. The three different batches of Ecuador cocoa liquor did not show significant differences in the triacylglycerol profile.

  1. Screening newborns for metabolic disorders based on targeted metabolomics using tandem mass spectrometry.

    PubMed

    Yoon, Hye-Ran

    2015-09-01

    The main purpose of newborn screening is to diagnose genetic, metabolic, and other inherited disorders, at their earliest to start treatment before the clinical manifestations become evident. Understanding and tracing the biochemical data obtained from tandem mass spectrometry is vital for early diagnosis of metabolic diseases associated with such disorders. Accordingly, it is important to focus on the entire diagnostic process, including differential and confirmatory diagnostic options, and the major factors that influence the results of biochemical analysis. Compared to regular biochemical testing, this is a complex process carried out by a medical physician specialist. It is comprised of an integrated program requiring multidisciplinary approach such as, pediatric specialist, expert scientist, clinical laboratory technician, and nutritionist. Tandem mass spectrometry is a powerful tool to improve screening of newborns for diverse metabolic diseases. It is likely to be used to analyze other treatable disorders or significantly improve existing newborn tests to allow broad scale and precise testing. This new era of various screening programs, new treatments, and the availability of detection technology will prove to be beneficial for the future generations.

  2. Detection of various freshwater cyanobacterial toxins using ultra-performance liquid chromatography tandem mass spectrometry.

    PubMed

    Oehrle, Stuart A; Southwell, Ben; Westrick, Judy

    2010-05-01

    Several freshwater cyanobacteria species have the capability to produce toxic compounds, frequently referred to as cyanotoxins. The most prevalent of these cyanotoxins is microcystin LR. Recognizing the potential health risk, France, Italy, Poland, Australia, Canada, and Brazil have set either standards or guidelines for the amount of microcystin LR permissible in drinking water based on the World Health Organization guideline of one microg/L of microcystin LR. Recently, the United States Environmental Protection Agency has begun to evaluate the occurrence and health effects of cyanotoxins and their susceptibility to water treatment under the Safe Drinking Water Act through the Contaminant Candidate List (CCL). A recent update of the Contaminant Candidate List focuses research and data collection on the cyanotoxins microcystin LR, anatoxin-a, and cylindrospermopsin. Liquid Chromatography/Tandem-Mass Spectrometry (LC/MS/MS) is a powerful tool for the analysis of various analytes in a wide variety of matrices because of its sensitivity and selectivity. The use of smaller column media (sub 2 microm particles) was investigated to both improve the speed, sensitivity and resolution, and to quantify the CCL cyanotoxins, in a single analysis, using Ultra-Performance Liquid Chromatography (UPLC) combined with tandem mass spectrometry. Natural waters and spiked samples were analyzed to show proof-of-performance. The presented method was able to clearly resolve each of the cyanotoxins in less than eight minutes with specificity and high spike recoveries.

  3. Quantitation of tetrahydrocannabinol in hair using immunoassay and liquid chromatography with tandem mass spectrometric detection.

    PubMed

    Coulter, Cynthia; Taruc, Margaux; Tuyay, James; Moore, Christine

    2009-05-01

    A quantitative analytical procedure for the determination of Delta(9)-tetrahydrocannabinol (THC) in hair has been developed and validated using liquid chromatography with tandem mass spectral detection (LC-MS/MS). Specimens that were determined as containing cannabinoids following immunoassay testing were quantified using solid-phase extraction followed by liquid chromatographic separation and tandem mass spectral detection in positive electrospray ionization mode. For confirmation, two transitions were monitored and one ratio determined. Samples being reported as positive were required to have both transitions present, the ratio of quantifying transition to qualifying transition being within 20% of that determined from known calibration standards. The limit of quantitation and the limit of detection was 10 pg/mg. The percentage recovery of the THC from hair at 20 pg/mg was 56% and a matrix effect of the hair showed an ion suppression percentage of -51%. The immunochemical screening method was performed following a rapid aqueous extraction, requiring only 10 mg of hair; the confirmatory procedure required 20 mg of hair. The methods were applied to proficiency specimens from the Society of Hair Testing, which had been received in August 2008.

  4. Determination of bromate in drinking water by ultraperformance liquid chromatography-tandem mass spectrometry.

    PubMed

    Alsohaimi, Ibrahim Hotan; Alothman, Zeid Abdullah; Khan, Mohammad Rizwan; Abdalla, Mohammad Abulhassan; Busquets, Rosa; Alomary, Ahmad Khodran

    2012-10-01

    Bromate is a byproduct formed as a result of disinfection of bromide-containing source water with ozone or hypochlorite. The International Agency for Research on Cancer has recognized bromate as a possible human carcinogen, thus it is essential to determine in drinking water. Present work highlights a development of sensitive and fast analytical method for bromate determination in drinking water by using ultraperformance liquid chromatography-tandem mass spectrometry. The quality parameters of the developed method were established, obtaining very low limit of detection (0.01 ng/mL), repeatability and reproducibility have been found to be less than 3% in terms of relative standard deviation when analyzing a bromate standard at 0.05 μg/mL with 0.4 min analysis time. Developed method was applied for the analysis of metropolitan and bottled water from Saudi Arabia; 22 samples have been analyzed. Bromate was detected in the metropolitan water samples (from desalinization source) at concentrations ranging between 3.43 and 75.04 ng/mL and in the bottled water samples at concentrations ranging between 2.07 and 21.90 ng/mL. Moreover, in comparison to established analytical methods such as liquid chromatography-tandem mass spectrometry, the proposed method was found to be very sensitive, selective and rapid for the routine analysis of bromate at low level in drinking water.

  5. Determination of parabens in serum by liquid chromatography-tandem mass spectrometry: Correlation with lipstick use.

    PubMed

    Tahan, Gabriella Padovani; Santos, Nayara de Kássia Souza; Albuquerque, Ana Carolina; Martins, Isarita

    2016-08-01

    Parabens are the most widely used preservative and are considered to be relatively safe compounds. However, studies have demonstrated that they may have estrogenic activity, and there is ongoing debate regarding the safety and potential cancer risk of using products containing these compounds. In the present work, liquid chromatography-tandem mass spectrometry was applied to determine methylparaben and propylparaben concentrations in serum, and the results were correlated with lipstick application. Samples were analyzed using liquid-liquid extraction, followed by liquid chromatography-tandem mass spectrometry. The validation results demonstrated the linearity of the method over a range of 1-20 ng/mL, in addition to the method's precision and accuracy. A statistically significant difference was demonstrated between serum parabens in women who used lipstick containing these substances compared with those not using this cosmetic (p = 0.0005 and 0.0016, respectively), and a strong association was observed between serum parabens and lipstick use (Spearman correlation = 0.7202).

  6. Precursor ion scan profiles of acylcarnitines by atmospheric pressure thermal desorption chemical ionization tandem mass spectrometry.

    PubMed

    Paglia, Giuseppe; D'Apolito, Oceania; Corso, Gaetano

    2008-12-01

    The fatty acyl esters of L-carnitine (acylcarnitines) are useful biomarkers for the diagnosis of some inborn errors of metabolism analyzed by liquid chromatography/tandem mass spectrometry. In this study the acylcarnitines were analyzed by atmospheric pressure thermal desorption chemical ionization using a commercial tandem mass spectrometer (APTDCI-MS/MS). The method is based on the precursor ion scan mode determination of underivatized acylcarnitines desorbed from samples by a hot desolvation gas flow and ionized by a corona pin discharge. During desorption/ionization step the temperature induces the degradation of acylcarnitines; nevertheless, the common fragment to all acylcarnitines [MH-59](+) is useful for analyzing their profile. APTDCI parameters, including angle of collection and incidence, gas flows and temperatures, were optimized for acylcarnitines. The experiments were performed drying 2 microL of an equimolar mixture of acylcarnitine standards on a glass slide. The specificity was evaluated by comparing product ion spectra and the precursor ion spectra of 85 m/z of acylcarnitines obtained by the APTDCI method and by electrospray ionization flow injection analysis (ESI-FIA). The method was also employed to analyze acylcarnitines extracted from a pathological dried blood spot and a control. The method enables analysis of biological samples and recognition of some acylcarnitines that are diagnostic markers of inherited metabolic diseases. The intrinsic high-throughput analysis of the ambient desorption ionization methods offers a new opportunity either for its potential application in clinical chemistry and for the expanded screening of some inborn errors of metabolism.

  7. Quantitation of Free Metanephrines in Plasma by Liquid Chromatography-Tandem Mass Spectrometry.

    PubMed

    Heideloff, Courtney; Payto, Drew; Wang, Sihe

    2016-01-01

    Plasma metanephrines are measured to aid in the diagnosis of pheochromocytomas. In patients with pheochromocytomas there is excessive production of catecholamines and metanephrines. Measurement of plasma free metanephrines is one of the first-line clinical tests that are used for the diagnosis and follow-up of pheochromocytoma. We describe here a liquid chromatography-tandem mass spectrometry method to measure free metanephrines in plasma. Free metanephrine and normetanephrine are extracted via solid-phase extraction. After extraction and evaporation, the reconstituted supernatant is analyzed by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The MS/MS is set to selective reaction monitoring mode (180.1 → 148.1 m/z for metanephrine, 183.1 → 168.1 for d3-metanephrine, 166.1 → 134.1 m/z for normetanephrine, and 169.1 → 137.2 m/z for d3-normetanephrine) with positive electrospray ionization. Quantitation is based on peak area ratio of the analyte to its respective deuterated internal standard. The assay is linear from 5.9 to 4090.0 pg/mL for metanephrine and 22.0 to 4386.7 pg/mL for normetanephrine with precision of <6 % over the ranges.

  8. Steroid hormone levels in pregnancy and 1 year postpartum using isotope dilution tandem mass spectrometry

    PubMed Central

    Soldin, Offie P.; Guo, Tiedong; Weiderpass, Elisabete; Tractenberg, Rochelle E.; Hilakivi-Clarke, Leena; Soldin, Steven J.

    2013-01-01

    Objective To establish normal, trimester-specific reference intervals for serum 17β-estradiol, progesterone (P), 17α-hydroxyprogesterone, cortisol, 11-deoxycortisol, androstenedione, DHEA, and DHEAS, measured simultaneously using isotope dilution tandem mass spectrometry. Design Sequential cohort study. Patient(s) Healthy women undergoing a normal pregnancy (age, 25–38 years; mean, 30 years) attending a prenatal well clinic at gestation weeks 12, 22, and 32 and approximately 1 year postpartum. Main Outcome Measure(s) Trimester-specific reference intervals of endogenous steroid hormones analyzed using an isotope dilution tandem mass spectrometer equipped with an atmospheric pressure photoionization source with deuterium-labeled internal standards. Result(s) Serum estradiol, P, 17α-hydroxyprogesterone, and 11-deoxycortisol increased throughout pregnancy; cortisol increased up to the second trimester and then remained steady, while androstenedione increased by 80 percent by gestation week 12, then remained constant. Serum DHEA-S decreased by 50% by the third trimester. Conclusion(s) Trimester-specific reference intervals are reported for eight serum steroids. The ratios of individual serum hormone concentrations during pregnancy relative to their 1-year postpartum concentrations illustrate the expected normal trends of changes in hormone concentrations during pregnancy. PMID:16169406

  9. Improved liquid chromatography-tandem mass spectrometry method for the analysis of eptifibatide in human plasma.

    PubMed

    Zhou, Zhi-Ling; Yu, Xi-Yong; Yang, Min; Mai, Li-Ping; Lin, Qiu-Xiong; Deng, Chun-Yu; Shan, Zhi-Xin; Kuang, Su-Juan; Zhu, Ping; Huang, Xiao-Zhong; Li, Xiao-Hong; Chen, Tie-Feng; Lin, Shu-Guang

    2010-08-01

    A rapid, selective and highly sensitive high performance liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed and validated for the determination and pharmacokinetic investigation of eptifibatide in human plasma. Eptifibatide and the internal standard (IS), EPM-05, were extracted from plasma samples using solid phase extraction. Chromatographic separation was performed on a C(18) column at a flow rate of 0.5 mL/min. Detection of eptifibatide and the IS was achieved by tandem mass spectrometry with an electrospray ionization (ESI) interface in positive ion mode. Traditional multiple reaction monitoring (MRM) using the transition of m/z 832.6-->m/z 646.4 and m/z 931.6-->m/z 159.4 was performed to quantify eptifibatide and the IS, respectively. The calibration curves were linear over the range of 1-1000 ng/mL with the lower limit of quantitation validated at 1 ng/mL. The intra- and inter-day precisions were within 13.3%, while the accuracy was within +/-7.6% of nominal values. The validated LC-MS/MS method was successfully applied for the evaluation of pharmacokinetic parameters of eptifibatide after intravenous (i.v.) administration of a 45 microg/kg bolus of eptifibatide to 8 healthy volunteers.

  10. Determination of biogenic amines in beer and wine by capillary electrophoresis-tandem mass spectrometry.

    PubMed

    Daniel, Daniela; Dos Santos, Vagner Bezerra; Vidal, Denis Tadeu Rajh; do Lago, Claudimir Lucio

    2015-10-16

    A capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) method for the simultaneous assessment of nine biogenic amines (spermine, spermidine, putrescine, cadaverine, histamine, phenylethylamine, tryptamine, tyramine, and urocanic acid) in commercial samples of beer and wine is introduced. The samples were submitted to a simple clean-up step with poly(vinylpolypyrrolidone) followed by filtration. Electrophoretic separation in a polyvinyl alcohol (PVA)-coated capillary using 0.5 mol L(-1) acetic acid (pH 2.5) as background electrolyte and detection by electrospray-tandem mass spectrometry was employed. The range of the correlation coefficients of the calibration curves of the analyzed compounds was 0.996-0.999, and the limits of detection and limits of quantification were in the range of 1-2 μg L(-1) and 3-8 μg L(-1), respectively. The recovery values for samples spiked at three concentration levels (0.2, 0.5, and 1.0 mg L(-1)) ranged from 87 to 113% with standard deviation not greater than 5.8%. The use of a PVA-coated silica capillary allows suppressing the electroosmotic flow and, consequently, increasing of the separation efficiency. The method was successfully used to determine biogenic amines in commercial samples of beer and wine.

  11. Determination of tolperisone in human plasma by liquid chromatography/tandem mass spectrometry for clinical application.

    PubMed

    Choi, Chang-Ik; Park, Jung-In; Lee, Hye-In; Lee, Yun-Jeong; Jang, Choon-Gon; Bae, Jung-Woo; Lee, Seok-Yong

    2012-12-12

    We have developed and validated a simple, rapid, and sensitive liquid chromatography analytical method employing tandem mass spectrometry (LC-MS/MS) for the determination of tolperisone, a centrally acting muscle relaxant, in human plasma. After liquid-liquid extraction with methyl t-butyl ether, chromatographic separation of tolperisone was performed using a reversed-phase Luna C(18) column (2.0mm×50mm, 5μm particles) with a mobile phase of 10mM ammonium formate buffer (pH 3.5) - methanol (12:88, v/v) and quantified by tandem mass detection in ESI positive ion mode. The flow rate of the mobile phase was 250μL/min and the retention times of tolperisone and the internal standard (IS, dibucaine) were both 0.6min. The calibration curves were linear over a range of 0.5-300ng/mL (r>0.999). The lower limit of quantification, using 200μL human plasma, was 0.5ng/mL. The mean accuracy and precision for intra- and inter-day validation of tolperisone were within acceptable limits. The LC-MS/MS method reported here showed improved sensitivity for quantification of tolperisone in human plasma compared with previously described analytical methods. Lastly, the validated method was successfully applied to a pharmacokinetic study in humans.

  12. Principles of electron capture and transfer dissociation mass spectrometry applied to peptide and protein structure analysis.

    PubMed

    Zhurov, Konstantin O; Fornelli, Luca; Wodrich, Matthew D; Laskay, Ünige A; Tsybin, Yury O

    2013-06-21

    This tutorial review describes the principles and practices of electron capture and transfer dissociation (ECD/ETD or ExD) mass spectrometry (MS) employed for peptide and protein structure analysis. ExD MS relies on interactions between gas phase peptide or protein ions carrying multiple positive charges with either free low-energy (~1 eV) electrons (ECD), or with reagent radical anions possessing an electron available for transfer (ETD). As a result of recent implementation on sensitive, high resolution, high mass accuracy, and liquid chromatography timescale-compatible mass spectrometers, ExD, more specifically, ETD MS has received particular interest in life science research. In addition to describing the fundamental aspects of ExD radical ion chemistry, this tutorial provides practical guidelines for peptide de novo sequencing with ExD MS, as well as reviews some of the current capabilities and limitations of these techniques. The merits of ExD MS are discussed primarily within the context of life science research.

  13. Hybrid quadrupole mass filter/quadrupole ion trap/time-of-flight-mass spectrometer for infrared multiple photon dissociation spectroscopy of mass-selected ions

    SciTech Connect

    Gulyuz, Kerim; Stedwell, Corey N.; Wang Da; Polfer, Nick C.

    2011-05-15

    We present a laboratory-constructed mass spectrometer optimized for recording infrared multiple photon dissociation (IRMPD) spectra of mass-selected ions using a benchtop tunable infrared optical parametric oscillator/amplifier (OPO/A). The instrument is equipped with two ionization sources, an electrospray ionization source, as well as an electron ionization source for troubleshooting. This hybrid mass spectrometer is composed of a quadrupole mass filter for mass selection, a reduced pressure ({approx}10{sup -5} Torr) quadrupole ion trap (QIT) for OPO irradiation, and a reflectron time-of-flight drift tube for detecting the remaining precursor and photofragment ions. A helium gas pulse is introduced into the QIT to temporarily increase the pressure and hence enhance the trapping efficiency of axially injected ions. After a brief pump-down delay, the compact ion cloud is subjected to the focused output from the continuous wave OPO. In a recent study, we implemented this setup in the study of protonated tryptophan, TrpH{sup +}, as well as collision-induced dissociation products of this protonated amino acid [W. K. Mino, Jr., K. Gulyuz, D. Wang, C. N. Stedwell, and N. C. Polfer, J. Phys. Chem. Lett. 2, 299 (2011)]. Here, we give a more detailed account on the figures of merit of such IRMPD experiments. The appreciable photodissociation yields in these measurements demonstrate that IRMPD spectroscopy of covalently bound ions can be routinely carried out using benchtop OPO setups.

  14. Reactivity and analytical performance of oxygen as cell gas in inductively coupled plasma tandem mass spectrometry

    NASA Astrophysics Data System (ADS)

    Virgilio, Alex; Amais, Renata S.; Amaral, Clarice D. B.; Fialho, Lucimar L.; Schiavo, Daniela; Nóbrega, Joaquim A.

    2016-12-01

    The reactivity and analytical performance of O2 as cell gas in inductively coupled plasma tandem mass spectrometry was investigated. Selected analytes in a wide mass range were divided in three groups according to their reactivity: G1 represents elements with high oxygen affinity (Ce, La, P, Sc, Ti, and Y), G2 contains elements that may partially react with oxygen (As, Ba, Mo, Si, Sr, and V), and G3 comprises elements expected to be less reactive towards oxygen (Al, Bi, Cu, Mg, Pb, and Pd). On-mass and mass-shift modes were evaluated by monitoring atomic and metal oxide ions, respectively. Analytical signal profiles, oxide percentages, sensitivities and limits of detection for oxygen flow rates varying from 0.1 to 1.0 mL min- 1 were also studied. Group 1 elements plus As and V presented better sensitivities and LODs when measuring oxides, which were the major species for all flow rates evaluated. Molybdenum and Si oxides presented intermediate behavior and MoO fraction was up to 47% and limit of detection was the same as that obtained in on-mass mode. For others G2 and G3 elements, on-mass mode presented higher sensitivity and better LODs, with estimated oxide contents lower than 10%. In most cases, increasing oxygen flow rates led to lower sensitivities and worse LODs.

  15. Characterization of four Phyllanthus species using liquid chromatography coupled to tandem mass spectrometry.

    PubMed

    Sprenger, Ricardo da Fontoura; Cass, Quezia Bezerra

    2013-05-24

    This paper reports a comparison of four Phyllanthus species (P. amarus, P. stipulatus, P. niruri and P. tenellus), commonly known as stone breaker, by the characterization of the chemical profile of their aqueous extracts. Such characterization was carried out using liquid chromatography coupled to ion trap tandem mass spectrometry (LC-IT-MS(n)) under reversed-phase gradient elution mode. The results of MS/MS and MS(3) on-line experiments, using the electrospray ionization source in the positive and negative mode, are extensively discussed. Furthermore, quercetin-3-O-β-d-glucuronopyranoside was isolated in multimilligram scale from the aqueous extract of P. stipulatus and characterized by mass spectrometry and NMR. Although it is an unusual flavonol in natural products, LC-IT-MS(n) experiments showed it to be present also in P. amarus.

  16. Recent advances of liquid chromatography-(tandem) mass spectrometry in clinical and forensic toxicology - An update.

    PubMed

    Remane, Daniela; Wissenbach, Dirk K; Peters, Frank T

    2016-09-01

    Liquid chromatography (LC) coupled to mass spectrometry (MS) or tandem mass spectrometry (MS/MS) is a well-established and widely used technique in clinical and forensic toxicology as well as doping control especially for quantitative analysis. In recent years, many applications for so-called multi-target screening and/or quantification of drugs, poisons, and or their metabolites in biological matrices have been developed. Such methods have proven particularly useful for analysis of so-called new psychoactive substances that have appeared on recreational drug markets throughout the world. Moreover, the evolvement of high resolution MS techniques and the development of data-independent detection modes have opened new possibilities for applications of LC-(MS/MS) in systematic toxicological screening analysis in the so called general unknown setting. The present paper will provide an overview and discuss these recent developments focusing on the literature published after 2010.

  17. Identification of Asp isomerization in proteins by ¹⁸O labeling and tandem mass spectrometry.

    PubMed

    Zhang, Jennifer; Katta, Viswanatham

    2012-01-01

    Isomerization of aspartic acid (Asp) to isoaspartic acid (isoAsp) via succinimide intermediate is a common route of degradation for proteins that can affect their structural integrity. As Asp/isoAsp is isobaric in mass, it is difficult to identify the site of modification by LC-MS/MS peptide mapping. Here, we describe an approach to label the Asp residue involved in isomerization at the protein level by hydrolyzing the succinimide intermediate in H₂¹⁸O. Tryptic digestion of this labeled protein will result in peptides containing the site of isomerization being 2 Da heavier than the ¹⁶O-containing counterparts, due to ¹⁸O incorporation during the hydrolysis process. Comparison of tandem mass spectra of isomerized peptides with and without ¹⁸O incorporation allows easy identification of the Asp residue involved. This method proved to be especially useful in identifying the sites when isomerization occurs in Asp-Asp motifs.

  18. Analysis of Non-Enzymatically Glycated Peptides: Neutral-Loss Triggered MS3 Versus Multi-Stage Activation Tandem Mass Spectrometry

    SciTech Connect

    Zhang, Qibin; Petyuk, Vladislav A.; Schepmoes, Athena A.; Orton, Daniel J.; Monroe, Matthew E.; Yang, Feng; Smith, Richard D.; Metz, Thomas O.

    2008-10-15

    Non-enzymatic glycation of tissue proteins has important implications in the development of complications of diabetes mellitus. While electron transfer dissociation (ETD) has been shown to outperform collision-induced dissociation (CID) in sequencing glycated peptides by tandem mass spectrometry, ETD instrumentation is not yet available in all laboratories. In this study, we evaluated different advanced CID techniques (i.e., neutral-loss triggered MS3 and multi-stage activation) during LC-MSn analyses of Amadori-modified peptides enriched from human serum glycated in vitro. During neutral-loss triggered MS3 experiments, MS3 scans triggered by neutral-losses of 3 H2O or 3 H2O + HCHO produced similar results in terms of glycated peptide identifications. However, neutral losses of 3 H2O resulted in significantly more glycated peptide identifications during multi-stage activation experiments. Overall, the multi-stage activation approach produced more glycated peptide identifications, while the neutral-loss triggered MS3 approach resulted in much higher specificity. Both techniques offer a viable alternative to ETD for identifying glycated peptides when that method is unavailable.

  19. The dissociation kinetics of NO on Rh(111) as studied by temperature programmed static secondary ion mass spectrometry and desorption

    NASA Astrophysics Data System (ADS)

    Borg, H. J.; Reijerse, J. F. C.-J. M.; van Santen, R. A.; Niemantsverdriet, J. W.

    1994-12-01

    Temperature programmed static secondary ion mass spectrometry (TPSSIMS) and temperature programmed desorption (TPD) have been used to study the kinetics of adsorption, dissociation, and desorption of NO on Rh(111). At 100 K, NO adsorption is molecular and proceeds via mobile precursor state kinetics with a high initial sticking probability. SSIMS indicates the presence of two distinct NO adsorption states, indicative of threefold adsorption at low coverage, and occupation of bridge sites at higher coverages. Three characteristic coverage regimes appear with respect to NO dissociation. At low coverages θNO<0.25 ML, NO dissociates completely at temperatures between 275 and 340 K. If we neglect lateral interactions and assume pure first order dissociation kinetics, we find effective values for the activation barrier and preexponential factor of 40±6 kJ/mol and 106±1 s-1 for the dissociation of 0.15-0.20 ML NO. However, if we assume that a NO molecule needs an ensemble of three to four vacant sites in order to dissociate, the preexponential factor and activation energy are ˜1011 s-1 and 65 kJ/mol, in better agreement with transition state theory expectations. The Nads and Oads dissociation products desorb as N2 and O2, respectively, with desorption parameters Edes=118±10 kJ/mol and νdes=1010.1±1.0 s-1 for N2 in the zero coverage limit. At higher coverages, the desorption kinetics of N2 is strongly influenced by the presence of coadsorbed oxygen. In the medium coverage range 0.25<θNO<0.50 ML, part of the NO desorbs molecularly, with an estimated desorption barrier of 113±10 kJ/mol and a preexponential of 1013.5±1.0 s-1. Dissociation of NO becomes progressively inhibited due to site blocking, the onset shifting from 275 K at 0.25 ML to 400 K, coinciding with the NO desorption temperature, at a coverage of 0.50 ML. The accumulation of nitrogen and oxygen atoms on the highly covered surface causes a destabilization of the nitrogen atoms, which results in an

  20. Method and apparatus for enhanced sequencing of complex molecules using surface-induced dissociation in conjunction with mass spectrometric analysis

    DOEpatents

    Laskin, Julia [Richland, WA; Futrell, Jean H [Richland, WA

    2008-04-29

    The invention relates to a method and apparatus for enhanced sequencing of complex molecules using surface-induced dissociation (SID) in conjunction with mass spectrometric analysis. Results demonstrate formation of a wide distribution of structure-specific fragments having wide sequence coverage useful for sequencing and identifying the complex molecules.

  1. Advanced Laser Architecture for Two-Step Laser Tandem Mass Spectrometer

    NASA Technical Reports Server (NTRS)

    Fahey, Molly E.; Li, Steven X.; Yu, Anthony W.; Getty, Stephanie A.

    2016-01-01

    Future astrobiology missions will focus on planets with significant astrochemical or potential astrobiological features, such as small, primitive bodies and the icy moons of the outer planets that may host diverse organic compounds. These missions require advanced instrument techniques to fully and unambiguously characterize the composition of surface and dust materials. Laser desorptionionization mass spectrometry (LDMS) is an emerging instrument technology for in situ mass analysis of non-volatile sample composition. A recent Goddard LDMS advancement is the two-step laser tandem mass spectrometer (L2MS) instrument to address the need for future flight instrumentation to deconvolve complex organic signatures. The L2MS prototype uses a resonance enhanced multi-photon laser ionization mechanism to selectively detect aromatic species from a more complex sample. By neglecting the aliphatic and inorganic mineral signatures in the two-step mass spectrum, the L2MS approach can provide both mass assignments and clues to structural information for an in situ investigation of non-volatile sample composition. In this paper we will describe our development effort on a new laser architecture that is based on the previously flown Lunar Orbiter Laser Altimeter (LOLA) laser transmitter for the L2MS instrument. The laser provides two discrete midinfrared wavelengths (2.8 m and 3.4 m) using monolithic optical parametric oscillators and ultraviolet (UV) wavelength (266 nm) on a single laser bench with a straightforward development path toward flight readiness.

  2. MS Amanda, a Universal Identification Algorithm Optimized for High Accuracy Tandem Mass Spectra

    PubMed Central

    2014-01-01

    Today’s highly accurate spectra provided by modern tandem mass spectrometers offer considerable advantages for the analysis of proteomic samples of increased complexity. Among other factors, the quantity of reliably identified peptides is considerably influenced by the peptide identification algorithm. While most widely used search engines were developed when high-resolution mass spectrometry data were not readily available for fragment ion masses, we have designed a scoring algorithm particularly suitable for high mass accuracy. Our algorithm, MS Amanda, is generally applicable to HCD, ETD, and CID fragmentation type data. The algorithm confidently explains more spectra at the same false discovery rate than Mascot or SEQUEST on examined high mass accuracy data sets, with excellent overlap and identical peptide sequence identification for most spectra also explained by Mascot or SEQUEST. MS Amanda, available at http://ms.imp.ac.at/?goto=msamanda, is provided free of charge both as standalone version for integration into custom workflows and as a plugin for the Proteome Discoverer platform. PMID:24909410

  3. Molecular resolution and fragmentation of fulvic acid by electrospray ionization/multistage tandem mass spectrometry

    USGS Publications Warehouse

    Leenheer, J.A.; Rostad, C.E.; Gates, Paul M.; Furlong, E.T.; Ferrer, I.

    2001-01-01

    Molecular weight distributions of fulvic acid from the Suwannee River, Georgia, were investigated by electrospray ionization/quadrupole mass spectrometry (ESI/QMS), and fragmentation pathways of specific fulvic acid masses were investigated by electrospray ionization/ion trap multistage tandem mass spectrometry (ESI/MST/MS). ESI/QMS studies of the free acid form of low molecular weight poly(carboxylic acid) standards in 75% methanol/25% water mobile phase found that negative ion detection gave the optimum generation of parent ions that can be used for molecular weight determinations. However, experiments with poly(acrylic acid) mixtures and specific high molecular weight standards found multiply charged negative ions that gave a low bias to molecular mass distributions. The number of negative charges on a molecule is dependent on the distance between charges. ESI/MST/MS of model compounds found characteristic water loss from alcohol dehydration and anhydride formation, as well as CO2 loss from decarboxylation, and CO loss from ester structures. Application of these fragmentation pathways to specific masses of fulvic acid isolated and fragmented by ESI/MST/MS is indicative of specific structures that can serve as a basis for future structural confirmation after these hypothesized structures are synthesized.

  4. Collision-induced dissociation of glycero phospholipids using electrospray ion-trap mass spectrometry.

    PubMed

    Larsen, A; Uran, S; Jacobsen, P B; Skotland, T

    2001-01-01

    Characterisation of phospholipids was achieved using collision-induced dissociation (CID) with an ion-trap mass spectrometer. The product ions were compared with those obtained with a triple quadrupole mass spectrometer. In the negative ion mode the product ions were mainly sn-1 and sn-2 lyso-phospholipids with neutral loss of ketene in combination with neutral loss of the polar head group. Less abundant product ions were sn-1 and sn-2 carboxylate anions. CID using a triple quadrupole mass spectrometer, however, gave primarily the sn-1 and sn-2 carboxylate anions together with lyso-phosphatidic acid with neutral loss of water. For the ion trap a charge-remote-type mechanism is proposed for formation of the lyso-phospholipid product ions by loss of alpha-hydrogen on the fatty acid moiety, electron rearrangement and neutral loss of ketene. A second mechanism involves nucleophilic attack of the phosphate oxygen on the sn-1 and sn-2 glycerol backbone to form carboxylate anions with neutral loss of cyclo lyso-phospholipids. CID (MS(3) and MS(4)) of the lyso-phospholipids using the ion-trap gave the same carboxylate anions as those obtained with a triple quadrupole instrument where multiple collisions in the collision cell are expected to occur. The data demonstrate that phospholipid species determination can be performed by using LC/MS(n) with an ion-trap mass spectrometer with detection of the lyso-phospholipid anions. The ion-trap showed no loss in sensitivity in full scan MS(n) compared to multiple reaction monitoring data acquisition. In combination with on-line liquid chromatography this feature makes the ion-trap useful in the scanning modes for rapid screening of low concentrations of phospholipid species in biological samples as recently described (Uran S, Larsen A, Jacobsen PB, Skotland T. J. Chromatogr. B 2001; 758: 265).

  5. Characterization of smokeless powders using multiplexed collision-induced dissociation mass spectrometry and chemometric procedures.

    PubMed

    Reese, Kristen L; Jones, A Daniel; Smith, Ruth Waddell

    2017-03-01

    This work demonstrates a non-targeted mass spectrometry approach for identification of organic compounds in smokeless powders. Unburned powders were removed from various commercial ammunitions of different brand, primer composition, caliber, and age. The unburned powders and corresponding fired residues were analyzed by liquid chromatography-atmospheric pressure chemical ionization-time-of-flight mass spectrometry (LC-APCI-TOFMS). Multiplexed collision-induced dissociation was performed at increasing collision potentials resulting in successive fragmentation that provided structural information for compound identification in a non-targeted manner. Nine compounds were identified in the powders, including akardite II, ethyl centralite, diphenylamine, N-nitrosodiphenylamine, and dibutyl phthalate. Multivariate statistical procedures were performed to first investigate association and discrimination of the unburned powders. Principal components analysis (PCA) of the chemical profiles suggested nine distinct groups of powders, according to the dominant organic compounds present. The clusters formed in hierarchical cluster analysis (HCA) were mostly in agreement with PCA groupings although HCA provided a metric to quantify the similarity. Finally, association of the fired residue to the corresponding unburned powder was possible although the success was highly dependent on the composition of the unburned powder and the extent of compound depletion as a result of firing.

  6. Detection of tyrosine phosphorylated peptides via skimmer collision-induced dissociation/ion trap mass spectrometry.

    PubMed

    Zolodz, Melissa D; Wood, Karl V

    2003-03-01

    Phosphorylation of proteins is an important post-translational protein modification in cellular response to environmental change and occurs in both prokaryotes and eukaryotes. Identification of the amino acid on individual proteins that become phosphorylated in response to extracellular stimulus is essential for understanding the mechanisms involved in the intracellular signals that these modifications facilitate. Most protein kinases catalyze the phosphorylation of proteins on serine, threonine or tyrosine. Although tyrosine phosphorylation is often the least abundant of the three major phosphorylation sites, it is important owing to its role in signal pathways. Currently available methods for the identification of phosphorylation sites can often miss low levels of tyrosine phosphorylations. This paper describes a method for the identification of phosphotyrosine-containing peptides using electrospray ionization on an ion trap mass spectrometer. Skimmer-activated collision-induced dissociation (CID) was used to generate the phosphotyrosine immonium ion at m/z 216. This method is gentle enough that the protonated molecule of the intact peptide is still observed. In-trap CID was employed for the verification of the phosphotyrosine immonium ion. Using this technique, low levels of phosphotyrosine-containing peptides can be identified from peptide mixtures separated by nanoflow micro liquid chromatography/mass spectrometry.

  7. GenoMass software: a tool based on electrospray ionization tandem mass spectrometry for characterization and sequencing of oligonucleotide adducts

    PubMed Central

    Sharma, Vaneet K; Glick, James; Liao, Qing; Shen, Chang; Vouros, Paul

    2012-01-01

    The analysis of DNA adducts is of importance in understanding DNA damage, and in the last few years mass spectrometry (MS) has emerged as the most comprehensive and versatile tool for routine characterization of modified oligonucleotides. The structural analysis of modified oligonucleotides, although routinely analyzed using mass spectrometry, is followed by a large amount of data, and a significant challenge is to locate the exact position of the adduct by computational spectral interpretation, which still is a bottleneck. In this report, we present an additional feature of the in-house developed GenoMass software, which determines the exact location of an adduct in modified oligonucleotides by connecting tandem mass spectrometry (MS/MS) to a combinatorial isomer library generated in silico for nucleic acids. The performance of this MS/MS approach using GenoMass software was evaluated by MS/MS data interpretation for an unadducted and its corresponding N-acetylaminofluorene (AAF) adducted 17-mer (5′OH-CCT ACC CCT TCC TTG TA-3′OH) oligonucleotide. Further computational screening of this AAF adducted 17-mer oligonucleotide (5′OH-CCT ACC CCT TCC TTG TA-3′OH) from a complex oligonucleotide mixture was performed using GenoMass. Finally, GenoMass was also used to identify the positional isomers of the AAF adducted 15-mer oligonucleotide (5′OH-ATGAACCGGAGGCCC-3′OH). GenoMass is a simple, fast, data interpretation software that uses an in silico constructed library to relate the MS/MS sequencing approach to identify the exact location of adduct on oligonucleotides. PMID:22689626

  8. Mining Large Scale Tandem Mass Spectrometry Data for Protein Modifications Using Spectral Libraries.

    PubMed

    Horlacher, Oliver; Lisacek, Frederique; Müller, Markus

    2016-03-04

    Experimental improvements in post-translational modification (PTM) detection by tandem mass spectrometry (MS/MS) has allowed the identification of vast numbers of PTMs. Open modification searches (OMSs) of MS/MS data, which do not require prior knowledge of the modifications present in the sample, further increased the diversity of detected PTMs. Despite much effort, there is still a lack of functional annotation of PTMs. One possibility to narrow the annotation gap is to mine MS/MS data deposited in public repositories and to correlate the PTM presence with biological meta-information attached to the data. Since the data volume can be quite substantial and contain tens of millions of MS/MS spectra, the data mining tools must be able to cope with big data. Here, we present two tools, Liberator and MzMod, which are built using the MzJava class library and the Apache Spark large scale computing framework. Liberator builds large MS/MS spectrum libraries, and MzMod searches them in an OMS mode. We applied these tools to a recently published set of 25 million spectra from 30 human tissues and present tissue specific PTMs. We also compared the results to the ones obtained with the OMS tool MODa and the search engine X!Tandem.

  9. [Measurement of free urinary cortisol and cortisone using liquid chromatography associated with tandem mass spectrometry method].

    PubMed

    Vieira, José Gilberto H; Nakamura, Odete H; Carvalho, Valdemir M

    2005-04-01

    Free urinary cortisol (UFF) measurement is one of the most useful screening tests for Cushing's syndrome. Immunoassays employed today by most clinical laboratories present limitations, specially concerning specificity. These limitations restrain a widespread application of the method, as well as the comparison of results obtained by the use of different methods. We present the development and characterization of a UFF and cortisone method based on liquid chromatography and tandem mass spectrometry (LC-MS/MS). A 200 microL aliquot from a 24 h urine sample is mixed with a solution containing a known quantity of deuterated cortisol and on-line extracted in solid phase (C18). The eluate is transferred to a second C18 column (Phenomenex Luna, 3 micro, 50 x 2 mm) and the isocratic mode elution profile is directly applied to a tandem mass spectrometer model Quattro Micro operating in positive mode atmospheric pressure chemical ionization (APCI). All process is automated and the quantification is performed by isotopic dilution, based on the analyte and the deuterated internal standard peak area ratios. The specificity study showed that all the steroids tested presented cross reactivity of <1% for cortisol and cortisone. Functional sensitivity is <1 microg/L for both steroids, and the interassay CV <8%. Recovery and linearity studies were satisfactory and comparison of results obtained using a RIA for UFF and the present method in 98 routine samples showed a correlation of r= 0.838, with the results obtained with LC-MS/MS significantly lower (medians of 22.0 vs. 49.4 microg/24 h for RIA) (P<0.0001). Reference values for cortisol were defined as values between 11 and 43 microg/24 h, compatible to those recently described for similar methods. The concomitant measurement of UF cortisone allows the study of the activity of the enzyme 11beta-HSD2 and the diagnosis of the apparent mineralocorticoid excess syndrome. The method represents the first steroid assay of a new generation

  10. Characterization of crude oil biomarkers using comprehensive two-dimensional gas chromatography coupled to tandem mass spectrometry.

    PubMed

    Mogollón, Noroska Gabriela Salazar; Prata, Paloma Santana; Dos Reis, Jadson Zeni; Neto, Eugênio Vaz Dos Santos; Augusto, Fabio

    2016-09-01

    Oil samples from Recôncavo basin (NE Brazil), previously analyzed by traditional techniques such as gas chromatography coupled to tandem mass spectrometry, were evaluated using comprehensive two-dimensional gas chromatography coupled to quadrupole mass spectrometry and comprehensive two-dimensional gas chromatography coupled to tandem mass spectrometry along with simplified methods of samples preparation to evaluate the differences and advantages of these analytical techniques to better understand the development of the organic matter in this basin without altering the normal distribution of the compounds in the samples. As a result, the geochemical parameters calculated by comprehensive two-dimensional gas chromatography coupled to tandem mass spectrometry described better the origin, maturity, and biodegradation of both samples probably by increased selectivity, resolution, and sensitivity inherent of the multidimensional technique. Additionally, the detection of the compounds such as, the C(14α-) homo-26-nor-17α-hopane series, diamoretanes, nor-spergulanes, C19 -C26 A-nor-steranes and 4α-methylsteranes resolved and detected by comprehensive two-dimensional gas chromatography coupled to tandem mass spectrometry were key to classify and differentiate these lacustrine samples according to their maturity and deposition conditions.

  11. Tandem Mass Spectrometry for Structural Identification of Sesquiterpene Alkaloids from the Stems of Dendrobium nobile Using LC-QToF.

    PubMed

    Wang, Yan-Hong; Avula, Bharathi; Abe, Naohito; Wei, Feng; Wang, Mei; Ma, Shuang-Cheng; Ali, Zulfiqar; Elsohly, Mahmoud A; Khan, Ikhlas A

    2016-05-01

    Dendrobium nobile is one of the fundamental herbs in traditional Chinese medicine. Sesquiterpene alkaloids are the main active components in this plant. Due to weak ultraviolet absorption and low content in D. nobile, these sesquiterpene alkaloids have not been extensively studied using chromatographic methods. Herein, tandem mass spectrometry combined with liquid chromatography separation provides a tool for the identification and characterization of the alkaloids from D. nobile. A total of nine sesquiterpene alkaloids were characterized by ultrahigh-performance liquid chromatography tandem mass spectrometry. These alkaloids can be classified into two subgroups that are represented by dendrobine and nobilonine. Tandem mass spectrometric studies revealed the fragmentation pathways of these two subgroup alkaloids that were used for the identification and characterization of other alkaloids in D. nobile. Characterization of these alkaloids using accurate mass and diagnostic fragments provided a reliable methodology for the analysis of D. nobile by ultrahigh-performance liquid chromatography tandem mass spectrometry. The limit of detection was defined as the signal-to-noise ratio equal to 3 : 1. Limits of detection of dendrobine and nobilonine were less than 30 ng/mL. The developed method was applied for the analysis of various Dendrobium species and related dietary supplements. Alkaloids were identified from D. nobile, but not detected from commercial samples including 13 other Dendrobium species and the 7 dietary supplements.

  12. One-color two-photon mass-analyzed threshold ionization spectroscopy of ethyl bromide through a dissociative intermediate state

    NASA Astrophysics Data System (ADS)

    Tang, Bifeng; Zhang, Song; Wang, Yanmei; Tang, Ying; Zhang, Bing

    2005-10-01

    Mass-analyzed threshold ionization (MATI) spectra of ethyl bromide were obtained using one-color two-photon ionization through a dissociative intermediate state. Accurate values for the adiabatic ionization energy have been obtained, 83099±5 and 85454±5cm-1 for the X˜1E2 and X˜2E2 states of the ethyl bromide cation, respectively, giving a splitting of 2355±10cm-1. Compared with conventional photoelectron data, the two-photon MATI spectrum exhibited a more extensive vibrational structure with a higher resolution, mainly containing the modes involving the dissociation coordinate. The observed modes were analyzed and discussed in terms of wave packet evolving on the potential-energy surface of the dissociative state.

  13. Online deuterium hydrogen exchange and protein digestion coupled with ion mobility spectrometry and tandem mass spectrometry.

    PubMed

    Donohoe, Gregory C; Arndt, James R; Valentine, Stephen J

    2015-05-19

    Online deuterium hydrogen exchange (DHX) and pepsin digestion (PD) is demonstrated using drift tube ion mobility spectrometry (DTIMS) coupled with linear ion trap (LTQ) mass spectrometry (MS) with electron transfer dissociation (ETD) capabilities. DHX of deuterated ubiquitin, followed by subsequent quenching and digestion, is performed within ∼60 s, yielding 100% peptide sequence coverage. The high reproducibility of the IMS separation allows spectral feature matching between two-dimensional IMS-MS datasets (undeuterated and deuterated) without the need for dataset alignment. Extracted ion drift time distributions (XIDTDs) of deuterated peptic peptides are mobility-matched to corresponding XIDTDs of undeuterated peptic peptides that were identified using collision-induced dissociation (CID). Matching XIDTDs allows a straightforward identification and deuterium retention evaluation for labeled peptides. Aside from the mobility separation, the ion trapping capabilities of the LTQ, combined with ETD, are demonstrated to provide single-residue resolution. Deuterium retention for the c- series ions across residues M(1)-L(15) and N(25)-R(42) are in good agreement with the known secondary structural elements within ubiquitin.

  14. Screening of dimethoate in food by isotope dilution and electrospray ionization tandem mass spectrometry.

    PubMed

    Mazzotti, Fabio; Di Donna, Leonardo; Macchione, Barbara; Maiuolo, Loredana; Perri, Enzo; Sindona, Giovanni

    2009-05-01

    Crop control is an important issue in both developed and developing countries. An environmentally friendly approach is represented by the so-called Integrated Pest Management (IPM), whereby synthetic pesticides are only applied as a last resort, under the strict control of suitable experts. European and US regulatory authorities, such as the US EPA, are constantly assessing the risks of exposure to the organophosphate (OP) class of pesticides and, among these, specifically dimethoate. The use of dimethoate is still allowed in many crops, including olives, which once was based in the Mediterranean area but now is expanding rapidly throughout the world. An important aspect of IPM protocols is represented by the availability of reliable and sensitive methods to detect pesticides residues. This paper describes an isotope dilution dimethoate assay based on the application of electrospray ionization tandem mass spectrometry (ESI-MS/MS) by means of a deuterium-labeled internal standard.

  15. Electrospray ionization-tandem mass spectrometry method for differentiating chlorine substitution in disinfection byproduct formation.

    PubMed

    Deng, Zhuo; Yang, Xin; Shang, Chii; Zhang, Xiangru

    2014-05-06

    An electrospray ionization-tandem mass spectrometry (ESI-tqMS) method was developed to identify the location of chlorine substitution during the chlorination of model organic compounds. The chlorine substitution in the aliphatic part and that in the benzene ring of an organic molecule can be differentiated by their corresponding ranges of optimum collision energies, 5-7 eV and over 15 eV, respectively, in the precursor ion scan of m/z 35. The method was applied to predict the structures of intermediates and reveal the transformation pathways during the chlorination of 4-amino-2-chlorobenzoic acid and phenylalanine as a function of reaction time and the chlorine-to-precursor ratio. In the case of phenylalanine, chlorine was found to replace one hydrogen atom attached to the aliphatic nitrogen; in the case of 4-amino-2-chlorobenzoic acid, chlorine was found to replace the hydrogen atoms attached to the aromatic rings.

  16. Characterization of protein N-glycosylation by tandem mass spectrometry using complementary fragmentation techniques

    SciTech Connect

    Ford, Kristina L.; Zeng, Wei; Heazlewood, Joshua L.; Bacic, Antony

    2015-08-28

    The analysis of post-translational modifications (PTMs) by proteomics is regarded as a technically challenging undertaking. While in recent years approaches to examine and quantify protein phosphorylation have greatly improved, the analysis of many protein modifications, such as glycosylation, are still regarded as problematic. Limitations in the standard proteomics workflow, such as use of suboptimal peptide fragmentation methods, can significantly prevent the identification of glycopeptides. The current generation of tandem mass spectrometers has made available a variety of fragmentation options, many of which are becoming standard features on these instruments. Lastly, we have used three common fragmentation techniques, namely CID, HCD, and ETD, to analyze a glycopeptide and highlight how an integrated fragmentation approach can be used to identify the modified residue and characterize the N-glycan on a peptide.

  17. Building and searching tandem mass (MS/MS) spectral libraries for peptide identification in proteomics.

    PubMed

    Lam, Henry; Aebersold, Ruedi

    2011-08-01

    Spectral library searching is an emerging approach in peptide identifications from tandem mass spectra, a critical step in proteomic data analysis. In spectral library searching, a spectral library is first meticulously compiled from a large collection of previously observed peptide MS/MS spectra that are conclusively assigned to their corresponding amino acid sequence. An unknown spectrum is then identified by comparing it to all the candidates in the spectral library for the most similar match. This review discusses the basic principles of spectral library building and searching, describes its advantages and limitations, and provides a primer for researchers interested in adopting this new approach in their data analysis. It will also discuss the future outlook on the evolution and utility of spectral libraries in the field of proteomics.

  18. Determination of Metabolic Viability and Cell Mass Using a Tandem Resazurin/Sulforhodamine B Assay.

    PubMed

    Silva, Filomena S G; Starostina, Irina G; Ivanova, Vilena V; Rizvanov, Albert A; Oliveira, Paulo J; Pereira, Susana P

    2016-05-04

    The identification of rapid, reliable, and highly reproducible biological assays that can be standardized and routinely used in preclinical tests constitutes a promising approach to reducing drug discovery costs and time. This unit details a tandem, rapid, and reliable cell viability method for preliminary screening of chemical compounds. This assay measures metabolic activity and cell mass in the same cell sample using a dual resazurin/sulforhodamine B assay, eliminating the variation associated with cell seeding and excessive manipulations in assays that test different cell samples across plates. The procedure also reduces the amount of cells, test compound, and reagents required, as well as the time expended in conventional tests, thus resulting in a more confident prediction of toxic thresholds for the tested compounds. © 2016 by John Wiley & Sons, Inc.

  19. Determination of dapsone in meat and milk by liquid chromatography tandem mass spectrometry.

    PubMed

    Hadjigeorgiou, M; Papachrysostomou, Ch; Theodorou, Z; Kanari, P; Constantinou, S

    2009-04-01

    Within the EU the use of dapsone (4,4-diaminodiphenylsulfone) is prohibited in food-producing animals and consequently it's included in the Annex IV of the Directive 90/2377/EC. A quantitative confirmatory method has been developed and validated according to the criteria defined in the Commission Decision 2002/657/EC, for the determination of dapsone in meat and milk. Samples, after homogenization in alkaline conditions and organic solvent extraction, were purified on silica gel solid phase extraction cartridges. The eluate was evaporated and redissolved in mobile phase and was analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS) in positive electrospray ionisation (ESI) using deuterium labelled Sulphadimidine-d7 as internal standard. The calculated value for, decision limit, CCalpha is 0.12 microgkg(-1), and the detection capability; CCbeta value is 0.16 microgkg(-1).

  20. Determination of glufosfamide in rat plasma by liquid chromatography/tandem mass spectrometry.

    PubMed

    Chen, Xiaoyan; Sun, Yuming; Cao, Xueqin; Jin, Fengdan; Zhong, Dafang

    2005-01-01

    A sensitive and selective high-performance analytical method based on liquid chromatography with tandem mass spectrometric detection (LC/MS/MS) was developed for the quantification of glufosfamide in rat plasma. Zidovudine was employed as internal standard. Glufosfamide was determined after methanol-mediated plasma protein precipitation using LC/MS/MS with an electrospray ionization interface in negative ion mode. Two sets of standard curves were developed, from 0.005 to 1.0 microg/mL and from 1.0 to 50.0 microg/mL. The assay was accurate (% deviations from nominal concentrations < 5%), precise and reproducible (intra- and inter-day coefficients of variation < 10%). Glufosfamide in rat plasma was stable over three freeze/thaw cycles, and at ambient temperatures, for at least 2 h. The validated method was successfully applied to the determination of glufosfamide plasma concentrations in rats for 24 h following an intravenous administration of 25 mg/kg.

  1. Determination of carbocysteine in human plasma by liquid chromatography/tandem mass spectrometry employing precolumn derivatization.

    PubMed

    Chen, Xiaoyan; Zhong, Dafang; Han, Ying; Xie, Zhiyong

    2003-01-01

    A sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed to determine carbocysteine in human plasma using 2-pyridylacetic acid as the internal standard (IS). The method employed derivatization with 10 M hydrochloric acid/methanol, which significantly improved the ionization efficiency of carbocysteine. After methanol-induced protein precipitation of plasma samples, carbocysteine and the IS were derivatized and subjected to LC/MS/MS analysis using atmospheric pressure chemical ionization. The method has a lower limit of quantitation of 20 ng/mL for a 0.2-mL plasma aliquot. The intra- and inter-day precision (RSD), calculated from quality control (QC) samples, was less than 7%. The accuracy, determined using QC samples, was within +/- 1%. The method offered increased sensitivity, selectivity and speed of analysis over existing methods. The method was utilized to support clinical pharmacokinetic studies of carbocysteine in volunteers following oral administration.

  2. Plasma lipid analysis by hydrophilic interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

    PubMed

    Sonomura, Kazuhiro; Kudoh, Shinobu; Sato, Taka-Aki; Matsuda, Fumihiko

    2015-06-01

    A novel method for the analysis of endogenous lipids and related compounds was developed employing hydrophilic interaction liquid chromatography with electrospray ionization tandem mass spectrometry. A hydrophilic interaction liquid chromatography with carbamoyl stationary phase achieved clear separation of phosphatidylcholine, lysophosphatidylcholine, sphingomyelin, ceramide, and mono-hexsosyl ceramide groups with good peak area repeatability (RSD% < 10) and linearity (R(2) > 0.99). The established method was applied to human plasma assays and a total of 117 endogenous lipids were successfully detected and reproducibly identified. In addition, we investigated the simultaneous detection of small polar metabolites such as amino and organic acids co-existing in the same biological samples processed in a single analytical run with lipids. Our results show that hydrophilic interaction liquid chromatography is a useful tool for human plasma lipidome analysis and offers more comprehensive metabolome coverage.

  3. Pressurized liquid extraction followed by liquid chromatography with tandem mass spectrometry to determine pharmaceuticals in mussels.

    PubMed

    Núñez, Mireia; Borrull, Francesc; Pocurull, Eva; Fontanals, Núria

    2016-02-01

    An analytical method based on pressurized liquid extraction and solid-phase extraction with a mixed-mode Oasis(®) MAX sorbent as cleanup, followed by liquid chromatography with electrospray ionization and tandem mass spectrometry was developed and validated for the determination of seven widely used pharmaceuticals in mussel species. The optimization of the pressurized liquid extraction and the solid-phase extraction parameters is described. The method provided extraction recoveries ranging from 61 to 90%, and limits of detection ranging from 2 to 50 ng/g (dry weight). The repeatability and reproducibility of the method, expressed as relative standard deviation, were lower than 15 and 19%, respectively. The method was successfully applied to the analysis of mussel samples from different locations. The analyses showed that salicylic acid was present in mussels at concentrations up to 177 ng/g (dry weight).

  4. Determination of prostaglandin analogs in cosmetic products by high performance liquid chromatography with tandem mass spectrometry.

    PubMed

    Wittenberg, James B; Zhou, Wanlong; Wang, Perry G; Krynitsky, Alexander J

    2014-09-12

    A method was developed and validated for the determination of 16 prostaglandin analogs in cosmetic products. The QuEChERS (Quick, Easy, Cheap, Efficient, Rugged, Safe) liquid-liquid extraction method, typically used for pesticide residue analysis, was utilized as the sample preparation technique. The prostaglandin analogs were chromatographically separated and quantified using high performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). Thirty-one cosmetic products were surveyed, and 13 products were determined to contain a prostaglandin analog with amounts ranging from 27.4 to 297μg/g. The calculated concentrations for the cosmetic products were in a similar range when compared to the concentrations of three different prostaglandin analog-containing prescription products.

  5. Multidetection of antibiotics in liver tissue by ultra-high-pressure-liquid-chromatography-tandem mass spectrometry.

    PubMed

    Freitas, Andreia; Barbosa, Jorge; Ramos, Fernando

    2015-01-22

    A multiresidue quantitative screening method covering 39 antibiotics from 7 different families by ultra-high-pressure-liquid-chromatography-tandem mass spectrometry (UHPLC-MS/MS) is described. Sulfonamides, trimethoprim, tetracyclines, macrolides, quinolones, penicillins and chloramphenicol are simultaneously detected in liver tissue. A simple sample treatment method consisting of extraction with a mixture of acetonitrile and ethylenediaminetetraacetic acid (EDTA) followed by solid-phase extraction (SPE) with a hydrophilic-lipophilic balanced (HLB) cartridge was developed. The methodology was validated, in accordance with Decision 2002/657/EC, by evaluating the following required parameters: decision limit (CCα), detection capability (CCβ), specificity, repeatability and reproducibility. The precision, in terms of the relative standard deviation, was under 22% for all of the compounds, and the recoveries were between 80% and 110%. The CCα and CCβ were determined according to the maximum residue limit (MRL) or the minimum required performance limit (MRPL), when established.

  6. Analysis of bromate in drinking water using liquid chromatography-tandem mass spectrometry without sample pretreatment.

    PubMed

    Kosaka, Koji; Asami, Mari; Takei, Kanako; Akiba, Michihiro

    2011-01-01

    An analytical method for determining bromate in drinking water was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The (18)O-enriched bromate was used as an internal standard. The limit of quantification (LOQ) of bromate was 0.2 µg/L. The peak of bromate was separated from those of coexisting ions (i.e., chloride, nitrate and sulfate). The relative and absolute recoveries of bromate in two drinking water samples and in a synthesized ion solution (100 mg/L chloride, 10 mg N/L nitrate, and 100 mg/L sulfate) were 99-105 and 94-105%, respectively. Bromate concentrations in 11 drinking water samples determined by LC-MS/MS were <0.2-2.3 µg/L. The results of the present study indicated that the proposed method was suitable for determining bromate concentrations in drinking water without sample pretreatment.

  7. Analysis of Total Human Urinary Glycosaminoglycan Disaccharides by Liquid Chromatography-Tandem Mass Spectrometry.

    PubMed

    Sun, Xiaojun; Li, Lingyun; Overdier, Katherine H; Ammons, Lee Anne; Douglas, Ivor S; Burlew, Clay Cothren; Zhang, Fuming; Schmidt, Eric P; Chi, Lianli; Linhardt, Robert J

    2015-06-16

    The determination of complex analytes, present at low concentrations, in biological fluids poses a difficult challenge. This study relies on an optimized method of recovery, enzymatic treatment, and disaccharide analysis by liquid chromatography-tandem mass spectrometry to rapidly determine low concentrations of glycosaminoglycans in human urine. The approach utilizes multiple reaction monitoring (MRM) of glycosaminoglycan disaccharides obtained from treating urine samples with recombinant heparin lyases and chondroitin lyase. This rapid and sensitive method allows the analysis of glycosaminoglycan content and disaccharide composition in urine samples having concentrations 10- to 100-fold lower than those typically analyzed from patients with metabolic diseases, such as mucopolysaccharidosis. The current method facilitates the analysis low (ng/mL) levels of urinary glycosaminoglycans present in healthy individuals and in patients with pathological conditions, such as inflammation and cancers, that can subtly alter glycosaminoglycan content and composition.

  8. Uncommonly thorough hydrolysis of peptides during ripening of Ragusano cheese revealed by tandem mass spectrometry.

    PubMed

    Gagnaire, Valérie; Carpino, Stefania; Pediliggieri, Concetta; Jardin, Julien; Lortal, Sylvie; Licitra, Giuseppe

    2011-12-14

    Ragusano is a pasta filata cheese produced from raw milk in Sicily. The proteolysis was extensively analyzed after stretching (day 0), at 4 and 7 months of ripening through soluble nitrogen, urea-PAGE, and peptide identification by tandem mass spectrometry. After stretching, 123 peptides were identified: 72 arising from β-casein, 34 from α(s1)-casein, and 17 from α(s2)-casein. The main protein splitting corresponded to the action of plasmin, chymosin, cathepsin D, cell envelope proteinase, and peptidase activities of lactic acid bacteria. Unlike other types of cheeses, <10% residual β- and α(s)-caseins remained intact at 7 months, indicating original network organization based on large casein fragments. The number of identified soluble peptides also dramatically decreased after 4 and 7 months of ripening, to 47 and 25, respectively. Among them, bioactive peptides were found, that is, mineral carrier, antihypertensive, and immunomodulating peptides and phosphopeptides.

  9. Characterization of protein N-glycosylation by tandem mass spectrometry using complementary fragmentation techniques

    DOE PAGES

    Ford, Kristina L.; Zeng, Wei; Heazlewood, Joshua L.; ...

    2015-08-28

    The analysis of post-translational modifications (PTMs) by proteomics is regarded as a technically challenging undertaking. While in recent years approaches to examine and quantify protein phosphorylation have greatly improved, the analysis of many protein modifications, such as glycosylation, are still regarded as problematic. Limitations in the standard proteomics workflow, such as use of suboptimal peptide fragmentation methods, can significantly prevent the identification of glycopeptides. The current generation of tandem mass spectrometers has made available a variety of fragmentation options, many of which are becoming standard features on these instruments. Lastly, we have used three common fragmentation techniques, namely CID, HCD,more » and ETD, to analyze a glycopeptide and highlight how an integrated fragmentation approach can be used to identify the modified residue and characterize the N-glycan on a peptide.« less

  10. Targeted quantitative analysis of eicosanoid lipids in biological samples using liquid chromatography-tandem mass spectrometry

    PubMed Central

    Mesaros, Clementina; Lee, Seon Hwa; Blair, Ian A.

    2009-01-01

    The eicosanoids are a large family of arachidonic acid oxidation products that contain twenty carbon atoms. Cyclooxygenase (COX)-derived eicosanoids have important roles as autacoids involved in the regulation of cardiovascular function and tumor progression. Lipoxygenase (LO)-derived eicosanoids have been implicated as important mediators of inflammation, asthma, cardiovascular disease and cancer. Cytochrome P-450 (P450)-derived eicosanoids are both vasodilators and vasoconstrictors. There is intense interest in the analysis of reactive oxygen species (ROS)-derived isoprostanes (isoPs) because of their utility as biomarkers of oxidative stress. Enzymatic pathways of eicosanoid formation are regioselective and enantioselective, whereas ROS-mediated eicosanoid formation proceeds with no stereoselectivity. Many of the eicosanoids are also present in only pM concentrations in biological fluids. This presents a formidable analytical challenge because methodology is required that can separate enantiomers and diastereomers with high sensitivity and specificity. However, the discovery of atmospheric pressure ionization (API)/MS methodology of electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and electron capture (EC) APCI has revolutionized our ability to analyze endogenous eicosanoids. LC separations of eicosanoids can now be readily coupled with API ionization, collision induced dissociation (CID) and tandem MS (MS/MS). This makes it possible to efficiently conduct targeted eicosanoid analyses using LC-multiple reaction motoring (MRM)/MS. Several examples of targeted eicosanoid lipid analysis using conventional LC-ESI/MS have been discussed and some new data on the analysis of eicosanoids using chiral LC-ECAPCI/MS has been presented. PMID:19345647

  11. Determination of azatadine in human plasma by liquid chromatography/tandem mass spectrometry.

    PubMed

    Zhu, Yan-Rong; Jia, Yan-Yan; Jiang, Ling; Wang, Chao; Ding, Li-Kun; Yang, Jing; Li, Liang; Zhao, Pei-Xi; Liu, Wen-Xin; Yi-Ding; Wang, Li; Wen, Ai-Dong

    2011-08-01

    A sensitive method using liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) was developed and validated for the analysis of antihistamine drug azatadine in human plasma. Loratadine was used as internal standard (IS). Analytes were extracted from human plasma by liquid/liquid extraction using ethyl acetate. The organic phase was reduced to dryness under a stream of nitrogen at 30 °C and the residue was reconstituted with the mobile phase. 5 μL of the resulting solution was injected onto the LC-MS/MS system. A 4.6 mm × 150 mm, I.D. 5 μm, Agilent TC-C(18) column was used to perform the chromatographic analysis. The mobile phase consisted of ammonium formate buffer 0.010 M (adjusted to pH 4.3 with 1M formic acid)/acetonitrile (20:80, v/v) The chromatographic run time was 5 min per injection and flow rate was 0.6 mL/min. The retention time was 2.4 and 4.4 min for azatadine and IS, respectively. The tandem mass spectrometric detection mode was achieved with electrospray ionization (ESI) iron source and the multiple reaction monitoring (MRM) (291.3 → 248.2m/z for azatadine, 383.3 → 337.3m/z for IS) was operated in positive ion modes. The low limit of quantitation (LLOQ) was 0.05 ng/mL. The intra-day and inter-day precision of the quality control (QC) samples was 8.93-11.57% relative standard deviation (RSD). The inter-day accuracy of the QC samples was 96.83-105.07% of the nominal values.

  12. Determination of salivary efavirenz by liquid chromatography coupled with tandem mass spectrometry.

    PubMed

    Theron, Anri; Cromarty, Duncan; Rheeders, Malie; Viljoen, Michelle

    2010-10-15

    A novel and robust screening method for the determination of the non-nucleoside reverse transcriptase inhibitor, efavirenz (EFV), in human saliva has been developed and validated based on high performance liquid chromatography tandem mass spectrometry (LC-MS/MS). Sample preparation of the saliva involved solid-phase extraction (SPE) on C18 cartridges. The analytes were separated by high performance liquid chromatography (Phenomenex Kinetex C18, 150mm×3mm internal diameter, 2.6μm particle size) and detected with tandem mass spectrometry in electrospray positive ionization mode with multiple reaction monitoring. Gradient elution with increasing methanol (MeOH) concentration was used to elute the analytes, at a flow-rate of 0.4mL/min. The total run time was 8.4min and the retention times for the internal standard (reserpine) was 5.4min and for EFV was 6.5min. The calibration curves showed linearity (r(2), 0.989-0.992) over the concentration range of 3.125-100μg/L. Mean intra- and inter-assay relative standard deviation, accuracy, mean extraction recovery, limit of detection (LOD) and limit of quantification (LOQ) were 0.46-9.43%, 80-120%, 60% (±7.95), 1.84 and 6.11μg/L respectively. The working range was defined as 6.25-100μg/L. This novel LC-MS/MS assay is suitable for reliable detection of low EFV concentrations in saliva and can be used as a screening tool for monitoring EFV compliance.

  13. Determination of 23 phthalic acid esters in food by liquid chromatography tandem mass spectrometry.

    PubMed

    Xu, Dunming; Deng, Xiaojun; Fang, Enhua; Zheng, Xianghua; Zhou, Yu; Lin, Liyi; Chen, Luping; Wu, Ming; Huang, Zhiqiang

    2014-01-10

    A rapid and sensitive method was developed for the determination of 23 phthalates in food samples including milk-based products, distilled liquor, wine, beverage, grain, meat, oil, biscuit (cookie), and canned food by liquid chromatography tandem mass spectrometry (LC-MS/MS). Liquid samples were exacted by acetonitrile, while solid samples were prepared by QuEChERS or glass-based SPE methods. The 23 phthalates were separated on Poroshell 120 EC-C18 column and followed by positive electrospray ionization as well as multi-reaction monitoring provided by a triple-quadrupole tandem mass spectrometer. To reduce contamination, the plastic materials were avoided in sample handling and preparation . The LODs were between 0.8 and 15 μg kg(-1) and LOQs were between 10 and 100 μg kg(-1). By using different concentrations: 100, 500, and 1000 μg kg(-1)) for DINP and DIDP; 50, 100, and 1000 μg kg(-1) for other 21 phthalate compounds, the spiked recoveries were within 75.5-115.2% and the relative standard deviations (RSDs) were in the range of 3.2-18.9%. The proposed protocol was then applied to the analysis of 623 real samples collected from the two sides of the Taiwan Straits, and the DEHP was found in almost all samples tested in this study, with levels ranging from 0.02 to 2685 mg kg(-1). The present study demonstrated a rapid, sensitive, and accurate method for determining 23 phthalates in foodstuffs.

  14. Determination of eptifibatide concentration in human plasma utilizing the liquid chromatography-tandem mass spectrometry method.

    PubMed

    Liu, Jia; Duan, Xiaotao; Chen, Xiaoyan; Zhong, Dafang

    2009-02-15

    A sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to determine the concentration of eptifibatide in human plasma. Following protein precipitation, the analyte was separated on a reversed-phase C(18) column. Acetonitrile:5mM ammonium acetate:acetic acid (30:70:0.1, v/v/v) was used at a flow-rate of 0.5mL/min with the isocratic mobile phase. An API 4000 tandem mass spectrometer equipped with a Turbo IonSpray ionization source was used as the detector and was operated in the positive ion mode. "Truncated" multiple reaction monitoring using the transition of m/z 832.6-->m/z 832.6 and m/z 931.3-->m/z 931.3 was performed to quantify eptifibatide and the internal standard (EPM-05), respectively. The method had a lower limit of quantification of 4.61ng/mL for eptifibatide. The calibration curve was demonstrated to be linear over the concentration range of 4.61-2770ng/mL. The intra- and inter-day precisions were less than 10.5% for each QC level, and the inter-day relative errors were 2.0%, 5.6%, and 2.8% for 9.22, 184, and 2490ng/mL, respectively. The validated method was successfully applied to the quantification of eptifibatide concentration in human plasma after intravenous (i.v.) administration of a 270-microg/kg bolus of eptifibatide and i.v. administration of eptifibatide at a constant rate of infusion of 2microg/(kgmin) for 18h in order to evaluate the pharmacokinetics.

  15. Plasma free and total carnitine measured in children by tandem mass spectrometry.

    PubMed

    Osorio, J H; Pourfarzam, M

    2002-11-01

    Free and total carnitine quantification is important as a complementary test for the diagnosis of unusual metabolic diseases, including fatty acid degradation disorders. The present study reports a new method for the quantification of free and total carnitine in dried plasma specimens by isotope dilution electrospray tandem mass spectrometry with sample derivatization. Carnitine is determined by looking for the precursor of ions of m/z = 103 of N-butylester derivative, and the method is validated by comparison with radioenzymatic assay. We obtained an inter- and intra-day assay coefficient of variation of 4.3 and 2.3, respectively. Free and total carnitine was analyzed in 309 dried plasma spot samples from children ranging in age from newborn to 14 years using the new method, which was found to be suitable for calculating reference age-related values for free and total carnitine (less than one month: 19.3 +/- 2.4 and 23.5 +/- 2.9; one to twelve months: 28.8 +/- 10.2 and 35.9 +/- 11.4; one to seven years: 30.7 +/- 10.3 and 38.1 +/- 11.9; seven to 14 years: 33.7 +/- 11.6, and 43.1 +/- 13.8 micro M, respectively). No difference was found between males and females. A significant difference was observed between neonates and the other age groups. We compare our data with reference values in the literature, most of them obtained by radioenzymatic assay. However, this method is laborious and time consuming. The electrospray tandem mass spectrometry method presented here is a reliable, rapid and automated procedure for carnitine quantitation.

  16. Antioxidant activity guided separation of major polyphenols of marjoram (Origanum majorana L.) using flash chromatography and their identification by liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

    PubMed

    Hossain, Mohammad B; Camphuis, Gabriel; Aguiló-Aguayo, Ingrid; Gangopadhyay, Nirupama; Rai, Dilip K

    2014-11-01

    Marjoram extracts have been separated into polar and nonpolar parts using liquid-liquid extraction. Both polar and nonpolar parts of the extracts were further fractionated by flash chromatography. The obtained fractions (90 polar and 45 nonpolar fractions) were investigated for their antioxidant activities by 2,2-diphenylpicrylhydrazyl and ferric ion reducing antioxidant power assays. A direct, positive, and linear relationship between antioxidant activity and total phenolic content of the fractions was observed. Based on antioxidant and total phenolic content data, the three fractions with the high antioxidant activities from polar and nonpolar part of the extract were analyzed for their constituent polyphenols by liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Compounds were identified by matching the mass spectral data and retention time with those of authentic standards. Identification of the compounds for which there were no "in-house" standards available was carried out by accurate mass measurement of the precursor ions and product ions generated from collision-induced dissociation. Rosmarinic acid was found to be the strongest antioxidant polyphenol conferring the highest antioxidant activity to fractions 47 and 17 of polar and nonpolar part of the extract, respectively. The identification of the rosmarinic acid was further confirmed by (1) H NMR spectroscopy.

  17. Stimulation of Slack K+ channels alters mass at the plasma membrane by triggering dissociation of a phosphatase-regulatory complex

    PubMed Central

    Fleming, Matthew R.; Brown, Maile R.; Kronengold, Jack; Zhang, Yalan; Jenkins, David P.; Barcia, Gulia; Nabbout, Rima; Bausch, Anne E.; Ruth, Peter; Lukowski, Robert; Navaratnam, Dhasakumar S.; Kaczmarek, Leonard K.

    2016-01-01

    Summary Human mutations in the cytoplasmic C-terminal domain of Slack sodium-activated potassium (KNa) channels result in childhood epilepsy with severe intellectual disability. Slack currents can be increased by pharmacological activators or by phosphorylation of a Slack C-terminal residue by protein kinase C. Using an optical biosensor assay, we find that Slack channel stimulation in neurons or transfected cells produces loss of mass near the plasma membrane. Slack mutants associated with intellectual disability fail to trigger any change in mass. The loss of mass results from the dissociation of the protein phosphatase 1 (PP1) targeting protein, Phactr-1, from the channel. Phactr1 dissociation is specific to wild-type Slack channels and is not observed when related potassium channels are stimulated. Our findings suggest that Slack channels are coupled to cytoplasmic signaling pathways, and that dysregulation of this coupling may trigger the aberrant intellectual development associated with specific childhood epilepsies. PMID:27545877

  18. Comparison of peptide mass mapping and electron capture dissociation as assays for histone posttranslational modifications

    NASA Astrophysics Data System (ADS)

    Zhang, Liwen; Freitas, Michael A.

    2004-05-01

    Posttranslational modifications of core histones play a critical role in the structure of chromatin and the regulation of gene activities. Improved techniques for determining these modification sites may lead to a better understanding of histone regulation at the molecular level. LC-MS peptide mass mapping was performed on pepsin, trypsin and Glu-C digests of bovine thymus H4 using a QqTOF instrument. The well established modification sites of H4 (acetylation of K8, 12, 16 and methylation of K20) were observed in addition to several recently discovered modifications including: methylation of K31, 44, 59 and acetylation of K20, 77, 79. For comparison, electron capture dissociation (ECD) was performed on intact H4 along with several peptides from enzymatic digestion. The results from the ECD experiments of histone H4 indicated the acetylation of K5, 12, 16, 31, 91 and the methylation of K20 and 59 in good agreement with the result from peptide mapping. The work is dedicated to Alan G. Marshall on his 60th birthday. His endeavors in the advancement of FT-ICR facilitated experiments reported herein.

  19. Determination of nitrofuran and chloramphenicol residues by high resolution mass spectrometry versus tandem quadrupole mass spectrometry.

    PubMed

    Kaufmann, A; Butcher, P; Maden, K; Walker, S; Widmer, M

    2015-03-03

    An ultra-high performance liquid chromatography based method, coupled to high resolution mass spectrometry (UHPLC-HRMS), was developed to permit the detection and quantification of various nitrofuran and chloramphenicol residues in a number of animal based food products. This method is based on the hydrolysis of covalently bound metabolites and derivatization with 2-nitrobenzaldehyde. Clean-up is achieved by a liquid/liquid and a reversed phase/solid phase extraction. Not only are the four conventional nitrofurans (nitrofurantoin, furazolidone, nitrofurazone and furaltadone) detected, but also nifursol, nitrovin and nifuroxazide. Furthermore, an underivatizable nitrofuran (nifurpirinol) and another banned drug (chloramphenicol) can be quantified as well. The compounds are detected in the form of their precursor ions, [M+H](+) and [M-H](-), respectively. The mass resolving power of 70,000 FWHM, and the applied mass window ensure sufficient selectivity and sensitivity. Confirmation is obtained by monitoring the HRMS resolved product ions which were derived from the unit-mass resolved precursor ions. The multiplexing capability of the utilized Orbitrap instrument provides not only highly selective, but also sensitive confirmatory signals. This method has been validated according to the CD 2002/657/EC for the following matrices: muscle, liver, kidney, fish, honey, eggs and milk.

  20. False sugar sequence ions in electrospray tandem mass spectrometry of underivatized sialyl-Lewis-type oligosaccharides

    NASA Astrophysics Data System (ADS)

    Ernst, Beat; Müller, Dieter R.; Richter, Wilhelm J.

    1997-01-01

    Formation of "false" sugar sequence ions from branched tetrasaccharides of the sialyl-Lewis-type by migration of fucose towards sialic acid residues is shown to occur in [M + H]+ and [M + NH4]+ ions produced by electrospray ionization and subjected to low energy collision induced dissociation (CID). For the verification of their composition and sequence, such irregular ions were produced in the orifice region of the ion source, mass selected in Q1, and subjected to a second CID step in Q2 of a triple quadrupole analyser. When produced and analysed in the same "double CID" fashion, the branched B3 ions still containing all four sugar subunits show such migration to only a minor extent. The analysis of Bn fragment ions with high numbers for n may thus have advantages over the analysis of M-like species

  1. Liquid chromatography coupled to tandem mass spectrometry for the analysis of acrylamide in typical Spanish products.

    PubMed

    Bermudo, E; Moyano, E; Puignou, L; Galceran, M T

    2008-07-15

    This paper describes the use of liquid chromatography coupled to tandem mass spectrometry for the determination of acrylamide in several typical foods produced and consumed in Spain. Christmas sweets, olives, traditionally made potato crisps, pastry products, sweet fritters ("churros") and one of Spain's most famous dishes, Spanish omelette, were selected. Using the mass spectra information provided by an ion trap analyzer in combination with the accurate mass measurements from time-of-flight (TOF) spectrometry a co-extractive interference present in some potato products was identified as valine. A porous graphitic carbon column, which enabled the co-extractive and acrylamide to be separated, and ion trap or triple quadrupole analyzers, depending on the acrylamide concentration, were used to determine this genotoxic compound in foodstuffs. The highest values were found in potato products, sweet fritters, Christmas sweets and pastry products, with values ranging between 70 and 2000 microg/g. Spanish omelette presented relatively low levels, similar to those obtained for dried fruits.

  2. Ultra-high performance liquid chromatography tandem mass spectrometry for comprehensive analysis of urinary acylcarnitines.

    PubMed

    Zuniga, Azeret; Li, Liang

    2011-03-09

    We report an enabling mass spectrometric method for the analysis of lipid metabolites in order to define better the lipid metabolome in terms of chemical diversity and generate fragment ion spectra of these metabolites as a potential resource for unknown metabolite identification. This work focuses on the analysis of one important class of lipid metabolites, the acylcarnitines. Current analytical methods have only detected and identified a limited number of these metabolites. The method described herein provides the most comprehensive acylcarnitine profile in urine of healthy individuals up to date. It involves an optimized solid phase extraction technique for selective analyte extraction using cartridges containing both lipophilic and cation-exchange properties. The captured analytes are then subjected to ultra-high performance liquid chromatography (UPLC) separation, followed by tandem mass spectrometry (MS/MS) analysis using information-dependent acquisitions and selected reaction monitoring (SRM). The urine of six healthy individuals was analyzed using this method. A total of 355 acylcarnitines were detected; only 43 of them have been previously reported in the urine of healthy individuals. Detection of this large number of acylcarnitines illustrates the great diversity of the lipid metabolome as well as the usefulness of the method for profiling acylcarnitines. Furthermore, the MS/MS spectra of the 355 acylcarnitines will be uploaded to a public human metabolome database as a mass spectrometric resource for unknown metabolite identification.

  3. Characterization of N,N-dimethyl amino acids by electrospray ionization-tandem mass spectrometry.

    PubMed

    Naresh Chary, V; Sudarshana Reddy, B; Kumar, Ch Dinesh; Srinivas, R; Prabhakar, S

    2015-05-01

    Methylation is an essential metabolic process for a number of critical reactions in the body. Methyl groups are involved in the healthy function of the body life processes, by conducting methylation process involving specific enzymes. In these processes, various amino acids are methylated, and the occurrence of methylated amino acids in nature is diverse. Nowadays, mass-spectrometric-based identification of small molecules as biomarkers for diseases is a growing research. Although all dimethyl amino acids are metabolically important molecules, mass spectral data are available only for a few of them in the literature. In this study, we report synthesis and characterization of all dimethyl amino acids, by electrospray ionization-tandem mass spectrometry (MS/MS) experiments on protonated molecules. The MS/MS spectra of all the studied dimethyl amino acids showed preliminary loss of H2O + CO to form corresponding immonium ions. The other product ions in the spectra are highly characteristic of the methyl groups on the nitrogen and side chain of the amino acids. The amino acids, which are isomeric and isobaric with the studied dimethyl amino acids, gave distinctive MS/MS spectra. The study also included MS/MS analysis of immonium ions of dimethyl amino acids that provide information on side chain structure, and it is further tested to determine the N-terminal amino acid of the peptides.

  4. Analyzing Protease Specificity and Detecting in Vivo Proteolytic Events Using Tandem Mass Spectrometry

    SciTech Connect

    Gupta, Nitin; Hixson, Kim K.; Culley, David E.; Smith, Richard D.; Pevzner, Pavel A.

    2010-07-01

    While trypsin remains the most commonly used protease in mass spectrometry, other proteases may be employed for increasing peptide-coverage or generating overlapping peptides. Knowledge of the accurate specifcity rules of these proteases is helpful for database search tools to detect peptides, and becomes crucial when mass spectrometry is used to discover in vivo proteolytic cleavages. In this study, we use tandem mass spectrometry to analyze the specifcity rules of selected proteases and describe MS- Proteolysis, a software tool for identifying putative sites of in vivo proteolytic cleavage. Our analysis suggests that the specifcity rules for some commonly used proteases can be improved, e.g., we find that V8 protease cuts not only after Asp and Glu, as currently expected, but also shows a smaller propensity to cleave after Gly for the conditions tested in this study. Finally, we show that comparative analysis of multiple proteases can be used to detect putative in vivo proteolytic sites on a proteome-wide scale.

  5. A proteomics search algorithm specifically designed for high-resolution tandem mass spectra.

    PubMed

    Wenger, Craig D; Coon, Joshua J

    2013-03-01

    The acquisition of high-resolution tandem mass spectra (MS/MS) is becoming more prevalent in proteomics, but most researchers employ peptide identification algorithms that were designed prior to this development. Here, we demonstrate new software, Morpheus, designed specifically for high-mass accuracy data, based on a simple score that is little more than the number of matching products. For a diverse collection of data sets from a variety of organisms (E. coli, yeast, human) acquired on a variety of instruments (quadrupole-time-of-flight, ion trap-orbitrap, and quadrupole-orbitrap) in different laboratories, Morpheus gives more spectrum, peptide, and protein identifications at a 1% false discovery rate (FDR) than Mascot, Open Mass Spectrometry Search Algorithm (OMSSA), and Sequest. Additionally, Morpheus is 1.5 to 4.6 times faster, depending on the data set, than the next fastest algorithm, OMSSA. Morpheus was developed in C# .NET and is available free and open source under a permissive license.

  6. Simultaneous determination of estrogens and progestogens in honey using high performance liquid chromatography-tandem mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This work describes the development and validation of a method for the simultaneous determination of 13 estrogens and progestogens in honey by high performance liquid chromatography-tandem mass spectrometry. The target compounds were preconcentrated by solid phase extraction. Pretreatment variables ...

  7. Simplified analysis of glyphosate and aminomethylphosphonic acid in water, vegetation, and soil by liquid chromatography-tandem mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A simple, fast, efficient, and sensitive method was developed for analysis of glyphosate and its degradate, aminomethylphosphonic acid (AMPA), in water, vegetation, and soil. Aqueous extracts were passed through reverse phase and cation exchange columns and directly injected into a tandem mass spect...

  8. An Undergraduate Experiment for the Measurement of Perfluorinated Surfactants in Fish Liver by Liquid Chromatography-Tandem Mass Spectrometry

    ERIC Educational Resources Information Center

    Stock, Naomi L.; Martin, Jonathan W.; Ye, Yun; Mabury, Scott A.

    2007-01-01

    A laboratory experiment that provides students a hands-on introduction to the specific techniques of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and electrospray ionization is presented. The students can thus practice the analytical principles of sample extraction, detection, quantification, and quality control using a fresh fish…

  9. Structural analysis of a heteropolysaccharide from Saccharina japonica by electrospray mass spectrometry in tandem with collision-induced dissociation tandem mass spectrometry (ESI-CID-MS/MS).

    PubMed

    Jin, Weihua; Wang, Jing; Ren, Sumei; Song, Ni; Zhang, Quanbin

    2012-10-01

    A fucoidan extracted from Saccharina japonica was fractionated by anion exchange chromatography. The most complex fraction F0.5 was degraded by dilute sulphuric acid and then separated by use of an activated carbon column. Fraction Y1 was fractionated by anion exchange and gel filtration chromatography while Fraction Y2 was fractionated by gel filtration chromatography. The fractions were determined by ESI-MS and analyzed by ESI-CID-MS/MS. It was concluded that F0.5 had a backbone of alternating 4-linked GlcA and 2-linked Man with the first Man residue from the nonreducing end accidentally sulfated at C6. In addition, F0.5 had a 3-linked glucuronan, in accordance with a previous report by NMR. Some other structural characteristics included GlcA 1→3 Man 1→4 GlcA, Man 1→3 GlcA 1→4 GlcA, Fuc 1→4 GlcA and Fuc 1→3 Fuc. Finally, it was shown that fucose was sulfated at C2 or C4 while galactose was sulfated at C2, C4 or C6.

  10. Investigation of an enhanced resolution triple quadrupole mass spectrometer for high-throughput liquid chromatography/tandem mass spectrometry assays.

    PubMed

    Yang, Liyu; Amad, Ma'an; Winnik, Witold M; Schoen, Alan E; Schweingruber, Hans; Mylchreest, Iain; Rudewicz, Patrick J

    2002-01-01

    Triple quadrupole mass spectrometers, when operated in multiple reaction monitoring (MRM) mode, offer a unique combination of sensitivity, specificity, and dynamic range. Consequently, the triple quadrupole is the workhorse for high-throughput quantitation within the pharmaceutical industry. However, in the past, the unit mass resolution of quadrupole instruments has been a limitation when interference from matrix or metabolites cannot be eliminated. With recent advances in instrument design, triple quadrupole instruments now afford mass resolution of less than 0.1 Dalton (Da) full width at half maximum (FWHM). This paper describes the evaluation of an enhanced resolution triple quadrupole mass spectrometer for high-throughput bioanalysis with emphasis on comparison of selectivity, sensitivity, dynamic range, precision, accuracy, and stability under both unit mass (1 Da FWHM) and enhanced (mass resolution, the transmitted precursor ion from the first quadrupole contained not only protonated molecules from mometasone, but also PPG interference. At enhanced resolution only selected mometasone peaks were transmitted, and no interference from PPG was detected. Sensitivity of the instrument was demonstrated with 10 femtograms of descarboethoxyloratadine injected on-column, for which a signal-to-noise (S/N) ratio of 24 was obtained for MRM chromatograms at both unit and enhanced resolution. Absolute signals obtained at enhanced resolution were about one-third those obtained at unit mass resolution. However, S/N was maintained at enhanced resolution due to the proportional decrease in noise level. Finally, the stability of the instrument operating at enhanced resolution was demonstrated during an overnight 17 h period that was used to validate a liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay for

  11. Tandem Mass Spectrometric Characterization of Thiol Peptides Modified by the Chemoselective Cationic Sulfhydryl Reagent (4-Iodobutyl)Triphenylphosphonium—. Effects of a Cationic Thiol Derivatization on Peptide Fragmentation

    NASA Astrophysics Data System (ADS)

    Wang, Jing; Zhang, Jie; Arbogast, Brian; Maier, Claudia S.

    2011-10-01

    Fixed charge chemical modifications on peptides and proteins can impact fragmentation behaviors in tandem mass spectrometry (MS/MS). In this study, we employed a thiol-specific cationic alkylation reagent, (4-iodobutyl)triphenylphosphonium (IBTP), to selectively modify cysteine thiol groups in mitochondrial proteome samples. Tandem mass spectrometric characteristics of butyltriphenylphosphonium (BTP)-modified peptides were evaluated by comparison to their carbamidomethylated (CAM) analogues using a quadrupole time-of-flight (Q-TOF) instrument under low energy collision-induced dissociation (CID) conditions. Introduction of the fixed charge modification resulted in the observation of peptide and fragment (bn and yn) ions with higher charge states than those observed for CAM-modified analogues. The charged BTP moiety had a significant effect on the neighboring amide bond fragmentation products. A decrease in relative abundances of the product ions at the corresponding cleavage sites was observed compared with those from the CAM-modified derivatives. This effect was particularly noticeable when an Xxx-Pro bond was in the vicinity of a BTP group. We hypothesized that the presence of a phosphonium moiety will reduce the tendency for protonation of the proximal amide bonds in the peptide backbone. Indeed, calculations indicated that proton affinities of backbone amide bonds close to the modified cysteine residues were generally 20-50 kcal/mol lower for BTP-modified peptides than for the unmodified or CAM-modified analogues with the sequence motif -Ala-Cys-Alan-Ala2-, -Ala-Cys-Alan-Pro-Ala-, and -Ala-Pro-Alan-Cys-Ala-, n = 0-3.

  12. Occurrence of C-Terminal Residue Exclusion in Peptide Fragmentation by ESI and MALDI Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Dupré, Mathieu; Cantel, Sonia; Martinez, Jean; Enjalbal, Christine

    2012-02-01

    By screening a data set of 392 synthetic peptides MS/MS spectra, we found that a known C-terminal rearrangement was unexpectedly frequently occurring from monoprotonated molecular ions in both ESI and MALDI tandem mass spectrometry upon low and high energy collision activated dissociations with QqTOF and TOF/TOF mass analyzer configuration, respectively. Any residue localized at the C-terminal carboxylic acid end, even a basic one, was lost, provided that a basic amino acid such arginine and to a lesser extent histidine and lysine was present in the sequence leading to a fragment ion, usually depicted as (bn-1 + H2O) ion, corresponding to a shortened non-scrambled peptide chain. Far from being an epiphenomenon, such a residue exclusion from the peptide chain C-terminal extremity gave a fragment ion that was the base peak of the MS/MS spectrum in certain cases. Within the frame of the mobile proton model, the ionizing proton being sequestered onto the basic amino acid side chain, it is known that the charge directed fragmentation mechanism involved the C-terminal carboxylic acid function forming an anhydride intermediate structure. The same mechanism was also demonstrated from cationized peptides. To confirm such assessment, we have prepared some of the peptides that displayed such C-terminal residue exclusion as a C-terminal backbone amide. As expected in this peptide amide series, the production of truncated chains was completely suppressed. Besides, multiply charged molecular ions of all peptides recorded in ESI mass spectrometry did not undergo such fragmentation validating that any mobile ionizing proton will prevent such a competitive C-terminal backbone rearrangement. Among all well-known nondirect sequence fragment ions issued from non specific loss of neutral molecules (mainly H2O and NH3) and multiple backbone amide ruptures (b-type internal ions), the described C-terminal residue exclusion is highly identifiable giving raise to a single fragment ion in

  13. Identification of two meridianins from the crude extract of the tunicate Aplidium meridianum by tandem mass spectrometry.

    PubMed

    Seldes, A M; Brasco, M F Rodriguez; Franco, L Hernandez; Palermo, J A

    2007-05-20

    As part of the development of an analytical technique for the detection of meridianins and related compounds in biological fluids, a crude extract of the tunicate Aplidium meridianum was analysed using neutral loss tandem mass spectrometry. The 41 u neutral loss-EI(+) mass spectrum showed molecular ions corresponding to two previously undetected alkaloids. We report herein the isolation and structure elucidation of these alkaloids, meridianins F and G.

  14. Mass disasters: rapid molecular screening of human remains by means of short tandem repeats typing.

    PubMed

    Corach, D; Sala, A; Penacino, G; Sotelo, A

    1995-09-01

    Human remains identification represents a challenging situation and constitutes a difficult task associated with mass disasters. The only highly efficient means for individual and family group reconstruction is that based on DNA typing. On July 18, 1994 an explosion destroyed the A.M.I.A. (Argentine Israeli Association). Over 100 people died; however, the exact number of victims is still being investigated. Our Service received over 70 remains to be characterized by DNA typing in order to determine the number of victims and to try to reconstruct the family groups to which they belonged. DNA was extracted by a cetyltrimethylammonium bromide (CTAB) based protocol, a rapid molecular screening of all samples was carried out by multiplex STR amplifications including HUMTH01, HUMFABP, HUMHPRTB, HUMRENA4, HUMVWA, HUMFES/FPS and Y27H39LR. Samples with identical genotypes were HaeIII-digested. Southern blotted and probed with YNH-24 (D2S44). PH-30 (D4S139). LH-1 (D5S110) and MS-1 (D1S7) for variable number of tandem repeats (VNTR) evaluation. The minisatellite variant repeat (MVR) approach was used in those cases in which band or profile shift were detected in Southern blot assays. Kinship between victims and putative relatives was initially evaluated by comparison of short tandem repeat (STR) profiles and then confirmed by VNTR with the above probes. The high identification efficiency attained in this case is, in part, supported by a previous experience, the DNA-based molecular characterization of human remains caused by the explosion of the Israeli Embassy in Buenos Aires, March 1992.

  15. Simultaneous determination of eight illegal dyes in chili products by liquid chromatography-tandem mass spectrometry.

    PubMed

    Li, Juan; Ding, Xiao-Ming; Liu, Dan-Dan; Guo, Fei; Chen, Yu; Zhang, Yan-Bing; Liu, Hong-Min

    2013-12-30

    A sensitive and accurate method based on the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the simultaneous determination of eight illegal synthetic dyes (Sudan (I-IV), Para Red, Rhodamine B, Chrysoidin and Auramine O) in chili products. A simple sample treatment procedure entailing the use of an extraction step with acetonitrile/H2O (9/1) without further cleanup was developed. HPLC was performed on a C18 column using a multistep gradient elution with 5mM ammonium acetate (pH 3.0 with formic acid) and methanol as the mobile phase. Mass spectral acquisition was done in multiple reaction monitoring (MRM) mode using positive electrospray ionization (ESI). Linear calibrations were obtained with correlation coefficients R(2)>0.99. Limit of detection (LOD) and limit of quantification (LOQ) for the studied dyes were in the ranges of 0.05-0.6μgkg(-1) and 0.3-3.0μgkg(-1) depending on matrices, respectively. The recoveries of the eight synthetic dyes in five matrices ranged from 70.5% to 119.2%. The intra- and inter-day precisions (RSDs) were between 2.3-15.8% and 5.7-15.6%, respectively. The applicability of the method to the determination of eight banned dyes in chili products was demonstrated.

  16. Quantitative analysis of phenibut in rat brain tissue extracts by liquid chromatography-tandem mass spectrometry.

    PubMed

    Grinberga, Solveiga; Zvejniece, Liga; Liepinsh, Edgars; Dambrova, Maija; Pugovics, Osvalds

    2008-12-01

    Phenibut (3-phenyl-4-aminobutyric acid) is a gamma-aminobutyric acid mimetic drug, which is used clinically as a mood elevator and tranquilizer. In the present work, a rapid, selective and sensitive liquid chromatography-tandem mass spectrometry method for quantification of phenibut in biological matrices has been developed. The method is based on protein precipitation with acidic acetonitrile followed by isocratic chromatographic separation using acetonitrile-formic acid (0.1% in water; 8:92, v/v) mobile phase on a reversed-phase column. Detection of the analyte was performed by electrospray ionization mass spectrometry in multiple reaction monitoring mode with the precursor-to-product ion transition m/z 180.3 --> m/z 117.2. The calibration curve was linear over the concentration range 50-2000 ng/mL. The lower limit of quantification for phenibut in rat brain extracts was 50 ng/mL. Acceptable precision and accuracy were obtained over the whole concentration range. The validated method was successfully applied in a pharmacological study to analyze phenibut concentration in rat brain tissue extract samples.

  17. Chiral purity assay for Flindokalner using tandem mass spectrometry: method development, validation, and benchmarking.

    PubMed

    Young, Brandy L; Cooks, R G; Madden, Michelle C; Bair, Michael; Jia, Jingpin; Aubry, Anne-Françoise; Miller, Scott A

    2007-04-11

    The present work demonstrates the application and validation of a mass spectrometry method for quantitative chiral purity determination. The particular compound analyzed is Flindokalner, a Bristol-Myers Squibb drug candidate for post-stroke neuroprotection. Chiral quantification of Flindokalner was achieved using tandem mass spectrometry (MS/MS) and the kinetic method, a gas phase method used for thermochemical and chiral determinations. The MS/MS method was validated and benchmarked against two separate chromatographic techniques, chiral high performance liquid chromatography with ultra-violet detection (LC/UV) and achiral high performance liquid chromatography with circular dichroism detection (LC/CD). The chiral purity determination of Flindokalner using MS/MS proved to be rapid (3 min run time for each sample) and to have accuracy and precision comparable to the chiral LC/UV and achiral LC/CD methods. This method represents an alternative to commonly used chromatographic techniques as a means of chiral purity determination and is particularly useful in rapid screening experiments.

  18. Isomerization of 4-vinylcyclohexene radical cation. A tandem mass spectrometry study

    SciTech Connect

    Vollmer, D.; Rempel, D.L.; Gross, M. L. ); Williams, F. )

    1995-02-08

    Investigation by matrix-isolation ESR has shown that 4-vinylcyclohexene, 1, surprisingly undergoes isomerization to the bicyclo[3.2.1]oct-2-ene ion, 3. Here we demonstrate the occurrence of this isomerization in the gas phase by use of tandem (MS/MS) sector and Fourier transform (FT) mass spectrometries. The radical cations of 4-vinylcyclohexene (IE = 8.93 eV) or bicyclo[3.2.1]oct-2-ene (approximately 14 kcal/mol more stable than that of 4-vinylcyclohexene) were formed, in separate trials, in a chemical ionization (CI) source by electron ionization (EI). The radical cations were then studied by obtaining their collisionally activated decomposition (CAD) spectra. The CAD spectra are similar, indicating that the isomerization has occurred. Both the sector and the FT mass spectrometer results reflect those obtained in the matrix-isolation ESR investigation. That is isomerizes to 3 at high internal energy, but is stable at low internal energy. Two mechanisms explain this rearrangement. The second mechanism is questionable because the most stable olefin radical cation formed from 5 is that of bicyclo[2.2.2]-2-octene, which gives different ESR and CAD spectra than those of 1 or 3. The CAD spectrum of bicyclo[2.2.2]-2-octene radical cation indicates that the retro-Diels-Alder loss of ethylene is more facile than that from 1 or 3. 18 refs., 3 figs.

  19. Tandem mass spectrometric analysis of a complex triterpene saponin mixture of Chenopodium quinoa.

    PubMed

    Madl, Tobias; Sterk, Heinz; Mittelbach, Martin; Rechberger, Gerald N

    2006-06-01

    A nano-HPLC electrospray ionization multi-stage tandem mass spectrometry (nLC-ESI-MS/MS) approach was applied to a complex crude triterpene saponin extract of Chenopodium quinoa seed coats. In ESI-MS/MS spectra of triterpene saponins, characteristic fragmentation reactions are observed and allow the determination of aglycones, saccharide sequences, compositions, and branching. Fragmentation of aglycones provided further structural information. The chemical complexity of the mixture was resolved by a complete profiling. Eighty-seven triterpene saponins comprising 19 reported and 68 novel components were identified and studied by MS. In addition to four reported, five novel triterpene aglycones were detected and characterized according to their fragmentation reactions in ESI-MS/MS and electron ionization mass spectrometry (EI-MS). As a novelty fragmentation pathways were proposed and analyzed based upon quantum chemical calculations using a hybrid Hartree-Fock density functional method. Accuracy of the assignment procedure was proven by isolation and structure determination of a novel compound. As the relative distribution and composition of saponins varies between different cultivars and soils, the presented strategy allows a rapid and complete analysis of Chenopodium quinoa saponin distribution and composition, and is particularly suitable for quality control and screening of extracts designated for pharmaceutical, agricultural, and industrial applications.

  20. Redefining the Breast Cancer Exosome Proteome by Tandem Mass Tag Quantitative Proteomics and Multivariate Cluster Analysis.

    PubMed

    Clark, David J; Fondrie, William E; Liao, Zhongping; Hanson, Phyllis I; Fulton, Amy; Mao, Li; Yang, Austin J

    2015-10-20

    Exosomes are microvesicles of endocytic origin constitutively released by multiple cell types into the extracellular environment. With evidence that exosomes can be detected in the blood of patients with various malignancies, the development of a platform that uses exosomes as a diagnostic tool has been proposed. However, it has been difficult to truly define the exosome proteome due to the challenge of discerning contaminant proteins that may be identified via mass spectrometry using various exosome enrichment strategies. To better define the exosome proteome in breast cancer, we incorporated a combination of Tandem-Mass-Tag (TMT) quantitative proteomics approach and Support Vector Machine (SVM) cluster analysis of three conditioned media derived fractions corresponding to a 10 000g cellular debris pellet, a 100 000g crude exosome pellet, and an Optiprep enriched exosome pellet. The quantitative analysis identified 2 179 proteins in all three fractions, with known exosomal cargo proteins displaying at least a 2-fold enrichment in the exosome fraction based on the TMT protein ratios. Employing SVM cluster analysis allowed for the classification 251 proteins as "true" exosomal cargo proteins. This study provides a robust and vigorous framework for the future development of using exosomes as a potential multiprotein marker phenotyping tool that could be useful in breast cancer diagnosis and monitoring disease progression.

  1. Support Vector Machines for Improved Peptide Identification from Tandem Mass Spectrometry Database Search

    SciTech Connect

    Webb-Robertson, Bobbie-Jo M.

    2009-05-06

    Accurate identification of peptides is a current challenge in mass spectrometry (MS) based proteomics. The standard approach uses a search routine to compare tandem mass spectra to a database of peptides associated with the target organism. These database search routines yield multiple metrics associated with the quality of the mapping of the experimental spectrum to the theoretical spectrum of a peptide. The structure of these results make separating correct from false identifications difficult and has created a false identification problem. Statistical confidence scores are an approach to battle this false positive problem that has led to significant improvements in peptide identification. We have shown that machine learning, specifically support vector machine (SVM), is an effective approach to separating true peptide identifications from false ones. The SVM-based peptide statistical scoring method transforms a peptide into a vector representation based on database search metrics to train and validate the SVM. In practice, following the database search routine, a peptides is denoted in its vector representation and the SVM generates a single statistical score that is then used to classify presence or absence in the sample

  2. De Novo Sequencing of Peptides from Top-Down Tandem Mass Spectra

    SciTech Connect

    Vyatkina, Kira; Wu, Si; Dekker, Lennard J. M.; VanDuijn, Martijn M.; Liu, Xiaowen; Tolić, Nikola; Dvorkin, Mikhail; Alexandrova, Sonya; Luider, Theo M.; Paša-Tolić, Ljiljana; Pevzner, Pavel A.

    2015-11-06

    De novo sequencing of proteins and peptides is one of the most important problems in mass spectrometry-driven proteomics. A variety of methods have been developed to accomplish this task from a set of bottom-up tandem (MS/MS) mass spectra. However, a more recently emerged top-down technology, now gaining more and more popularity, opens new perspectives for protein analysis and characterization, implying a need in efficient algorithms for processing this kind of MS/MS data. Here we describe a method that allows to retrieve from a set of top-down MS/MS spectra long and accurate sequence fragments of the proteins contained in a sample. To this end, we outline a strategy for generating high-quality sequence tags from top-down spectra, and introduce the concept of a T-Bruijn graph by adapting to the case of tags the notion of an A-Bruijn graph widely used in genomics. The output of the proposed approach represents the set of amino acid strings spelled out by optimal paths in the connected components of a T-Bruijn graph. We illustrate its performance on top-down datasets acquired from carbonic anhydrase 2 (CAH2) and the Fab region of alemtuzumab.

  3. Identification of metabolites of lobeline in the rat urine by liquid chromatography-tandem mass spectrometry

    NASA Astrophysics Data System (ADS)

    Song, Wei; Peng, Zhihong; Ge, Baoying; Han, Fengmei; Chen, Yong

    2008-01-01

    This is a report about the analysis of lobeline and its metabolites in rat urine by using high-performance liquid chromatography-electrospray ionization ion trap tandem mass spectrometric method (LC/MSn). The urine of healthy rat were sampled from 0 to 24 h after administered a single dose of lobeline (3 mg/kg) by oral gavage, then centrifuged at 10,000 rpm for 10 min to get the supernatants. The supernatants were purified by solid-phase extraction (SPE) with a C18 cartridge. After the above purified process, the purified urine were injected into a reversed-phase C18 column with mobile phase of methanol/water (70:30, v/v, adjusted to pH 3.5 with formic acid) and detected by an on-line MSn system. The identification and structural elucidation of the metabolites were performed by comparing their changes in molecular mass ([Delta]M), full-scan MSn spectra with those of the parent drug. Ten metabolites of lobeline were found in rat urine. All the metabolites were reported for the first time.

  4. Predicting molecular formulas of fragment ions with isotope patterns in tandem mass spectra.

    PubMed

    Zhang, Jingfen; Gao, Wen; Cai, Jinjin; He, Simin; Zeng, Rong; Chen, Runsheng

    2005-01-01

    A number of different approaches have been proposed to predict elemental component formulas (or molecular formulas) of molecular ions in low and medium resolution mass spectra. Most of them rely on isotope patterns, enumerate all possible formulas for an ion, and exclude certain formulas violating chemical constraints. However, these methods cannot be well generalized to the component prediction of fragment ions in tandem mass spectra. In this paper, a new method, FFP (Fragment ion Formula Prediction), is presented to predict elemental component formulas of fragment ions. In the FFP method, the prediction of the best formulas is converted into the minimization of the distance between theoretical and observed isotope patterns. And, then, a novel local search model is proposed to generate a set of candidate formulas efficiently. After the search, FFP applies a new multiconstraint filtering to exclude as many invalid and improbable formulas as possible. FFP is experimentally compared with the previous enumeration methods, and shown to outperform them significantly. The results of this paper can help to improve the reliability of de novo in the identification of peptide sequences.

  5. Dynamic Bayesian Network for Accurate Detection of Peptides from Tandem Mass Spectra.

    PubMed

    Halloran, John T; Bilmes, Jeff A; Noble, William S

    2016-08-05

    A central problem in mass spectrometry analysis involves identifying, for each observed tandem mass spectrum, the corresponding generating peptide. We present a dynamic Bayesian network (DBN) toolkit that addresses this problem by using a machine learning approach. At the heart of this toolkit is a DBN for Rapid Identification (DRIP), which can be trained from collections of high-confidence peptide-spectrum matches (PSMs). DRIP's score function considers fragment ion matches using Gaussians rather than fixed fragment-ion tolerances and also finds the optimal alignment between the theoretical and observed spectrum by considering all possible alignments, up to a threshold that is controlled using a beam-pruning algorithm. This function not only yields state-of-the art database search accuracy but also can be used to generate features that significantly boost the performance of the Percolator postprocessor. The DRIP software is built upon a general purpose DBN toolkit (GMTK), thereby allowing a wide variety of options for user-specific inference tasks as well as facilitating easy modifications to the DRIP model in future work. DRIP is implemented in Python and C++ and is available under Apache license at http://melodi-lab.github.io/dripToolkit .

  6. Liquid Chromatography-Tandem Mass Spectrometry for Analysis of Intestinal Permeability of Loperamide in Physiological Buffer

    PubMed Central

    Rubelt, Miriam S.; Amasheh, Salah; Grobosch, Thomas; Stein, Christoph

    2012-01-01

    Analysis of in vitro samples with high salt concentrations represents a major challenge for fast and specific quantification with liquid chromatography-tandem mass spectrometry (LC-MS/MS). To investigate the intestinal permeability of opioids in vitro employing the Ussing chamber technique, we developed and validated a fast, sensitive and selective method based on LC–MS/MS for the determination of loperamide in HEPES-buffered Ringer's solution. Chromatographic separation was achieved with an Atlantis dC18 column, 2.1 mm×20 mm, 3 µm particle size and a gradient consisting of methanol/0.1% formic acid and ammonium acetate. The flow rate was 0.7 ml/min, and the total run time was 3 min. For quantification, two mass transitions for loperamide and a deuterated internal standard (methadone-d3) were used. The lower limit of loperamide quantification was 0.2 ng/ml. This new LC-MS/MS method can be used for the detection of loperamide in any experimental setup using HEPES-buffered Ringer's solution as a matrix compound. PMID:23144895

  7. Increased Throughput of Proteomics Analysis by Multiplexing High-resolution Tandem Mass Spectra

    PubMed Central

    Ledvina, A. R.; Savitski, M. M.; Zubarev, A. R.; Good, D. M.; Coon, J. J.; Zubarev, R. A.

    2014-01-01

    High-resolution and accuracy Fourier-transform mass spectrometry (FTMS) is becoming increasingly attractive due to its specificity. However, the speed of tandem FTMS analysis severely limits the competitive advantage of this approach relative to faster low-resolution quadrupole ion trap MS/MS instruments. Here we demonstrate an entirely FTMS-based analysis method with a 2.5–3.0 fold greater throughput than a conventional FT MS/MS approach. The method consists of accumulating together the MS/MS fragments ions from multiple precursors, with subsequent high-resolution analysis of the mixture. Following acquisition, the multiplexed spectrum is deconvoluted into individual MS/MS datasets which are separately submitted for peptide identification to a search engine. The method is tested both in silico using a database of MS/MS spectra as well as in situ using a modified LTQ-Orbitrap mass spectrometer. The performance of the method in the experiment was consistent with theoretical expectations. PMID:21913643

  8. Increased throughput of proteomics analysis by multiplexing high-resolution tandem mass spectra.

    PubMed

    Ledvina, A R; Savitski, M M; Zubarev, A R; Good, D M; Coon, J J; Zubarev, R A

    2011-10-15

    High-resolution and high-accuracy Fourier transform mass spectrometry (FTMS) is becoming increasingly attractive due to its specificity. However, the speed of tandem FTMS analysis severely limits the competitive advantage of this approach relative to faster low-resolution quadrupole ion trap MS/MS instruments. Here we demonstrate an entirely FTMS-based analysis method with a 2.5-3.0-fold greater throughput than a conventional FT MS/MS approach. The method consists of accumulating together the MS/MS fragments ions from multiple precursors, with subsequent high-resolution analysis of the mixture. Following acquisition, the multiplexed spectrum is deconvoluted into individual MS/MS spectra which are then combined into a single concatenated file and submitted for peptide identification to a search engine. The method is tested both in silico using a database of MS/MS spectra as well as in situ using a modified LTQ Orbitrap mass spectrometer. The performance of the method in the experiment was consistent with theoretical expectations.

  9. Analysis of 2-methylthio-derivatives of isoprenoid cytokinins by liquid chromatography-tandem mass spectrometry.

    PubMed

    Tarkowski, Petr; Václavíková, Kateřina; Novák, Ondřej; Pertry, Ine; Hanuš, Jan; Whenham, Robert; Vereecke, Danny; Šebela, Marek; Strnad, Miroslav

    2010-11-08

    A sensitive and reliable high-performance liquid chromatographic method with tandem mass spectrometric detection has been developed and used for the determination of 2-methylthio-cytokinin derivatives produced by the phytopathogenic actinomycete Rhodococcus fascians. The cultivation medium containing secreted cytokinins was concentrated and subjected to a solid-phase extraction (C18 and ion-exchange). The purified samples were further separated and analyzed by HPLC-ESI-MS/MS. This allowed to achieve chromatographic resolution of six highly hydrophobic cytokinin species including 2-methylthio-isopentenyladenine, 2-methylthio-isopentenyladenosine, 2-methylthio-trans-zeatin and 2-methylthio-trans-zeatin riboside and their cis-isomers when a reversed-phase chromatographic column (C4) and a mobile phase consisting of acetonitrile and 20 mM ammonium formate, pH 5, were used. Quantification was performed by a standard isotope dilution method using a multiple-reaction monitoring (MRM) mode. In the MRM mode, limits of detection reached 20-30 fmol and linear ranges spanned four orders of magnitude. Recovery values were between 35% and 65% and the analytical accuracy between 95% and 149%. The proposed bioanalytical method, which takes advantage of effective chromatographic separation of six 2-methyltio-derivatives (including isomers of zeatin-type cytokinins) and sensitive mass spectrometric detection, may become useful for plant biologists studying the significance of these substances in plant-microbe interactions.

  10. Characterization of the limonene oxidation products with liquid chromatography coupled to the tandem mass spectrometry

    NASA Astrophysics Data System (ADS)

    Witkowski, Bartłomiej; Gierczak, Tomasz

    2017-04-01

    Composition of the secondary organic aerosol (SOA) generated during ozonolysis of limonene was investigated with liquid chromatography coupled to the negative electrospray ionization (ESI), quadrupole tandem mass spectrometry (MS/MS) as well as high resolution Time-of-Flight mass spectrometry. Aerosol was generated in the flow-tube reactor. HR-MS/MS analysis allowed for proposing structures for the several up-to-date unknown limonene oxidation products. In addition to the low MW limonene oxidation products, significant quantities of oligomers characterized by elemental compositions: C19H30O5, C18H28O6, C19H28O7, C19H30O7 and C20H34O9 were detected in the SOA samples. It was concluded that these compounds are most likely esters, aldol reaction products and/or hemiacetals. In addition to detailed study of the limonene oxidation products, the reaction time as well as initial ozone concentration impact on the limonene SOA composition was investigated. The relative intensities of the two esters of the limonic acid and 7-hydroxy limononic acid increased as a result of lowering the initial ozone concentration and shortening the reaction time, indicating that esterification may be an important oligomerization pathway during limonene SOA formation.

  11. [Determination of glyphosate and aminomethylphosphonic acid in rice using liquid chromatography-tandem mass spectrometry].

    PubMed

    Cao, Zhaoyun; Mou, Renxiang; Chen, Mingxue

    2010-08-01

    A method was developed for the determination of glyphosate (GLY) and aminomethylphosphonic acid (AMPA) in rice using liquid chromatography tandem mass spectrometry (LC-MS/MS). The sample was extracted with water followed by a simple cleanup with a C18 solid phase extraction (SPE) cartridge, and then GLY and AMPA were derivatized using 9-fluorenylmethoxycarbonyl (FMOC-Cl) in borate buffer. The derivatives of GLY and AMPA were separated on a C18 column with gradient elution with the mobile phase of acetonitrile and 5 mmol/L ammonium acetate (pH 9), and finally detected with negative ion electrospray ionization-mass spectrometry (ESI-MS) in multiple reaction monitoring (MRM) mode. The results showed that the linearities of GLY and AMPA were in the concentration range of 0.000 50 to 1.0 mg/L with the correlation coefficients of 0.999 7 and 0.999 9, respectively. The mean spiked recoveries of GLY and AMPA at 3 spiked levels ranged from 72.5% to 113.6% with the relative standard deviations (RSD, n = 5) of 3.8% - 16.2%. The limits of detection were 2.0 and 3.0 microg/kg for GLY and AMPA, respectively. This method is rapid, sensitive, and suitable for simultaneous determination of GLY and AMPA in rice.

  12. Simultaneous quantitative analysis of isobars by tandem mass spectrometry from unresolved chromatographic peaks.

    PubMed

    Kushnir, Mark M; Rockwood, Alan L; Nelson, Gordon J

    2004-05-01

    A method was developed for the simultaneous quantitation of isobars from unresolved chromatographic peaks. The method is based on differences in branching ratios of ion abundances in their tandem mass spectra and an assumption that the product ion mass spectra of a mixture can be considered as a linear combination of the spectra of individual constituents. We present analytical equations and a matrix-based approach for deconvoluting the concentration of individual components from the total peak intensity for two and three isobars and also a matrix-based generalization to any number of compounds. The feasibility of the simultaneous analysis of mixtures containing two compounds was assessed. The approach was evaluated for the analysis of structural isomers of methylmalonic and succinic acids in human plasma and urine samples for a group of 270 samples. The linear regression equation, standard error and correlation coefficient for the agreement with a traditional method utilizing chromatographic separation of the isomers were y = 0.999x - 0.005, 0.024 micro mol l(-1), and 0.985, respectively. The utility of a spectral contrast angle as a predictor of analysis feasibility was evaluated.

  13. Multidimensional Liquid Chromatography Coupled with Tandem Mass Spectrometry for Identification of Bioactive Fatty Acyl Derivatives

    PubMed Central

    Divito, Erin B.; Kroniser, Kristin M.; Cascio, Michael

    2016-01-01

    Recognition of the contributions of lipids to cellular physiology, both as structural components of the membrane and as modulatory ligands for membrane proteins, has increased in recent years with the development of the biophysical and biochemical tools to examine these effects. Their modulatory roles in ion channels and transporters function have been extensively characterized, with the molecular mechanisms of these activities being the subject of intense scrutiny. The physiological significance of lipids in biochemistry is expanding as numerous fatty acyls are discovered to possess signaling properties. These bioactive lipids are often found in quantities of pmol/g of tissue and are co-extracted with numerous lipophilic molecules, making their detection and identification challenging. Common analytical methodologies involve chromatographic separation and mass spectrometric techniques; however, a single chromatographic step is typically ineffective due to the complexity of the biological samples. It is, therefore, essential to develop approaches that incorporate multiple dimensions of separation. Described in this manuscript are normal phase and reversed phase separation strategies for lipids that include detection of the bioactive primary fatty acid amides and N-acyl glycines via tandem mass spectrometry. Concerted utilization of these approaches are then used to separate and sensitively identify primary fatty acid amides extracted from homogenized tissue, using mouse brains as a test case. PMID:28018237

  14. Identification of palmatine and its metabolites in rat urine by liquid chromatography/tandem mass spectrometry.

    PubMed

    Zhu, Mingming; Han, Fengmei; Chen, Huaixia; Peng, Zhihong; Chen, Yong

    2007-01-01

    Palmatine is an isoquinoline alkaloid that has been widely used in China for the treatment of various inflammatory diseases such as gynecological inflammation, bacillary dysentery, enteritis, respiratory tract infection, urinary infection, etc. In the study reported in this paper, a simple and rapid high-performance liquid chromatography/electrospray ionization (ESI) tandem mass spectrometric method (MS/MS) was developed for elucidation of the structures of metabolites of palmatine in rat urine after administration of a single dose (20 mg/kg). The rat urine samples were collected and purified through C18 solid-phase extraction cartridges, and then injected onto a reversed-phase C18 column with 60:40 (v/v) methanol/0.01% triethylamine solution (2 mM, adjusted to pH 3.5 with formic acid) as mobile phase and detected by on-line MS/MS. Identification of the metabolites and elucidation of their structures were performed by comparing changes in molecular masses (DeltaM), retention times and spectral patterns of product ions with those of the parent drug. As a result, six phase I metabolites, the parent drug palmatine and two phase II metabolites were identified in rat urine for the first time.

  15. The inclusion of ADA-SCID in expanded newborn screening by tandem mass spectrometry.

    PubMed

    la Marca, Giancarlo; Giocaliere, Elisa; Malvagia, Sabrina; Funghini, Silvia; Ombrone, Daniela; Della Bona, Maria Luisa; Canessa, Clementina; Lippi, Francesca; Romano, Francesca; Guerrini, Renzo; Resti, Massimo; Azzari, Chiara

    2014-01-01

    Severe combined immunodeficiency due to adenosine-deaminase defect (ADA-SCID) is usually deadly in childhood because of severe recurrent infections. When clinical diagnosis is done, permanent damages due to infections or metabolite accumulation are often present. Gene therapy, bone marrow transplantation or enzyme replacement therapy may be effective if started early. The aim of this study was to set-up a robust method suitable for screening with a minimized preparation process and with inexpensive running costs, for diagnosing ADA-SCID by tandem mass spectrometry. ADA-SCID satisfies all the criteria for inclusion in a newborn screening program. We describe a protocol revised to incorporate adenosine and 2-deoxyadenosine testing into an expanded newborn screening program. We assessed the effectiveness of this approach testing dried blood spots from 4 genetically confirmed early-onset and 5 delayed-onset ADA-SCID patients. Reference values were established on 50,000 healthy newborns (deoxyadenosine <0.09μmol/L, adenosine <1.61μmol/L). We also developed a second tier test to distinguish true positives from false positives and improve the positive predictive value of an initial abnormal result. In the first 18 months, the pilot project has identified a newborn with a genetically confirmed defect in adenosine deaminase (ADA) gene. The results show that the method having great simplicity, low cost and low process preparations can be fully applicable to a mass screening program.

  16. Determination of thalidomide concentration in human plasma by liquid chromatography-tandem mass spectrometry.

    PubMed

    Bai, Nan; Cui, Xiang-Yong; Wang, Jin; Sun, Chun-Guang; Mei, He-Kun; Liang, Bei-Bei; Cai, Yun; Song, Xiu-Jie; Gu, Jing-Kai; Wang, Rui

    2013-02-01

    A rapid, sensitive and specific analytical method based on high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the determination of thalidomide concentration in human plasma. The analyte and internal standard were extracted by liquid-liquid extraction with ether-dichloromethane (3:2, v/v) and separated on a TC-C(18) column using methanol-10 mM ammonium acetate-formic acid (60:40:0.04, v/v/v) as the mobile phase at a flow rate of 0.9 ml/min. The detection was performed using an API 4000 triple quadrupole mass spectrometer in the positive electrospray ionization (ESI) mode and completed within 3.0 min. The multiple reaction monitoring (MRM) transitions were m/z 259.1→84.0 for the analyte and m/z 195.9→138.9 for temozolomide. The calibration curve exhibited a linear dynamic range of 2-1500 ng/ml (r>0.9991). The intra-and inter-day precisions (as relative standard deviation; RSD) were 6.8-13.5% and 4.3-5.0% respectively and the accuracy (as relative error; RE) was 2.0-3.5%. The recoveries and matrix effects were satisfactory in all the biological matrices examined. This method was successfully used in a pharmacokinetic study of thalidomide in healthy male volunteers receiving an oral administration of a 200-mg dose.

  17. Poiseuille flow-induced vibrations of two tandem circular cylinders with different mass ratios

    NASA Astrophysics Data System (ADS)

    Jiang, Ren-Jie; Lin, Jian-Zhong

    2016-06-01

    Flow-induced vibrations of two tandem circular cylinders with different mass ratios confined between two parallel walls are numerically studied via a lattice Boltzmann method. With fixed Reynolds number Re = 100 and blockage ratio β = 1/4, the effects of mass ratio m* = [0.0625, 16] and streamwise separation between two cylinders S/D = [1.125, 10] on the cylinder motions and vortex wake modes are investigated. A variety of distinct cylinder motion regimes involving the symmetric periodic vibration, biased quasi-periodic vibration, beating vibration, and steady regimes, with the corresponding wake structures, e.g., two rows of alternately rotating vortices, a single row of same-sign vortices, and steady wake, are observed. For each current case, the cylinder motion type is exclusive and in the binary oscillation regime, both cylinders always vibrate at a common primary frequency. The lighter cylinder usually oscillates at a larger amplitude than the heavier one, while the heavier cylinder undergoes larger lift force than the lighter one. The lift force and cylinder displacement always behave as an out-of-phase state. In the gap-interference region, large-amplitude oscillations could be produced extensively and in the wake-interference region, the cylinder motions and fluid flows are mainly dependent on the upstream cylinder. When the separation is large enough, both cylinders behave as two isolated ones. The mechanisms for the excitations of cylinder vibrations have also been analysed.

  18. Detection and characterization of N-alpha-chloramines by electrospray tandem mass spectrometry.

    PubMed

    Raftery, Mark J

    2007-07-15

    Hypochlorous acid (HOCl) is a major product of activated neutrophils and may be important in antimicrobial activities of cells by oxidation or chlorination of susceptible amino acids. Three major peaks separated using C18 reverse phase-high-performance liquid chromatography RP-HPLC after incubation of leucine enkephalin (LeuEnk) with HOCl. Electrospray mass spectrometry showed masses of m/z 556.2, 590.2, and 624.4 corresponding to unmodified LeuEnk and peptides altered by addition of one or two chlorines (Cl). Formation of stable N-alpha-chloramines was indicated because the chlorinated peptides were readily reduced with the physiological reductants glutathione and ascorbic acid to LeuEnk (m/z 556.2) within 10 min. Sequence-specific ions observed in product ion spectra of single-charged monochlorinated and dichlorinated peptides were consistent with modification of the N-terminal amine. There was no evidence for chlorination of the Tyr aromatic ring in any spectra. Similar RP-HPLC profiles were obtained after oxidation of des-Tyr1-LeuEnk (GGFL) with the masses of the major products being m/z 393.3, 427.2, and 461.1. These were identified as unmodified GGFL, N-alpha-Cl-GGFL, and N-alpha-Cl2-GGFL based on comparison of tandem mass spectra. Oxidation of Met and formation of disulfide dimers was observed after incubation of either N-alpha-Cl-LeuEnk or N-alpha-Cl2-LeuEnk with a protein, indicating that both peptide N-alpha-chloramines were able to readily modify sulfur-containing amino acids within proteins. These data indicate initial formation of stable N-alpha-chorinated peptides after incubation with HOCl and suggest that N-alpha-chlorinated peptides may exist for some hours in the absence of physiological reducing agents or sulfur-containing amino acids.

  19. Tandem mass spectrometry of poly(ethylene imine)s by electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI).

    PubMed

    Altuntaş, Esra; Knop, Katrin; Tauhardt, Lutz; Kempe, Kristian; Crecelius, Anna C; Jäger, Michael; Hager, Martin D; Schubert, Ulrich S

    2012-01-01

    In this contribution, linear poly(ethylene imine) (PEI) polymers, which are of importance in gene delivery, are investigated in detail by using electrospray ionization-quadrupole-time of flight (ESI-Q-TOF) and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS). The analyzed PEIs with different end groups were synthesized using the polymerization of substituted 2-oxazoline via a living cationic ring-opening polymerization (CROP) and a subsequent hydrolysis under acidic conditions. The main goal of this study was to identify linear PEI polymers in a detailed way to gain information about their fragmentation pathways. For this purpose, a detailed characterization of three different linear PEIs was performed by using ESI-Q-TOF and MALDI-TOF MS in combination with collision-induced dissociation (CID) experiments. In ESI-MS as well as MALDI-MS analysis, the obtained spectra of PEIs resulted in fitting mass distributions for the investigated PEIs. In the tandem MS analysis, a 1,2-hydride shift with a charge-remote rearrangement via a four-membered cyclic transition state, as well as charge-induced fragmentation reactions, was proposed as the main fragmentation mechanisms according to the obtained fragmentation products from the protonated parent peaks. In addition, heterolytic and homolytic cleavages were proposed as alternative fragmentation pathways. Moreover, a 1,4-hydrogen elimination was proposed to explain different fragmentation products obtained from the sodiated parent peaks.

  20. Structural analysis of diacyl peroxides by electrospray tandem mass spectrometry with ammonium acetate: bond homolysis of peroxide-ammonium and peroxide-proton adducts.

    PubMed

    Yin, H; Hachey, D L; Porter, N A

    2000-01-01

    Organic peroxides have significant implications in organic chemistry and biological processes. The weak O-O bond makes them extremely difficult to characterize by conventional analytical methods. Diacyl peroxides are one of the major radical sources in polymerization and organic synthesis. It is well known that diacyl peroxides are thermal labile and thus are not amenable to study by gas chromatography/mass spectrometry (GC/MS). Electrospray tandem mass spectrometry (ESI-MS/MS) has been applied to the structural analysis of diacyl peroxides by formation of ammonium adducts. Collision induced dissociation (CID) studies of the ammonium adducts of the peroxide [M + NH(4)](+) give collision energy dependent fragments. For most diacyl peroxides, homolysis of the peroxy bond predominates the fragmentation pathways of the peroxide-ammonium adducts. Deuterated substrates have been employed to provide evidence for typical fragmentation pathways. The CID studies were also used to locate the O-18 in some O-18 specifically labeled diacyl peroxides. For branched alkyl or alkoxy substrates, McLafferty rearrangement and decarboxylation become a major pathway. By comparison with some anhydride analogues, ESI-MS/MS can also be used to study this class of compounds.

  1. Identification of membrane proteins by tandem mass spectrometry of protein ions.

    PubMed

    Carroll, Joe; Altman, Matthew C; Fearnley, Ian M; Walker, John E

    2007-09-04

    The most common way of identifying proteins in proteomic analyses is to use short segments of sequence ("tags") determined by mass spectrometric analysis of proteolytic fragments. The approach is effective with globular proteins and with membrane proteins with significant polar segments between membrane-spanning alpha-helices, but it is ineffective with other hydrophobic proteins where protease cleavage sites are either infrequent or absent. By developing methods to purify hydrophobic proteins in organic solvents and by fragmenting ions of these proteins by collision induced dissociation with argon, we have shown that partial sequences of many membrane proteins can be deduced easily by manual inspection. The spectra from small proteolipids (1-4 transmembrane alpha-helices) are dominated usually by fragment ions arising from internal amide cleavages, from which internal sequences can be obtained, whereas the spectra from larger membrane proteins (5-18 transmembrane alpha-helices) often contain fragment ions from N- and/or C-terminal parts yielding sequences in those regions. With these techniques, we have, for example, identified an abundant protein of unknown function from inner membranes of mitochondria that to our knowledge has escaped detection in proteomic studies, and we have produced sequences from 10 of 13 proteins encoded in mitochondrial DNA. They include the ND6 subunit of complex I, the last of its 45 subunits to be analyzed. The procedures have the potential to be developed further, for example by using newly introduced methods for protein ion dissociation to induce fragmentation of internal regions of large membrane proteins, which may remain partially folded in the gas phase.

  2. Rapid determination of methandrostenolone in equine urine by isotope dilution liquid chromatography-tandem mass spectrometry.

    PubMed

    Edlund, O; Bowers, L; Henion, J; Covey, T R

    1989-12-29

    Urine samples were spiked with [17-methyl-2H3]methandrostenolone as internal standard and extracted with a mixture of dichloromethane and cyclohexane. The organic phase was concentrated and injected onto a short octyl-silica column (30 mm x 4.6 mm I.D.) for separation of methandrostenolone and 17-epimethandrostenolone. The effluent from the column was connected to a Sciex TAGA 6000E triple quadrupole mass spectrometer equipped with an atmospheric pressure ion source for sampling of ions generated by a heated pneumatic nebulizer with corona discharge ionization. This ion source produced abundant [M + H]+ ions and a weak fragment ion due to loss of water. The protonated molecular ions at m/z 301 and 304 for methandrostenolone, 17-epimethandrostenolone and the internal standard were transmitted to the second quadrupole for collision-induced dissociation. Quantification was obtained by selected reaction monitoring of three daughter ions. Methandrostenolone and 17-epimethandrostenolone were separated by liquid chromatography, but gave identical mass spectra. The method detection limit by injection of a urine extract corresponding to 2.8 ml urine was 180 pg/ml at the 99% confidence level. The precision (relative standard deviation) was 3% at the 16 ng/ml level and the linear dynamic range was at least 3 orders of magnitude. Screening for unknown metabolites in urine after administration of methandrostenolone to horses and humans was accomplished by a parent ion scan of m/z 121, a fragment corresponding to the intact A-ring of the steroids.

  3. Surface-induced dissociation of ion mobility-separated noncovalent complexes in a quadrupole/time-of-flight mass spectrometer.

    PubMed

    Zhou, Mowei; Huang, Chengsi; Wysocki, Vicki H

    2012-07-17

    A custom in-line surface-induced dissociation (SID) device has been incorporated into a commercial ion mobility quadrupole/time-of-flight mass spectrometer in order to provide an alternative and potentially more informative activation method than the commonly used collision-induced dissociation (CID). Complicated sample mixtures can be fractionated by ion mobility (IM) and then dissociated by CID or SID for further structural analysis. Interpretation of SID spectra for cesium iodide clusters was greatly simplified with IM prior to dissociation because products originating from different precursors and overlapping in m/z but separated in drift time can be examined individually. Multiple conformations of two protein complexes, source-activated transthyretin tetramer and nativelike serum amyloid P decamer, were separated in ion mobility and subjected to CID and SID. CID spectra of the mobility separated conformations are similar. However, drastic differences can be observed for SID spectra of different conformations, implying different structures in the gas phase. This work highlights the potential of utilizing IM-SID to study quaternary structures of protein complexes and provides information that is complementary to our recently reported SID-IM approach.

  4. Matrix effect on the determination of synthetic corticosteroids and diuretics by liquid chromatography-tandem mass spectrometry

    NASA Astrophysics Data System (ADS)

    Dikunets, M. A.; Appolonova, S. A.; Rodchenkov, G. M.

    2009-04-01

    This work presents a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) procedure for selective and reliable screening of corticosteroids and diuretics in human urine. Sample preparation included the extraction, evaporation of the organic extract under nitrogen, and solution of the dry residue. The extract was analyzed by HPLC combined with tandem mass spectrometry using electro-spraying ionization at atmospheric pressure with negative ion recording. The mass spectra of all compounds were recorded, and the characteristic ions, retention times, and detection limits were determined. The procedure was validated by evaluating the degree of the matrix suppression of ionization, extraction of analytes from human biological liquid, and the selectivity and specificity of determination.

  5. Chemical constituents of Meconopsis horridula and their simultaneous quantification by high-performance liquid chromatography coupled with tandem mass spectrometry.

    PubMed

    Liu, Jiajia; Wu, Haimei; Zheng, Feng; Liu, Wenyuan; Feng, Feng; Xie, Ning

    2014-09-01

    Meconopsis horridula Hook.f. Thoms has been used as a traditional Tibetan medicine to clear away heat, relieve pain, and mobilize static blood. In this study, a reliable method based on high-performance liquid chromatography with diode array detection and electrospray ionization quadrupole time-of-flight tandem mass spectrometry was established for the identification of components in this herb. A total of 40 compounds (including 17 flavonoids, 15 alkaloids, and eight phenylpropanoids) were identified or tentatively identified. Among them, 17 components were identified in the herb for the first time. Compound 39 appears to be a novel compound, which is confirmed as 3-(kaempferol-8-yl)-2,3-epoxyflavanone by NMR spectroscopy and mass spectrometry. Moreover, seven major constituents were simultaneously quantified by the developed high-performance liquid chromatography with tandem triple-quadrupole mass spectrometry method. The quantitative method was validated and quality parameters were established. The study provides a comprehensive approach for understanding this herbal medicine.

  6. Hadamard Transform Time-of-Flight Mass Spectrometry

    DTIC Science & Technology

    2010-01-26

    collisional activation is similar to the 11 more common collision - induced dissociation (CID), however in SID the...Hines, et al. (1993). "Low-mass ions produced from peptides by high- energy collision - induced dissociation in tandem mass spectrometry." Journal of the...biomolecules." Science 246(4926): 64-72. Galhena, A. S., S. Dagan, et al. (2008). "Surface- Induced Dissociation of Peptides and Protein Complexes in

  7. Characterization of N-Succinylation of L-Lysylphosphatidylglycerol in Bacillus subtilis Using Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Atila, Metin; Katselis, George; Chumala, Paulos; Luo, Yu

    2016-10-01

    Phospholipids generally dominate in bacterial lipids. The negatively charged nature of phospholipids renders bacteria susceptible to cationic antibiotic peptides. In comparison with Gram-negative bacteria, Gram-positive bacteria in general have much less zwitterionic phosphatidylethanolamine. However, they are known for producing aminoacylated phosphatidylglycerol (PG), especially positively charged l-lysyl-PG, which is catalyzed by lysyl-PG synthase MprF, which appears to have a broad range of specificity for l-aminoacyl transfer RNAs. In addition, many Gram-positive bacteria also have a dlt-gene-coded d-alanylation pathway for lipoteichoic acids and wall teichoic acids covalently attached to a glycolipid or peptidoglycan. d-Alanylation also masks the dominant negative charge of the phosphate-rich polymers of teichoic acids. Using mass spectrometry, we have recently observed that precursor scans in negative mode for deprotonated amino acid fragments were most sensitive for ester-linked amino acids. Such a scan for precursors generating an m/ z 145 lysyl anion revealed lysyl-PG as well as an additional species 100 m/ z units greater than lysyl-PG. This unexpected species corresponded precisely to the expected mass of N-succinylated lysyl-PG. Tandem mass spectrometry revealed a precise match to the fragmentation pattern of this putative new species. PG, lysyl-PG, and N-succinyl-lysyl-PG may form a complete loop of charge reversal from -1 to +1 and then back to -1. Analogous charge reversal by N-succinylation of lysine residues in the bacterial as well as eukaryotic proteomes has been recently discovered as a major posttranslational modification. Such modification in bacterial lipids is possibly catalyzed by an enzyme homologous to the enzymes that modify lysine residues in proteins.

  8. Characterization of thin film tandem solar cells by radiofrequency pulsed glow discharge - Time of flight mass spectrometry.

    PubMed

    Fernandez, Beatriz; Lobo, Lara; Reininghaus, Nies; Pereiro, Rosario; Sanz-Medel, Alfredo

    2017-04-01

    Beside low production costs and the use of nontoxic and abundant raw materials, silicon based thin-film solar cells have the advantage to be built up as multi junction devices like tandem or triple junction solar cells. Silicon thin film modules made of tandem cells with hydrogenated amorphous silicon (a-Si:H) top cell and microcrystalline (μc) Si:H bottom cell are available on the market. In this work, the analytical potential of state-of-the art radiofrequency (rf) pulsed glow discharge (PGD) time of flight mass spectrometry (TOFMS) commercial instrumentation is investigated for depth profiling analysis of tandem-junctions solar cells on 2mm thick glass substrate with 1µm thick ZnO:Al. Depth profile characterization of two thin film tandem photovoltaic devices was compared using millisecond and sub-millisecond rf-PGD regimes, as well as the so-called "low mass mode" available in the commercial instrument used. Two procedures for sample preparation, namely using flat or rough cell substrates, were compared and the distribution of dopant elements (phosphorous, boron and germanium) was investigated in both cases. Experimental results obtained by rf-PGD-TOFMS as well as electrical measurements of the samples showed that a worse depth resolution of dopant elements in the silicon layers (e.g. distribution of boron in a thicker region that suggests a diffusion of this dopant in the coating of the sample) found using a rough sample substrate was related to a higher power conversion efficiency.

  9. Multiclass determination of sunscreen chemicals in water samples by liquid chromatography-tandem mass spectrometry.

    PubMed

    Rodil, Rosario; Quintana, José Benito; López-Mahía, Purificación; Muniategui-Lorenzo, Soledad; Prada-Rodríguez, Darío

    2008-02-15

    A novel analytical method based on solid-phase extraction (SPE) and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) for the determination of UV sunscreen agents in the water environment is presented. After a thorough investigation of SPE and LC-MS/MS conditions, it permits the enrichment and determination of nine of these compounds in a single methodology, including three very polar sulfonates (e.g., 2-phenylbenzimidazole-5-sulfonic acid, PBSA) and six other less polar compounds (e.g., benzophenone-3, BP-3; octocrylene, OC,...). Other important matters of concern in the determination of UV filters at trace levels in water, i.e., adsorption on glassware and blank contamination problems, have also been discussed and minimized. This methodology affords detection limits between 7 and 46 ng L-1 and SPE recoveries in the range 63-102% from different real water matrixes, except for butylmethoxydibenzoylmethane (BM-DBM), which was not determinable in wastewater samples due to adsorption problems. The application of the method allowed reporting the levels of benzophenone-4 (BP-4) in environmental water samples for the first time, where it was identified as one of the most important in concentration among the UV filters studied, particularly in wastewater (237-1481 ng L-1).

  10. Differentiating Isobaric Steroid Hormone Metabolites Using Multi-Stage Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Tedmon, Lauren; Barnes, Jeremy S.; Nguyen, Hien P.; Schug, Kevin A.

    2013-03-01

    Steroid hormones and their metabolites are currently undergoing clinical trials as potential therapeutics for traumatic brain injury (TBI). To support this work, it is necessary to develop improved procedures for differentiating isobaric species in this compound class. Equilin sulfate (E-S), estrone sulfate (E1-S), 17α-dihydroequilin sulfate (ADHE-S), and 17β-dihydroequilin sulfate (BDHE-S) are primary constituents in hormone replacement therapies, such as Premarin, which are among pharmaceuticals being investigated for TBI treatment. The latter three compounds are isomers and can be difficult to differentiate in trace analytical determinations. In this work, a systematic study of the fragmentation of ADHE-S, BDHE-S, E1-S, and E-S under different stages of higher order tandem mass spectrometry (MSn) and variation of collision energy, allowed optimization of conditions for distinguishing the isomeric structures. For epimeric variants (e.g., ADHE-S versus BDHE-S; α- versus β-stereoisomerization in the C-17 position), differentiation was achieved at MS4 and fragmentation was demonstrated through MS5. Computational analysis was performed to further explore differences in the fragmentation pathways due to changes in stereochemistry.

  11. [Determination of seven toxaphene congeners in ginseng and milkvetch root by gas chromatography tandem mass spectrometry].

    PubMed

    Tian, Shaoqiong; Mao, Xiuhong; Miao, Shui; Jia, Zhengwei; Wang, Ke; Ji, Shen

    2012-01-01

    A novel method for the determination of representative toxaphene congeners in traditional Chinese herbal medicines was developed. Ginseng and Milkvetch Root were selected as the samples and seven toxaphene congeners were selected as the monitoring objects. The samples were extracted by accelerated solvent extraction with cyclohexane-acetone (9:1, v/v), then cleaned-up by Florisil solid phase extraction with hexane as the eluent and the residues were detected by gas chromatography-electron ionization tandem mass spectrometry (GC-EI-MS/MS) in multiple reaction monitoring (MRM) mode. The performance was demonstrated by the analysis of Ginseng and Milkvetch Root samples spiked with toxaphene congeners at three concentration levels of 0.005, 0.01 and 0.1 mg/kg. The recoveries ranged from 72.4% to 105% with the relative standard deviations (RSDs) of 0.96%-10.4%. The limits of detection (LODs) were 0.2-1.7 microg/kg. This method is sensitive and efficient in the aspect of extraction, and can be applied to monitor the residue of toxaphene congeners in Ginseng and Milkvetch Root.

  12. Evaluation of 3-methylcrotonyl-CoA carboxylase deficiency detected by tandem mass spectrometry newborn screening.

    PubMed

    Koeberl, D D; Millington, D S; Smith, W E; Weavil, S D; Muenzer, J; McCandless, S E; Kishnani, P S; McDonald, M T; Chaing, S; Boney, A; Moore, E; Frazier, D M

    2003-01-01

    Since the addition of tandem mass spectrometry (MS/MS) to the North Carolina Newborn Screening Program, 20 infants with two consecutive elevated 3-hydroxyisovalerylcarnitine (C5OH) levels have been evaluated for evidence of inborn errors of metabolism associated with this metabolite. Ten of these 20 infants had significant concentrations of both 3-hydroxyisovaleric acid and 3-methylcrotonylglycine in their urine, suggestive of 3-methylcrotonyl-CoA carboxylase (3-MCC) deficiency. Four of these 10 were infants whose abnormal metabolites were found to be of maternal origin. Of 8 patients with probable 3-MCC deficiency, 7 have been tested and found to have the enzyme deficiency confirmed in lymphoblasts or cultured fibroblasts; one of these 7 infants had only marginally decreased 3-MCC activity in lymphocytes but deficient 3-MCC in fibroblasts. We estimate the incidence of 3-MCC deficiency at 1:64000 live births in North Carolina. We conclude that MS/MS newborn screening will detect additional inborn errors of metabolism, such as 3-MCC deficiency, not traditionally associated with newborn screening. The evaluation of newborns with two abnormally elevated C5OH levels on MS/MS newborn screening should include, at least, urine organic acid analysis by capillary GC-MS and a plasma acylcarnitine profile by MS/MS. Long-term follow-up is needed to determine the outcome of presymptomatically diagnosed patients with 3-MCC deficiency by MS/MS newborn screening.

  13. High performance liquid chromatography tandem mass spectrometry determination of perfluorinated acids in cow milk.

    PubMed

    Capriotti, Anna Laura; Cavaliere, Chiara; Cavazzini, Alberto; Foglia, Patrizia; Laganà, Aldo; Piovesana, Susy; Samperi, Roberto

    2013-12-06

    A new and sensitive liquid chromatography/electrospray-tandem mass spectrometric (LC/ESI-MS/MS) method for the determination of 12 perfluorinated compounds (PFCs) in cow milk is described. Milk samples were extracted with acetone and cleaned-up by a graphitized carbon black solid-phase extraction cartridge, optimizing the entire procedure by using a screening experimental design. LC/ESI-MS/MS was performed in negative ion mode using multiple reaction monitoring mode. The performance of the method was evaluated under the optimized conditions in terms of matrix effects, range of linearity, accuracy, and repeatability. For all compounds, linearity in matrix was observed in the range LOQ-10μgL(-1), and coefficients of determination R(2) ranged from 0.9982 to 0.9999. The analytical recoveries, relative to the isotopic internal standard, measured at 10 and 50ngL(-1) were in the range of 91-105%, with relative standard deviations below 6% and method detection limit, based on the blank value +3 times the standard deviation of the blank, ranged from 0.5 to 3ngL(-1). The final method developed was used to determine the concentration of PFCs in 15 retail milk samples. None of these compounds were detected in cow milk analyzed samples.

  14. Glycerophospholipid analysis of Eastern red bat (Lasiurus borealis) hair by electrospray ionization tandem mass spectrometry.

    PubMed

    Pannkuk, Evan L; McGuire, Liam P; Gilmore, David F; Savary, Brett J; Risch, Thomas S

    2014-03-01

    Pilosebaceous units found in the mammalian integument are composed of a hair follicle, the proximal portion of the hair shaft, a sebaceous gland, and the erector pili muscle. Pilosebaceous units release protective oils, or sebum, by holocrine secretion onto skin and hair through rupturing of sebocytes. Sebum is composed largely of polar and neutral lipids including glycerolipids, free fatty acids, sterols, wax esters, sterol esters, and squalene. In addition to these lipid classes, there is a small proportion of ionic/anionic glycerophospholipids (GPs). Composition of GPs on hair is rarely addressed despite their broad biological activities as signaling molecules and membrane stability. Furthermore, knowledge on GP composition in bats is lacking. Bat GP composition is important to document due to GP roles ranging from decreasing drag during migration to interaction with the integumentary microbiome. In this study, we analyzed GP molecular composition with liquid chromatography electrospray ionization tandem mass spectrometry and compared GP content to previous literature. A total of 152 GPs were detected. Broad GP classes identified include lysophosphatidylcholine, phosphatidylcholine (PC), lysophosphatidylethanolamine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidic acid, and phosphatidylglycerol, with PC being the most abundant class. The acyl components were consistent with fatty acid methyl esters and triacylglyceride moieties found in Eastern red bat sebum. Glycerophospholipid proportions of the hair surface were different from a previous study on bat lung surfactants. This study determined the broad class and molecular species of bat sebum GPs that may be used in future ecological studies in vespertilionid bats.

  15. Analysis of acrylamide in coffee and cocoa by isotope dilution liquid chromatography-tandem mass spectrometry.

    PubMed

    Aguas, Patricia C; Fitzhenry, Matthew J; Giannikopoulos, Georgina; Varelis, Peter

    2006-08-01

    An accurate and precise method for the quantification of acrylamide using stable isotope dilution liquid chromatography-tandem mass spectrometry was developed and used to measure acrylamide in coffee and cocoa samples. The sample preparation involved extraction of the analyte and its internal standard, 13C3-acrylamide, into water and subsequent defatting of the aqueous extract with dichloromethane. An aliquot of the resulting aqueous extract was then azeotropically dried under reduced pressure and subsequently purified using an aminopropyl-bonded silica cartridge. The purified extracts were then chromatographed on a 5-microm 2.1 x 150 mm Hypercarb column, the effluent of which was monitored for the analyte and its internal standard using positive-ion APCI-selected reaction monitoring. The intra-laboratory reproducibility of the method, expressed as a relative coefficient of variation (%, n=5), was determined at four levels of concentration (12.3, 42.3, 139.3 and 464.8 microg kg(-1)) and was found to vary between 0.6-2.5%. The accuracy of the method was assessed using a reference sample of coffee. The average result obtained using our method differed from the assigned value of the reference material by less than 1%. An analysis of a cocoa sample revealed that the method is capable of precisely estimating acrylamide in challenging matrices down to a level of at least 12.3 microg kg(-1).

  16. Determination of homocitrulline in urine of patients with HHH syndrome by liquid chromatography tandem mass spectrometry.

    PubMed

    Al-Dirbashi, Osama Y; Al-Hassnan, Zuhair N; Rashed, Mohamed S

    2006-12-01

    A liquid chromatography tandem mass spectrometric method is described for the analysis of homocitrulline in human urine, a key metabolite in the differential diagnosis of hyperammonemia, hyperornithinemia, homocitrullinuria (HHH) syndrome. Urine samples were prepared by mere five-fold dilution with a mixture of internal standards (2H2-citrulline and 2H3-creatinine) used for the simultaneous quantification of creatinine. Analytes were separated on a cyano column and eluted isocratically within seven min. Detection was achieved by monitoring transitions of 190 > 84 and 190 > 127 for homocitrulline, 178 > 115 for 2H2-citrulline, 114 > 44 for creatinine and 117 > 47 for 2H3-creatinine. Calibration curves were linear up to 100 micromol/L. Intraday (n = 7) and interday (n = 6) variations were less than 10%. In urine samples from three siblings confirmed to have HHH syndrome, homocitrulline levels were at 13.3 (74), 21.1 (50) and 108.2 (103) mmol/mol creatinine (micromol/L). Control values were 0-9 mmol/mol creatinine (n = 120). The current method solves specificity issues in homocitrulline determination often encountered with some ninhydrin-based systems (coelution with methionine) and some o-phthalaldehyde-based ones (coelution with taurine), and presents an attractive alternative with a relatively high throughput.

  17. Structural elucidation of isocyanate-peptide adducts using tandem mass spectrometry.

    PubMed

    Hettick, Justin M; Ruwona, Tinashe B; Siegel, Paul D

    2009-08-01

    Diisocyanates are highly reactive chemical compounds widely used in the manufacture of polyurethanes. Although diisocyanates have been identified as causative agents of allergic respiratory diseases, the specific mechanism by which these diseases occur is largely unknown. To better understand the chemical species produced when isocyanates are reacted with model peptides, tandem mass spectrometry was employed to unambiguously identify the binding site of four commercially-relevant isocyanates on model peptides. In each case, the isocyanates react preferentially with the N-terminus of the peptide. No evidence of side-chain/isocyanate adduct formation exclusive of the N-terminus was observed. However, significant intra-molecular diisocyanate crosslinking was observed between the N-terminal amine and a side-chain amine of arginine, when Arg was located within two residues of the N-terminus. Addition of multiple isocyanates to the peptide occurs via polymerization of the isocyanate at the N-terminus, rather than via addition of multiple isocyanate molecules to varied residues within the peptide. The direct observation of isocyanate binding to the N-terminus of peptides under these experimental conditions is in good agreement with previous studies on the relative reaction rate of isocyanate with amino acid functional groups.

  18. Determination of PF-04928473 in human plasma using liquid chromatography with tandem mass spectrometry

    PubMed Central

    Jain, Lokesh; Gardner, Erin R.; Venitz, Jürgen; Giaccone, Giuseppe; Houk, Brett E.; Figg, William D.

    2010-01-01

    A simple, rapid and sensitive liquid chromatography/tandem mass spectrometric (LC/MS/MS) analytical method was developed for quantification of Hsp90 inhibitor PF-04928473 in human plasma, following administration of its prodrug, PF-04929113. Sample processing involved protein precipitation by addition of 0.4 mL of methanol containing internal standard (PF-04972487) to 50 μL volume of plasma sample. Chromatographic separation of PF-04928473 and PF-04972487 was achieved on a Phenomenex® Luna C18(2) (2.0×50 mm, 5 μm) column using a gradient elution method with mobile phase solvents: methanol containing 0.1% formic acid and 0.1% formic acid at a flow rate of 0.25 mL/min. Detection was performed in electrospray positive ionization mode, monitoring the ion transitions from m/z 465.1→350.1 (PF-04928473) and m/z 447.0→329.1 (PF-04972487). The retention times for PF-04928473 and PF-04972487 were 1.86 and 2.85 minutes, respectively. Calibration curves were generated in the range of 2–2000 ng/mL. The accuracy and precision ranged from 94.1–99.0% and 86.7–97.6%, respectively, which were calculated using quality control samples of three different concentrations analyzed in quintuplicate on four different days. PMID:20951100

  19. Fully automated screening of veterinary drugs in milk by turbulent flow chromatography and tandem mass spectrometry

    PubMed Central

    Stolker, Alida A. M.; Peters, Ruud J. B.; Zuiderent, Richard; DiBussolo, Joseph M.

    2010-01-01

    There is an increasing interest in screening methods for quick and sensitive analysis of various classes of veterinary drugs with limited sample pre-treatment. Turbulent flow chromatography in combination with tandem mass spectrometry has been applied for the first time as an efficient screening method in routine analysis of milk samples. Eight veterinary drugs, belonging to seven different classes were selected for this study. After developing and optimising the method, parameters such as linearity, repeatability, matrix effects and carry-over were studied. The screening method was then tested in the routine analysis of 12 raw milk samples. Even without internal standards, the linearity of the method was found to be good in the concentration range of 50 to 500 µg/L. Regarding repeatability, RSDs below 12% were obtained for all analytes, with only a few exceptions. The limits of detection were between 0.1 and 5.2 µg/L, far below the maximum residue levels for milk set by the EU regulations. While matrix effects—ion suppression or enhancement—are obtained for all the analytes the method has proved to be useful for screening purposes because of its sensitivity, linearity and repeatability. Furthermore, when performing the routine analysis of the raw milk samples, no false positive or negative results were obtained. PMID:20379812

  20. [Analysis of mouse liver membrane proteins using multidimensional ion exchange chromatography and tandem mass spectrometry].

    PubMed

    Wang, Zhuowei; Peng, Fuli; Wang, Yuan; Tong, Wei; Ren, Yan; Xu, Ningzhi; Liu, Siqi

    2010-02-01

    The analysis of membrane proteins is still a technical obstacle in proteomic investigation. A fundamental question is how to allow the hydrophobic proteins fully solubilizing in a proper solvent environment. We propose that the denatured membrane proteins in high denaturant solution are fully ionized and separated through ion exchange chromatography. The membrane proteins prepared from a mouse liver were dissolved in 4 mol/L urea, 20 mmol/L Tris-HCl buffer (pH 9.0), and loaded onto a tandem chromatography coupled with Q-Sepharose FF and Sephacryl S-200HR. With a linear NaCl gradient elution, the bound proteins were eluted and collected followed by sodium-dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to further separate the eluted proteins. The protein bound on SDS-PAGE were excised and in-gel digested by trypsin, while the digested peptides were delivered to reversed-phase high performance liquid chromatography (HPLC) and ion-trap mass spectrometry for the peptide identifications. Of a total of 392 proteins identified, 306 were membrane proteins or membrane associated proteins reported by literature. Based on the calculation of hydrophobicity, the GRAVY (grand average of hydropathicity) scores of 83 proteins are over or equal to 0.00. Taking all the evidence, we have established an effective approach which is feasible in the investigation towards mouse liver membrane proteomics.

  1. [Determination of ribavirin and amantadine in chicken by ultra performance liquid chromatography-tandem mass spectrometry].

    PubMed

    Yun, Huan; Cui, Fengyun; Yan, Hua; Liu, Xin; He, Yue; Zhang, Zhaohui

    2013-08-01

    A method for the determination of ribavirin and amantadine in chicken has been developed by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/ MS). After extracted by 1% (volume percentage) trichloroacetic acid solution-acetonitrile (1:1, v/v) and purified by a Supelco LC-SCX cartridge, the samples were loaded onto an Acquity UPLC BEH Hillic column (150 mm x 2.1 mm, 1.7 microm) and separated with gradient elution. The electrospray was operated in the positive mode and the samples were monitored by the multiple reaction monitoring (MRM) mode. The limits of quantification (LOQs, S/N = 10) of ribavirin and amantadine were 4.0 microg/kg. The calibration curves showed good linearity in the range of 10.0 - 100.0 microg/L, and the correlation coefficients (r(2)) were not lower than 0.99. When the spiked levels were 4.0, 8.0 and 20.0 microg/kg, the recoveries of ribavirin and amantadine in chicken ranged from 78% to 102.5%, with the relative standard deviations (RSDs) of 2.2% - 7.6%. The results indicate that the method is simple, rapid, sensitive and suitable for the qualitative and quantitative analyses of ribavirin and amantadine in chicken samples.

  2. MS2Grouper: group assessment and synthetic replacement of duplicate proteomic tandem mass spectra.

    PubMed

    Tabb, David L; Thompson, Melissa R; Khalsa-Moyers, Gurusahai; VerBerkmoes, Nathan C; McDonald, W Hayes

    2005-08-01

    Shotgun proteomics experiments require the collection of thousands of tandem mass spectra; these sets of data will continue to grow as new instruments become available that can scan at even higher rates. Such data contain substantial amounts of redundancy with spectra from a particular peptide being acquired many times during a single LC-MS/MS experiment. In this article, we present MS2Grouper, an algorithm that detects spectral duplication, assesses groups of related spectra, and replaces these groups with synthetic representative spectra. Errors in detecting spectral similarity are corrected using a paraclique criterion-spectra are only assessed as groups if they are part of a clique of at least three completely interrelated spectra or are subsequently added to such cliques by being similar to all but one of the clique members. A greedy algorithm constructs a representative spectrum for each group by iteratively removing the tallest peaks from the spectral collection and matching to peaks in the other spectra. This strategy is shown to be effective in reducing spectral counts by up to 20% in LC-MS/MS datasets from protein standard mixtures and proteomes, reducing database search times without a concomitant reduction in identified peptides.

  3. Pharmacokinetic studies of novel berberine derivatives with ultra-performance liquid chromatography-tandem mass spectrometry.

    PubMed

    Wang, Wenchao; Shen, Qin; Liang, Hui; Hua, Changlong; Liu, Yuhui; Li, Fengzhi; Li, Qingyong

    2016-09-15

    An ultra-performance liquid chromatography with tandem mass spectrometric detection method was developed for the detection of berberine and its derivatives (A4, B4) in rat plasma and other organs. This validated method was successfully applied to our pharmacokinetic study of BBR derivatives in rats. At the same dose of administration, the Cmax of B4 was about eight times higher than BBR, and its half-life was approximately two times longer than BBR, according to the bigger areas under plasma concentration curves. Inversely, the pharmacokinetic parameter levels of A4 were all inferior to BBR, suggesting a tight structure-activity relationship of these compounds. Small dose of parenteral administration was used for the study of absolute oral bioavailability of A4, B4, and BBR, and the results calculated were 0.12%, 3.4% and 0.7%, respectively. The accumulations of B4 among all organs were intestine>liver>heart>kidney>lung>spleen>plasma, proving a deeply targeting property of B4, which met our experimental assumption. Together, the experimental results proved that compared with BBR and A4, the derivative B4 had higher absolute oral bioavailability and the ability of deeply targeting so that can be likely used in some organ-targeted diseases.

  4. Analysis of testosterone and dihydrotestosterone in mouse tissues by liquid chromatography-electrospray tandem mass spectrometry

    PubMed Central

    Weng, Yan; Xie, Fang; Xu, Li; Zagorevski, Dmitri; Spink, David C.; Ding, Xinxin

    2010-01-01

    A novel method was established for simultaneous quantitation of testosterone (T) and dihydrotestosterone (DHT) in murine tissue and serum samples. Endogenous T and DHT, together with the internal standards, 17α-methyl-T and 17α-methyl-DHT, were extracted from tissues, and then derivatized by reaction with 2-hydrazino-4-(trifluoromethyl)-pyrimidine (HTP). Analysis by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) resulted in production spectra of HTP derivatives of both T and DHT that showed analyte-specific fragmentations; the latter fragmentations were characterized by use of high-resolution Orbitrap MS/MS. These specific fragmentations enabled quantitation of T and DHT in the multiple-reaction monitoring (MRM) mode. The method was validated with charcoal-stripped serum as the matrix; the LLOQ was 0.10 ng/ml for T and 0.50 ng/ml for DHT. The method was then used for determination of serum and tissue levels of T and DHT in transgenic mice carrying a hypomorphic NADPH-cytochrome P450 reductase gene (Cpr-low mice). Remarkably, ovarian T levels in Cpr-low mice were found to be 25-fold higher than those in wild-type mice, a finding that at least partly explains the female infertility seen in the Cpr-low mice. In conclusion, our method provides excellent sensitivity and selectivity for determination of endogenous levels of T and DHT in mouse tissues. PMID:20361922

  5. Quantitative analysis of chemical warfare agent degradation products in beverages by liquid chromatography tandem mass spectrometry.

    PubMed

    Owens, Janel; Koester, Carolyn

    2009-09-23

    Though chemical warfare agents (CWAs) have been banned by the Chemical Weapons Convention, the threat that such chemicals may be used, including their deliberate addition to food, remains. In such matrixes, CWAs may hydrolyze to phosphonic acids, which are good surrogate markers of CWA contamination. The method described here details the extraction of five CWA degradation products, including methylphosphonic acid (MPA), ethyl-MPA, isopropyl-MPA, cyclohexyl-MPA, and pinacolyl-MPA, from five different beverages by strata-X solid phase extraction cartridges. Samples were analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS) with multiple reaction monitoring. The limit of quantitation ranged from 0.05 to 0.5 ng on-column, and the limit of detection was >0.02 ng on-column. Beverages were fortified with the five phosphonic acids at 1 microg/mL and 0.25 microg/mL and quantitated using both an internally standardized method and matrix-matched standards. Reasonable recoveries (>50%) were achieved for ethyl, isopropyl, cyclohexyl, and pinacolyl-MPA for most matrixes.

  6. Determination of the active metabolite of sibutramine by liquid chromatography-electrospray ionization tandem mass spectrometry.

    PubMed

    Chen, Jun; Lu, Wei; Zhang, Qizhi; Jiang, Xinguo

    2003-03-05

    A sensitive and specific method for the determination of the active primary amine metabolite of sibutramine, N-di-desmethylsibutramine (BTS 54,505), in human plasma was developed, based on high-performance liquid chromatography (HPLC)-electrospray ionization tandem mass spectrometry (MS-MS). The samples were extracted from plasma with methyl tert.-butyl ether, followed by separation and evaporation after addition of the internal standard, propranolol, and basification with sodium hydroxide. The residue was reconstituted in mobile phase and injected into the HPLC-MS-MS system. Chromatography was performed on an ODS MS column with a mobile phase consisting of acetonitrile (containing 0.1% trifluoroacetic acid, v/v)-0.1% trifluoroacetic acid (55:45, v/v) at a flow-rate of 0.3 ml/min. Multiple reaction monitoring using precursor-->product ion combinations at m/z 252.00-->125.00 and 260.00-->115.70 was applied to determine BTS 54,505 and propranolol, respectively. Linearity was confirmed in the concentration range 0.328-32.8 ng/ml in human plasma and the imprecision of this assay was less than 19.90% over the entire concentration range. The method is sufficiently sensitive and repeatable to be used in pharmacokinetic studies.

  7. Analysis of nerve agent metabolites from nail clippings by liquid chromatography tandem mass spectrometry.

    PubMed

    Appel, Amanda S; Logue, Brian A

    2016-09-15

    While several methods for the bioanalysis of nerve agents or their metabolites have been developed for the verification of nerve agent exposure, these methods are generally limited in the amount of time after an exposure that markers of exposure can be detected (due to rapid metabolism from biological matrices). In this study, a method for the analysis of nerve agent hydrolysis products from nail clippings was developed to allow evaluation of nails as a long-term repository of these markers. Pinacolyl methylphosphonic acid (PMPA) and isopropyl methylphosphonic acid (IMPA) were extracted from nail samples with N,N-dimethylformamide and subsequently analyzed by liquid chromatography-tandem mass spectrometry. Limits of detection for PMPA and IMPA were 0.3μg/kg and 7.5μg/kg and linear ranges were 0.75-300μg/kg and 30-1500μg/kg, respectively. Precision was within 10% and 8% for PMPA and IMPA, respectively, and accuracy was 100±12% for both analytes. The approach presented here is complementary to current methods for nerve agent exposure verification, and should allow for long-term determination of nerve agent poisoning.

  8. Partial enzymatic elimination and quantification of sarcosine from alanine using liquid chromatography-tandem mass spectrometry.

    PubMed

    Burton, Casey; Gamagedara, Sanjeewa; Ma, Yinfa

    2013-04-01

    Since sarcosine and D,L-alanine co-elute on reversed-phase high-performance liquid chromatography (HPLC) columns and the tandem mass spectrometer cannot differentiate them due to equivalent parent and fragment ions, derivatization is often required for analysis of sarcosine in LC/MS systems. This study offers an alternative to derivatization by employing partial elimination of sarcosine by enzymatic oxidation. The decrease in apparent concentration from the traditionally merged sarcosine-alanine peak associated with the enzymatic elimination has been shown to be proportional to the total sarcosine present (R(2) = 0.9999), allowing for determinations of urinary sarcosine. Sarcosine oxidase was shown to eliminate only sarcosine in the presence of D,L-alanine, and was consequently used as the selective enzyme. This newly developed technique has a method detection limit of 1 μg/L (parts per billion) with a linear range of 3 ppb-1 mg/L (parts per million) in urine matrices. The method was further validated through spiked recoveries of real urine samples, as well as the analysis of 35 real urine samples. The average recoveries for low, middle, and high sarcosine concentration spikes were 111.7, 90.8, and 90.1 %, respectively. In conclusion, this simple enzymatic approach coupled with HPLC/MS/MS is able to resolve sarcosine from D,L-alanine leading to underivatized quantification of sarcosine.

  9. Liquid chromatography/tandem mass spectrometry method for quantitation of cremophor el and its applications.

    PubMed

    Vijaya Bhaskar, V; Middha, Anil

    2013-01-01

    A rapid sensitive and selective MRM based method for the determination of Cremophor EL (CrEL) in rat plasma was developed using liquid chromatography/tandem mass spectrometry (LC-MS/MS). CrEL and polypropylene glycol (internal standard) were extracted from rat plasma with acetonitrile and analysed on C18 column (XBridge, 50 × 4.6 mm, 3.5  μ m). The most abundant molecular ions corresponding to PEG oligomers at m/z 828, 872, 916 and 960 with daughter ion at m/z 89 were selected for multiple reaction monitoring (MRM) in electrospray mode of ionisation. Plasma concentrations of CrEL were quantified after administration through oral and intravenous routes in male sprague dawley rats at a dose of 0.26 g/kg. The standard curve was linear (0.9972) over the concentration range of 1.00 to 200  μ g/mL. The lower limit of quantitation for CrEL was 1.00  μ g/mL using 50  μ L plasma. The coefficient of variation and relative error for inter and intra assay at three QC levels were 0.69 to 9.21 and -7.60 to 4.74 respectively. A novel proposal was conveyed to the scientific community, where formulation excipient can be analysed as qualifier in the analysis of NCEs to address the spiky plasma concentration profiles.

  10. Determination of ethyl glucuronide in human hair by hydrophilic interaction liquid chromatography-tandem mass spectrometry.

    PubMed

    Yaldiz, Fadile; Daglioglu, Nebile; Hilal, Ahmet; Keten, Alper; Gülmen, Mete Korkut

    2013-10-01

    Ethyl glucuronide (EtG) is a direct metabolite of ethanol and has been utilized as a marker for alcohol intake. This study presents development, validation and application of a new hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method for the analysis of EtG in human hair samples. The linearity was assessed in the range of 5-2000 pg/mg hair, with a correlation coefficient of >0.99. The method was selective and sensitive, with a limit of detection (LOD) and limit of quantitation (LOQ) of 0.05 pg/mg and 0.18 pg/mg in hair, respectively. Differently from the extraction procedures in the literature, a fast and simple liquid-liquid method was used and highest recoveries and cleanest extracts were obtained. The method was successfully applied to 30 human hair samples which were taken from those who state they consume alcohol. EtG concentrations in the hair samples of alcohol users participated in this study, ranged between 1.34 and 82.73 pg/mg. From the concentration of EtG in hair strands 20 of the 30 subjects can be considered regular moderate drinkers.

  11. Faster and more accurate graphical model identification of tandem mass spectra using trellises

    PubMed Central

    Wang, Shengjie; Halloran, John T.; Bilmes, Jeff A.; Noble, William S.

    2016-01-01

    Tandem mass spectrometry (MS/MS) is the dominant high throughput technology for identifying and quantifying proteins in complex biological samples. Analysis of the tens of thousands of fragmentation spectra produced by an MS/MS experiment begins by assigning to each observed spectrum the peptide that is hypothesized to be responsible for generating the spectrum. This assignment is typically done by searching each spectrum against a database of peptides. To our knowledge, all existing MS/MS search engines compute scores individually between a given observed spectrum and each possible candidate peptide from the database. In this work, we use a trellis, a data structure capable of jointly representing a large set of candidate peptides, to avoid redundantly recomputing common sub-computations among different candidates. We show how trellises may be used to significantly speed up existing scoring algorithms, and we theoretically quantify the expected speedup afforded by trellises. Furthermore, we demonstrate that compact trellis representations of whole sets of peptides enables efficient discriminative learning of a dynamic Bayesian network for spectrum identification, leading to greatly improved spectrum identification accuracy. Contact: bilmes@uw.edu or william-noble@uw.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27307634

  12. Determination of melatonin in Acyrthosiphon pisum aphids by liquid chromatography-tandem mass spectrometry.

    PubMed

    Escrivá, Laura; Manyes, Lara; Barberà, Miquel; Martínez-Torres, David; Meca, Guiseppe

    2016-03-01

    Melatonin is a hormone mainly involved in the regulation of circadian and seasonal rhythms in both invertebrates and vertebrates. Despite the identification of melatonin in many insects, its involvement in the insect seasonal response remains unclear. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed for melatonin analysis in aphids (Acyrthosiphon pisum) for the first time. After comparing two different procedures and five extraction solvents, a sample preparation procedure with a mixture of methanol/water (50:50) was selected for melatonin extraction. The method was validated by analyzing melatonin recovery at three spiked concentrations (5, 50 and 100 pg/mg) and showed satisfactory recoveries (75-110%), and good repeatability, expressed as relative standard deviation (<10%). Limits of detection (LOD) and quantitation (LOQ) were 1 pg/mg and 5 pg/mg, respectively. Eight concentration levels were used for constructing the calibration curves which showed good linearity between LOQ and 200 times LOQ. The validated method was successfully applied to 26 aphid samples demonstrating its usefulness for melatonin determination in insects. This is -to our knowledge- the first identification of melatonin in aphids by LC-MS/MS.

  13. Simultaneous determination of zidovudine and lamivudine in human serum using HPLC with tandem mass spectrometry.

    PubMed

    Kenney, K B; Wring, S A; Carr, R M; Wells, G N; Dunn, J A

    2000-07-01

    A method employing high performance liquid chromatography (HPLC) with tandem mass spectrometry (MS) has been developed and validated for the simultaneous determination of clinically relevant levels of zidovudine (AZT) and lamivudine (3TC) in human serum. The method incorporates a fully automated ultrafiltration sample preparation step that replaces the solid-phase extraction step typically used for HPLC with UV detection. The calibration range of the dual-analyte LC-MS/MS method is 2.5-2,500 and 2.5-5,000 ng ml-1 for AZT and 3TC, respectively, using 0.25 ml of human serum. The lower limit of quantification was 2.5 ng ml-1 for each analyte, with a chromatographic run time of approximately 6 min. Overall accuracy, expressed as bias, and inter- and intra-assay precision are < +/- 7 and < 10% for AZT, and < +/- 5 and < 12.1% for 3TC over the full concentration ranges. A cross-validation study demonstrated that the LC-MS/MS method afforded equivalent results to established methods consisting of a radioimmuno-assay for AZT and an HPLC-UV method for 3TC. Moreover, the LC-MS/MS was more sensitive, allowed markedly higher-throughput, and required smaller sample volumes (for 3TC only). The validated method has been used to support post-marketing clinical studies for Combivir a combination tablet containing AZT and 3TC.

  14. Proteomic profiling of sea bass muscle by two-dimensional gel electrophoresis and tandem mass spectrometry.

    PubMed

    Terova, Genciana; Pisanu, Salvatore; Roggio, Tonina; Preziosa, Elena; Saroglia, Marco; Addis, Maria Filippa

    2014-02-01

    In this study, the proteome profile of European sea bass (Dicentrarchus labrax) muscle was analyzed using two-dimensional electrophoresis (2-DE) and tandem mass spectrometry with the aim of providing a more detailed characterization of its specific protein expression profile. A highly populated and well-resolved 2-DE map of the sea bass muscle tissue was generated, and the corresponding protein identity was provided for a total of 49 abundant protein spots. Upon Ingenuity Pathway Analysis, the proteins mapped in the sea bass muscle profile were mostly related to glycolysis and to the muscle myofibril structure, together with other biological activities crucial to fish muscle metabolism and contraction, and therefore to fish locomotor performance. The data presented in this work provide important and novel information on the sea bass muscle tissue-specific protein expression, which can be useful for future studies aimed to improve seafood traceability, food safety/risk management and authentication analysis. This work is also important for understanding the proteome map of the sea bass toward establishing the animal as a potential model for muscular studies.

  15. Global Analysis of the Membrane Subproteome of Pseudomonas aeruginosa using Liquid Chromatography-Tandem Mass Spectrometry

    SciTech Connect

    Blonder, Josip; Goshe, Michael B.; Xiao, Wenzhong; Camp, David G.; Wingerd, Mark A.; Davis, Ronald W.; Smith, Richard D.

    2004-05-30

    Pseudomonas aeruginosa is one of the most significant opportunistic bacterial pathogens in humans causing infections and premature death in patients with cystic fibrosis, AIDS, severe burns, organ transplants or cancer. Liquid chromatography coupled online with tandem mass spectrometry (LC-MS/MS) was used for the large-scale proteomic analysis of the P. aeruginosa membrane subproteome. Concomitantly, an affinity labeling technique, using iodoacetyl-PEO biotin to tag cysteinyl-containing proteins, permitted the enrichment and detection of lower abundance membrane proteins. The application of these approaches resulted in the identification of 786 proteins. A total of 333 proteins (42%) had a minimum of one transmembrane domain (TMD; ranging from 1 to 14) and 195 proteins were classified as hydrophobic based on their positive GRAVY values (ranging from 0.01 to 1.32). Key integral inner and outer membrane proteins involved in adaptation and antibiotic resistance were conclusively identified, including the detection of 53% of all predicted opr-type porins (outer integral membrane proteins) and all the components of the mexA-mexB-oprM transmembrane protein complex. This work represents the most comprehensive qualitative proteomic analysis of the membrane subproteome of P. aeruginosa and for prokaryotes in general to date.

  16. Proteomic analysis of Taenia ovis metacestodes by high performance liquid chromatography-coupled tandem mass spectrometry.

    PubMed

    Zheng, Yadong

    2017-03-15

    Taenia ovis metacestodes reside in the muscle of sheep and goats, and may cause great economic loss due to condemnation of carcasses if not effectively controlled. Although advances have been made in the control of T. ovis infection, our knowledge of T. ovis biology is limited. Herein the protein profiling of T. ovis metacestodes was determined by liquid chromatography-linked tandem mass spectrometry. A total of 966 proteins were identified and 25.1% (188/748) were annotated to be associated with metabolic pathways. Consistently, GO analysis returned a metabolic process (16.27%) as one of two main biological process terms. Moreover, it was found that 24 proteins, including very low-density lipoprotein receptor, enolase, paramyosin and endophilin B1, were abundant in T. ovis metacestodes. These proteins may be associated with motility, metabolism, signaling, stress, drug resistance and immune responses. Furthermore, comparative analysis of 5 cestodes revealed the presence of Taenia-specific enolases. These data provide clues for better understanding of T. ovis biology, which is informative for effective control of infection.

  17. Accelerator mass spectrometry and radioisotope detection at the Argonne FN tandem facility

    SciTech Connect

    Henning, W.; Kutschera, W.; Paul, M.; Smither, R.K.; Stephenson, E.J.; Yntema, J.L.

    1980-01-01

    The Argonne FN tandem accelerator and standard components of its experimental heavy-ion research facility, have been used as a highly-sensitive mass spectrometer to detect several long-lived radioisotopes and measure their concentration by counting of accelerated ions. Background beams from isobaric nuclei have been eliminated by combining the dispersion from the energy loss in a uniform Al foil stack with the momentum resolution of an Enge split-pole magnetic spectrograph. Radioisotope concentrations in the following ranges have been measured: /sup 14/C//sup 12/C = 10/sup -12/ to 10/sup -13/, /sup 26/Al//sup 27/Al = 10/sup -10/ to 10/sup -12/, /sup 32/Si/Si = 10/sup -8/ to 10/sup -14/, /sup 36/Cl/Cl = 10/sup -10/ to 10/sup -11/. Particular emphasis was put on exploring to what extent the technique of identifying and counting individual ions in an accelerator beam can be conveniently used to determine nuclear quantities of interest when their measurement involves very low radioisotope concentrations. The usefulness of this method can be demonstrated by measuring the /sup 26/Mg(p,n)/sup 26/Al(7.2 x 10/sup 5/ yr) cross section at proton energies in the astrophysically interesting range just above threshold, and by determining the previously poorly known half life of /sup 32/Si.

  18. Quantification of folate metabolites in serum using ultraperformance liquid chromatography tandem mass spectrometry.

    PubMed

    Wang, Xiuwei; Zhang, Ting; Zhao, Xin; Guan, Zhen; Wang, Zhen; Zhu, Zhiqiang; Xie, Qiu; Wang, Jianhua; Niu, Bo

    2014-07-01

    Folate deficiency is considered a risk factor for many diseases such as cancer, congenital heart disease and neural tube defects (NTDs). There is a pressing need for more methods of detecting folate and its main metabolites in the human body. Here, we developed a simple, fast and sensitive ultraperformance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) method for the simultaneous quantifications of folate metabolites including folic acid, 5-methyltetrahydrofolate (5-MeTHF), 5-formyltetrahydrofolate (5-FoTHF), homocysteine (Hcy), S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). The method was validated by determining the linearity (r(2)>0.998), sensitivity (limit of detection ranged from 0.05 to 0.200ng/mL), intra- and inter-day precision (both CV<6%) and recovery (each analyte was >90%). The total analysis time was 7min. Serum samples of NTD-affected pregnancies and controls from a NTD high-risk area in China were analyzed by this method, the NTD serum samples showed lower concentrations of 5-MeTHF (P<0.05) and 5-FoTHF (P<0.05), and higher concentrations of Hcy (P<0.05) and SAH (P<0.05) compared with serum samples from controls, consistent with a previous study. These results showed that the method is sensitive and reliable for simultaneous determination of six metabolites, which might indicate potential risk factors for NTDs, aid early diagnosis and provide more insights into the pathogenesis of NTDs.

  19. Comprehensive characterization of anticoagulant rodenticides in sludge by liquid chromatography-tandem mass spectrometry.

    PubMed

    Gómez-Canela, Cristian; Lacorte, Silvia

    2016-08-01

    The occurrence of 10 commonly used anticoagulant rodenticides in centrifuged sludge of 27 wastewater treatment plants was evaluated using solid-liquid extraction (SLE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Activated carbon, alumina, and Florisil cartridges with methanol/dichloromethane as eluting solvents were tested in combination with primary-secondary amine (PSA) to optimize an efficient sample cleanup. PSA in combination with Florisil was the best methodology to extract anticoagulant rodenticides in sludge providing recoveries between 42 ± 0.5 and 100 ± 2 %. Warfarin, bromadiolone, ferulenol, and coumachlor were the most ubiquitous compounds in sludge at concentrations up to 84.2 ng g(-1) for the latter. Coumatetralyl, dicoumarol, and brodifacoum were detected sporadically at levels between 6.1 and 17.4 ng g(-1). On the contrary, acenocoumarol, difenacoum, and flocoumafen were not detected in any sample. Finally, we estimated the amount of anticoagulant rodenticides discharged via sludge in order to determine the potential impact to agricultural soil according to different sludge usage practices in the region investigated. This study demonstrates that anticoagulant rodenticides are accumulated in sludge during activated sludge treatment and that the application of sludge as fertilizers may pose a future environmental risk, if not controlled.

  20. Comprehensive characterization of rodenticides in wastewater by liquid chromatography-tandem mass spectrometry.

    PubMed

    Gómez-Canela, Cristian; Vázquez-Chica, Alberto; Lacorte, Silvia

    2014-01-01

    Rodenticides are used as pest control to eradicate rodents and have emerged as new environmental contaminants due to their widespread use in domestic and urban infrastructures. In this study, we have developed and validated an analytical methodology based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the determination of 13 anticoagulant rodenticides in wastewater. In a first step, ionization conditions were tested in electrospray mode, and positive ionization gave the highest sensitivity. Fragmentation patterns were determined and two selected reaction monitoring (SRM) transitions were selected for each compound. Using a Zorbax Eclipse XDB-C18 column and specific SRM transitions, 13 compounds were resolved. The LC-MS/MS method provided good linearity, sensitivity, intra- and inter-day precision, and good identification capabilities for these compounds in wastewaters. Thereafter miniaturized liquid-liquid extraction (LLE) and solid-phase extraction (SPE) were optimized. Oasis HLB and Strata WA SPE cartridges with methanol/dichloromethane as eluting solvents provided good recoveries and limits of detection ranged between 0.34 and 20 ng L(-1), whereas LLE failed to recover some compounds. Finally, the performance of both LLE and SPE methods was evaluated by analyzing rodenticides in a set of wastewaters. Warfarin was the only detected compound at nanogram per liter level, and good agreement was observed between LLE and SPE.

  1. Simultaneous determination of chlorpheniramine and pseudoephedrine in human plasma by liquid chromatography-tandem mass spectrometry.

    PubMed

    Chen, Xiaoyan; Zhang, Yong; Zhong, Dafang

    2004-05-01

    A sensitive and specific procedure for simultaneous quantitation of chlorpheniramine and pseudoephedrine in human plasma has been developed and validated. Analytes were extracted from plasma samples by liquid-liquid extraction, separated on a Diamonsil C18 column (250 x 4.6 mm i.d.) and detected by tandem mass spectrometry with an atmospheric pressure chemical ionization interface. Diphenhydramine was used as the internal standard. The method has a lower limit of quantitation of 0.2 and 2.0 ng/mL for chlorpheniramine and pseudoephedrine, respectively. The intra- and inter-day relative standard deviation, calculated from quality control (QC) samples were below 4.3% for chlorpheniramine and below 9.5% for pseudoephedrine. The inter-day relative error as determined from QC samples was within 4.7% for each analyte. The overall extraction recoveries of chlorpheniramine and pseudoephedrine were 77 and 61% on average, respectively. The method was successfully applied to pharmaockinetic study of chlorpheniramine and pseudoephedrine in volunteers receiving formulations containing 4 mg of chlorpheniramine maleate and 60 mg of pseudoephedrine hydrochloride.

  2. Determination of cotinine in pericardial fluid and whole blood by liquid chromatography-tandem mass spectrometry.

    PubMed

    Hegstad, S; Stray-Pedersen, A; Olsen, L; Vege, A; Rognum, T O; Mørland, J; Christophersen, A S

    2009-05-01

    Cotinine is the main metabolite of nicotine and is used as an indicator of exposure to tobacco smoke. A method has been developed for quantification of cotinine in pericardial fluid and whole blood collected from autopsy casework involving cases of infant death. Sample clean-up was achieved by solid-phase extraction with a mixed-mode column. Cotinine was quantified by liquid chromatography-tandem mass spectrometry. Positive ionization was performed in the multiple reaction monitoring mode. Two transitions were monitored for the analyte and one for the internal standard, cotinine-d(3). The calibration range was 0.9-176 ng/mL for cotinine in both matrixes. The recovery of the analyte ranged from 86 to 92%, and the between-assay precisions ranged from 4 to 6% relative standard deviation. Whole blood and pericardial fluid samples from 95 infant deaths obtained during autopsy were analyzed. A strong correlation (R(2) = 0.97) was found between the cotinine concentrations in pericardial fluid and blood. The correlation was not affected by the postmortem time interval. This study demonstrates that pericardial fluid may be an alternative specimen to blood for quantification of cotinine in forensic autopsies.

  3. Development of a dedicated peptide tandem mass spectral library for conservation science.

    PubMed

    Fremout, Wim; Dhaenens, Maarten; Saverwyns, Steven; Sanyova, Jana; Vandenabeele, Peter; Deforce, Dieter; Moens, Luc

    2012-05-30

    In recent years, the use of liquid chromatography tandem mass spectrometry (LC-MS/MS) on tryptic digests of cultural heritage objects has attracted much attention. It allows for unambiguous identification of peptides and proteins, and even in complex mixtures species-specific identification becomes feasible with minimal sample consumption. Determination of the peptides is commonly based on theoretical cleavage of known protein sequences and on comparison of the expected peptide fragments with those found in the MS/MS spectra. In this approach, complex computer programs, such as Mascot, perform well identifying known proteins, but fail when protein sequences are unknown or incomplete. Often, when trying to distinguish evolutionarily well preserved collagens of different species, Mascot lacks the required specificity. Complementary and often more accurate information on the proteins can be obtained using a reference library of MS/MS spectra of species-specific peptides. Therefore, a library dedicated to various sources of proteins in works of art was set up, with an initial focus on collagen rich materials. This paper discusses the construction and the advantages of this spectral library for conservation science, and its application on a number of samples from historical works of art.

  4. Multiclass analysis of antibiotic residues in honey by ultraperformance liquid chromatography-tandem mass spectrometry.

    PubMed

    Vidal, Jose Luis Martínez; Aguilera-Luiz, María Del Mar; Romero-González, Roberto; Frenich, Antonia Garrido

    2009-03-11

    A method has been developed and validated for the simultaneous analysis of different veterinary drug residues (macrolides, tetracyclines, quinolones, and sulfonamides) in honey. Honey samples were dissolved with Na(2)EDTA, and veterinary residues were extracted from the supernatant by solid-phase extraction (SPE), using OASIS HLB cartridges. The separation and determination was carried out by ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS), using an electrospay ionization source (ESI) in positive mode. Data acquisition under MS/MS was achieved by applying multiple reaction monitoring (MRM) of two ion transitions per compound to provide a high degree of sensitivity and specificity. The method was validated, and mean recoveries were evaluated at three concentration levels (10, 50, and 100 microg/kg), ranging from 70 to 120% except for doxycycline, erythromycin, and tylmicosin with recovery higher than 50% at the three levels assayed. Relative standard deviations (RSDs) of the recoveries were less than 20% within the intraday precision and less than 25% within the interday precision. The limits of quantification (LOQs) were always lower than 4 microg/kg. The developed procedure was applied to 16 honey samples, and erythromycin, sarafloxacin, and tylosin were found in a few samples.

  5. Assay reproducibility of serum androgen measurements using liquid chromatography-tandem mass spectrometry

    PubMed Central

    Trabert, Britton; Xu, Xia; Falk, Roni T.; Guillemette, Chantal; Stanczyk, Frank Z.; McGlynn, Katherine A.

    2015-01-01

    Background Valid and precise measures of androgen concentrations are needed for etiologic studies of hormonally-related cancers. We developed a high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with two sample preparations to measure 11 androgens, including adrenal and gonadal androgenic precursors and their 5α-reduced metabolites. Methods Androgen levels were measured in serum from 20 healthy volunteers (5 men, 10 premenopausal women, 5 postmenopausal women). Two blinded, randomized aliquots per individual were assayed in each of three batches. A fourth batch of samples was measured at an external laboratory using comparable methodology to measure 9 of the 11 androgens. Coefficients of variation (CV) and intraclass correlation coefficients (ICC) were calculated from the individual components of variance. Comparability of 9 androgens across laboratories was assessed using Spearman ranked correlations, Deming regression and bias plots. Results The laboratory CVs were <5% and ICCs were uniformly high (>95%) for all androgens measured across sex/menopausal status groups. Spearman ranked correlations for 9 hormones measured in the comparison laboratory were high (>0.85), suggesting good agreement. Conclusion Our high-performance LC-MS/MS assays of 11 androgens, including adrenal and gonadal androgenic precursors and their 5α-reduced metabolites demonstrated excellent laboratory reproducibility, and good comparability with an established method that measured 9 of the 11 hormones tested. The serum androgen metabolite assays are suitable for use in epidemiologic research. PMID:26416142

  6. Multiresidue analysis of environmental pollutants in edible vegetable oils by gas chromatography-tandem mass spectrometry.

    PubMed

    Zhou, Rui-Ze; Jiang, Jie; Mao, Ting; Zhao, Ya-Song; Lu, Yong

    2016-09-15

    A novel multiresidue determination of polycyclic aromatic hydrocarbons (PAHs), phthalate esters (PAEs) and alkylphenols (APs) in edible vegetable oils was developed. The samples were extracted with hexane-saturated acetonitrile, and after concentration, the extract was directly qualitatively and quantitatively analyzed by gas chromatography-tandem mass spectrometry (GC-MS/MS) with multiple reaction monitoring (MRM) in positive ion mode. The calibration curve displayed good linearity in the range of 2-100 μg/L, with correlation coefficients greater than 0.99. The mean recoveries were 70.0-110.8% by analysis of spiked oil, and the relative standard deviations (RSDs) were 2.1-10.2% (n=6), respectively. The limits of detection (LODs) for the 23 PAHs, 17 PAEs and 3 APs were 0.1-1.0 μg/kg, 0.1-4.0 μg/kg and 1.2-3.0 μg/kg, respectively. The established method effectively avoided interference from large amounts of lipids and pigments. It was applied to real sample and shown to be a rapid and reliable alternative for determination and confirmation in routine analysis.

  7. Determination of benidipine in human plasma using liquid chromatography-tandem mass spectrometry.

    PubMed

    Kang, Wonku; Yun, Hwi-Yeol; Liu, Kwang-Hyeon; Kwon, Kwang-Il; Shin, Jae-Gook

    2004-06-15

    We developed a method for determining benidipine, a dihydropyridine analogue calcium-channel blocker, in plasma using liquid chromatography-tandem mass spectrometry (LC-MS-MS). Benidipine and benidipine-d5, an internal standard, were extracted from plasma using diethyl ether in the presence of 5M NaOH. After drying the organic layer, the residue was reconstituted in acetonitrile and injected onto a reversed-phase C18 column. The isocratic mobile phase (acetonitrile-5mM ammonium acetate, 90:10, v/v) was eluted at 0.2 ml/min. The ion transitions monitored in multiple reaction-monitoring mode were m/z 506-174 for benidipine and m/z 511-179 for the internal standard. The coefficient of variation of the assay precision was less than 13%, and the accuracy exceeded 92%, except at the limit of quantification, 0.05 ng/ml with 1ml of plasma, when it was 85%. This method was used to measure the benidipine concentration in plasma from healthy subjects after a single 4-mg oral dose of benidipine. This method is a very simple, sensitive, and accurate way to determine the plasma benidipine concentration.

  8. Measurement of trimethylamine-N-oxide by stable isotope dilution liquid chromatography tandem mass spectrometry

    PubMed Central

    Wang, Zeneng; Levison, Bruce S.; Hazen, Jennie E.; Donahue, Lillian; Li, Xin-Min; Hazen, Stanley L.

    2014-01-01

    Trimethylamine-N-oxide (TMAO) levels in blood predict future risk for major adverse cardiac events including myocardial infarction, stroke and death. Thus, the rapid determination of circulating TMAO concentration is of clinical interest. Here we report a method to measure TMAO in biological matrices by stable isotope dilution liquid chromatography tandem mass spectrometry (LC/MS/MS) with lower and upper limits of quantification of 0.05 and >200 µM, respectively. Spike and recovery studies demonstrate an accuracy at low (0.5 µM), mid (5 µM) and high (100 µM) levels of 98.2%, 97.3% and 101.6%, respectively. Additional assay performance metrics include intra-day and inter-day coefficients of variance of < 6.4% and < 9.9%, respectively, across the range of TMAO levels. Stability studies reveal TMAO in plasma is stable both during storage at −80 °C for 5 years and to multiple freeze thaw cycles. Fasting plasma normal range studies among apparently healthy subjects (n=349) shows a range of 0.73 – 126 µM, median (interquartile range) levels of 3.45 (2.25–5.79) µM, and increasing values with age. The LC/MS/MS based assay reported should be of value for further studies evaluating TMAO as a risk marker and for examining the effect of dietary, pharmacologic and environmental factors on TMAO levels. PMID:24704102

  9. Identification of Microorganisms by High Resolution Tandem Mass Spectrometry with Accurate Statistical Significance

    NASA Astrophysics Data System (ADS)

    Alves, Gelio; Wang, Guanghui; Ogurtsov, Aleksey Y.; Drake, Steven K.; Gucek, Marjan; Suffredini, Anthony F.; Sacks, David B.; Yu, Yi-Kuo

    2016-02-01

    Correct and rapid identification of microorganisms is the key to the success of many important applications in health and safety, including, but not limited to, infection treatment, food safety, and biodefense. With the advance of mass spectrometry (MS) technology, the speed of identification can be greatly improved. However, the increasing number of microbes sequenced is challenging correct microbial identification because of the large number of choices present. To properly disentangle candidate microbes, one needs to go beyond apparent morphology or simple `fingerprinting'; to correctly prioritize the candidate microbes, one needs to have accurate statistical significance in microbial identification. We meet these challenges by using peptidome profiles of microbes to better separate them and by designing an analysis method that yields accurate statistical significance. Here, we present an analysis pipeline that uses tandem MS (MS/MS) spectra for microbial identification or classification. We have demonstrated, using MS/MS data of 81 samples, each composed of a single known microorganism, that the proposed pipeline can correctly identify microorganisms at least at the genus and species levels. We have also shown that the proposed pipeline computes accurate statistical significances, i.e., E-values for identified peptides and unified E-values for identified microorganisms. The proposed analysis pipeline has been implemented in MiCId, a freely available software for Microorganism Classification and Identification. MiCId is available for download at http://www.ncbi.nlm.nih.gov/CBBresearch/Yu/downloads.html.

  10. Analysis of amphetamines and metabolites in urine with ultra performance liquid chromatography tandem mass spectrometry.

    PubMed

    Ramírez Fernández, María del Mar; Wille, Sarah M R; di Fazio, Vincent; Gosselin, Matthias; Samyn, Nele

    2010-06-01

    A simple, rapid and sensitive ultra performance liquid chromatography tandem mass spectrometry method was developed and fully validated for the quantitative determination of seven amphetamines and metabolites in urine. The method was validated for selectivity, linearity, LOQ, LOD, imprecision, bias, analyte and processed sample stability, matrix effect, recovery, carryover and dilution integrity. A classic liquid-liquid extraction with ethyl acetate was used as sample preparation procedure. The compounds were separated on an Acquity UPLC HSS C18 column in 6.8 min. The linear dynamic range was established from 25 to 500 ng/mL. The limit of quantification was fixed to the lowest calibrator level and the limit of detection ranged from 0.125 to 2.5 ng/mL. The method presented an excellent intra- and inter-assay imprecision and bias (<10.7%) at each measured concentration of two external quality controls (QC) and three "in house" QC. No matrix effects were observed and good recoveries (>70%) were obtained for all the compounds. No carryover was observed after the analysis of high concentrated samples (8000 ng/mL). The method was subsequently applied to authentic samples.

  11. Determination of Cranberry Phenolic Metabolites in Rats by Liquid Chromatography–Tandem Mass Spectrometry

    PubMed Central

    Rajbhandari, Rajani; Peng, Ning; Moore, Ray; Arabshahi, Alireza; Wyss, J. Michael; Barnes, Stephen; Prasain, Jeevan K

    2011-01-01

    The glycosides of flavonoid, anthocyanins and A type proanthocyanidins in cranberry concentrate were characterized and quantified using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Cranberry concentrate (1 g/body weight) was orally gavaged to Fischer-344 rats (n = 6), and blood and urine samples were collected over 24 h periods. Quercetin, 3′-O-methylquercetin (isorhamnetin), myricetin, kaempferol, and proanthocyanidin dimer A2, together with thirteen conjugated metabolites of quercetin and methylquercetin and intact peonidin 3-O-galactoside and cyanidin 3-O-galactoside were identified in the rat urine after cranberry treatment. Very low levels of isorhamnetin (0.48 ± 0.09 ng/mL) and proanthocyanidin dimer A2 (0.541 ± 0.10 ng/mL) were found in plasma samples after 1 h of cranberry administration. Although no quercetin was detected in plasma, MRM analysis of the methanolic extract of urinary bladder showed that chronic administration of cranberry concentrate to rats resulted in accumulation of quercetin and isorhamnetin in the bladder. These results demonstrate that cranberry components undergo rapid metabolism and elimination into the urine of rats and are present in the urinary bladder tissue potentially allowing them to inhibit urinary bladder carcinogenesis. PMID:21634376

  12. Determination of cosmogenic Ca-41 in a meteorite with tandem accelerator mass spectrometry

    NASA Technical Reports Server (NTRS)

    Kubik, P. W.; Elmore, D.; Conard, N. J.; Nishiizumi, K.; Arnold, J. R.

    1986-01-01

    The first use of tandem accelerator mass spectrometry (TAMS) to measure the content of Ca-41 in a natural sample, the iron Bogou meteorite, is reported. Ca in the samples was extracted by hydroxide precipitation and purified by means of a caution exchange resin (AG 50W-X8). After adding 4 percent ammonium oxide, the precipitate was ignited to CaO in a quartz vial at about 1100 C. The Ca-41/Ca ratios were determined following acceleration by alternate measurements of the Ca-40 beam current in an image Faraday cup. Ca-41 particles were also measured using a gas counter. The measured Ca-41/Ca ratio was 3.8 + or -0.6 x 10 to the 12th, which corresponds to a Ca-41 activity of 6.9 + or -1.1 d.p.m. per kg. Calculation of the half-life of Ca-41 in the Bogou meteorite yielded an age of 103,000 years.

  13. [Determination of pesticides in Chinese dumplings using liquid chromatography-tandem mass spectrometry].

    PubMed

    Okamoto, You; Takatori, Satoshi; Kitagawa, Yoko; Okihashi, Masahiro; Fukui, Naoki; Murata, Hiroshi; Sumimoto, Tatsuo; Tanaka, Yukio; Obana, Hirotaka

    2009-02-01

    A rapid and easy multiresidue method for determination of pesticide residues in Chinese dumplings using liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed. Pesticide residues were extracted with ethyl acetate in the presence of anhydrous magnesium sulfate in a disposable tube using a homogenizer. The extract was concentrated and reconstituted in hexane, followed by acetonitrile-hexane partition to remove lipids. The acetonitrile layer was purified with a double-layered cartridge column (graphite carbon black/primary secondary amine silica gel). After removal of the solvent, the extract was resolved in methanol/water and analyzed with LC-MS/MS. Recovery tests of 99 pesticide residues from Chinese dumpling were performed at 20 and 100 ng/g, and 72 pesticides exhibited acceptable recoveries (70-120%) with low relative standard deviations (<20%) at both concentrations. The time for sample preparation with 12 samples to test solutions was approximately 6 hr. This method could be useful for determination of pesticide residues in the Chinese dumplings.

  14. [Determination of 25 quinolones in cosmetics by liquid chromatography-tandem mass spectrometry].

    PubMed

    Lin, Li; Zhang, Yi; Tu, Xiaoke; Xie, Liqi; Yue, Zhenfeng; Kang, Haining; Wu, Weidong; Luo, Yao

    2015-03-01

    An analytical method was developed for the simultaneous determination of 25 quinolones, including danofloxacin mesylate, enrofloxacin, flumequine, oxloinic acid, ciprofloxacin, sarafloxacin, nalidixic acid, norfloxacin, and ofloxacin etc in cosmetics using direct extraction and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Cosmetic sample was extracted by acidified acetonitrile, defatted by n-hexane and separated on Poroshell EC-C18 column with gradient elution program using acetonitrile and water (both containing 0. 1% formic acid) as the mobile phases and analyzed by LC-ESI-MS/MS under the positive mode using multiple reaction monitoring (MRM). The interference of matrix was reduced by the matrix-matched calibration standard curve. The method showed good linearities over the range of 1-200 mg/kg for the 25 quinolones with good linear correlation coefficients (r ≥ 0.999). The method detection limit of the 25 quinolones was 1.0 mg/kg, and the recoveries of all analytes in lotion, milky and cream cosmetics matrices ranged from 87.4% to 105% at the spiked levels of 1, 5 and 10 mg/kg with the relative standard deviations (RSD) of 4.54%-19.7% (n = 6). The results indicated that this method is simple, fast and credible, and suitable for the simultaneous determination of the quinolones in the above three types of cosmetics.

  15. Simultaneous determination of beta-blockers in human plasma using liquid chromatography-tandem mass spectrometry.

    PubMed

    Umezawa, Hironobu; Lee, Xiao-Pen; Arima, Yoshiko; Hasegawa, Chika; Izawa, Hikaru; Kumazawa, Takeshi; Sato, Keizo

    2008-07-01

    A detailed procedure for the analysis of four beta-blockers, acebutolol, labetalol, metoprolol and propranolol, in human plasma by high-performance liquid chromatography (LC)-tandem mass spectrometry (MS-MS) using an MSpak GF column, which enables direct injection of crude plasma samples, is presented. Protein and/or macromolecule matrix compounds were eluted first from the column, while the drugs were retained on the polymer stationary phase of the MSpak GF column. The analytes retained on the column were then eluted into an acetonitrile-rich mobile phase using a gradient separation technique. All drugs showed base peak ions due to [M + H]+ ions by LC-MS with positive ion electrospray ionization, and the product ions were produced from each [M + H]+ ion by LC-MS-MS. Quantification was performed by selected reaction monitoring. The recoveries of the four beta-blockers spiked into plasma were 73.5-89.9%. The regression equations for all compounds showed excellent linearity in the range 10-1000 ng/mL of plasma, with the exception of propranolol (10-800 ng/mL). The limits of detection and quantification for each drug were 1-3 and 10 ng/mL, respectively, of plasma. The intra- and inter-day coefficients of variation for all drugs in plasma were not greater than 10.9%.

  16. Tandem Mass Spectrometry for Characterization of Covalent Adducts of DNA with Anti-cancer Therapeutics

    PubMed Central

    Silvestri, Catherine; Brodbelt, Jennifer S.

    2012-01-01

    The chemotherapeutic activities of many anticancer and antibacterial drugs arise from their interactions with nucleic acid substrates. Some of these ligands interact with DNA in a way that causes conformational changes or damage to the nucleic acid targets, ultimately altering recognition by key DNA-specific enzymes, interfering with DNA transcription or prohibiting replication, and terminating cell growth and proliferation. The design and synthesis of ligands that bind to nucleic acids remains a dynamic field in medicinal chemistry and pharmaceutical research. The quest for more selective and efficacious DNA-interactive anti-cancer chemotherapeutics has likewise catalyzed the need for sensitive analytical methods that can provide structural information about the nature of the resulting DNA adducts and provide insight into the mechanistic pathways of the DNA/drug interactions and the impact on the cellular processes in biological systems. This review focuses on the array of tandem mass spectrometric strategies developed and applied for characterization of covalent adducts formed between DNA and anti-cancer ligands. PMID:23150278

  17. Determination of ractopamine in pig hair using liquid chromatography with tandem mass spectrometric detection.

    PubMed

    Wu, Junlin; Liu, Xiaoyun; Peng, Yunping

    2014-01-01

    A quantitative analytical procedure for the determination of ractopamine in pig hair has been developed and validated. The hair samples were washed and incubated at 75°C with isoxuprine and hair extraction buffer. The drug present was quantified using mixed solid-phase extraction and liquid chromatography with tandem mass spectrometric detection. The limit of quantization (LOQ) was 10pg/mg and the intra-day precision at 25pg/mg and 750pg/mg was 0.49% and 2.8% respectively. Inter-day precision was 0.88% and 3.52% at the same concentrations. The hair extraction percentage recovery at 25pg/mg and 50ng/mL was 99.47% and 103.83% respectively. The extraction percentage recovery at 25pg/mg and 50ng/mg was 93.52% and 100.26% respectively. Our results showed that ractopamine residues persist in hair in 24days of withdrawal and also showed the possibility to test ractopamine from pig hair samples.

  18. Determination of pyrolysis products of smoked methamphetamine mixed with tobacco by tandem mass spectrometry.

    PubMed

    Lee, M R; Jeng, J; Hsiang, W S; Hwang, B H

    1999-01-01

    This study examines the pyrolysis products of smoked methamphetamine mixed with tobacco that was trapped with a C8 adsorbent cartridge and then detected by gas chromatography-tandem mass spectrometry. According to the results, the mainstream smoke contains 2-methylpropyl-benzene, 2-chloropropyl-benzene, 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one, 3-ethyl-phenol, methamphetamine, dimethylamphetamine, hydroquinone, 3-methyl-5-(1-methylethyl)-methylcarbamate phenol, N-methyl-N-(2-phenylethyl)-acetamide, 4-(3-hydroxy-1-butenyl)-3,5,5-trimethyl-2-cyclohexene-1-one, propanoic acid, N-acetylmethamphetamine, phenyl ester, and furfurylmethylamphetamine. In addition, the compounds in sidestream smoke are 2-propenyl benzene, phenylacetone, methamphetamine, dimethylamphetamine, benzyl methyl ketoxime, 3,4-dihydro-2-naphthalenone, N-folmyamphetamine, N-acetylamphetamine, bibenzyl, N-folmylmethamphetamine, N-acetylmethamphetamine, N-propionymethamphetamine, and furfurylmethylamphetamine. Moreover, the presence of methamphetamine promotes the oxidation of the tobacco components.

  19. Quantification of plasma homocitrulline using hydrophilic interaction liquid chromatography (HILIC) coupled to tandem mass spectrometry.

    PubMed

    Jaisson, Stéphane; Gorisse, Laëtitia; Pietrement, Christine; Gillery, Philippe

    2012-02-01

    Homocitrulline (HCit), an amino acid formed by the carbamylation of ε-amino groups of lysine residues, is considered a promising biomarker for monitoring diseases such as chronic renal failure and atherosclerosis. This paper describes a tandem mass spectrometric method for total, protein-bound and free HCit measurement in plasma samples. HCit was separated from other plasma components by hydrophilic interaction liquid chromatography. Detection was achieved by monitoring transitions of 190.1 > 127.1 and 190.1 > 173.1 for HCit, and 183.1 > 120.2 for d(7)-citrulline used as internal standard. This method allowed HCit quantification within 5.2 min and was precise (inter-assay CV < 5.85%), accurate (mean recoveries ranging from 97% to 106%), and exhibited a good linearity from 10 nmol/L to 1.6 μmol/L. Plasma samples from control and uremic mice (n = 10) were analyzed. In control mice, mean total plasma HCit concentration was 0.78 ± 0.12 μmol/mol amino acids, whereas it was increased 2.7-fold in uremic mice plasma, reaching 2.10 ± 0.50 μmol/mol amino acids (p < 0.001). In conclusion, this method exhibits good analytical performances and meets the criteria of sensitivity suitable for HCit concentration assessment in plasma samples.

  20. Determination of sulfonamides in beeswax by liquid chromatography coupled to tandem mass spectrometry.

    PubMed

    Mitrowska, Kamila; Antczak, Maja

    2015-12-01

    The manuscript presents the development of a new method for the quantification of 16 sulfonamides in beeswax. Different sample preparation techniques were tested and modified to maximise the recovery of the target analytes and minimise the amount of coeluted impurities under conditions that provide reproducible results. The proposed method consisted of melting and dilution of beeswax in a mixture of n-hexane and isopropanol followed by extraction with 2% acetic acid. The extract was cleaned up by solid-phase extraction using strong cation exchange phase. Determination of the sulfonamides was achieved by liquid chromatography coupled to tandem mass spectrometry with the use of a pentafluorophenyl analytical column and applying a gradient elution with acetonitrile and 0.01% acetic acid as mobile phases. The limits of detection and limits of quantification ranged from 1 to 2μg/kg and from 2 to 5μg/kg, respectively. The recoveries varied between 65.2% and 117.8% while coefficient of variation of the method was less than 24.2% under intermediate precision conditions. Finally, the method was applied to the analysis of real samples of beeswax from beekeepers and commercial foundations manufacturers.

  1. Determination of domoic acid in seawater and phytoplankton by liquid chromatography-tandem mass spectrometry.

    PubMed

    Wang, Zhihong; King, Kristen L; Ramsdell, John S; Doucette, Gregory J

    2007-09-07

    Domoic acid (DA) is an algal neurotoxin produced by diatoms primarily of the genus Pseudo-nitzschia and is responsible for the human intoxication syndrome known as amnesic shellfish poisoning. A method has been developed to determine DA in seawater and phytoplankton matrices by liquid chromatography-tandem mass spectrometry for both quantitation and confirmation purposes. Sample extraction and clean-up was achieved on a C18 solid-phase extraction (SPE) cartridge. An acidic condition is critical for retaining hydrophilic DA on the cartridge. Direct injection of SPE eluate for analysis is recommended in order to avoid loss of DA by drying with heat prior to resuspension and injection. DA was quantified using the fragments produced from the protonated DA ion through multiple reaction monitoring (MRM). Recoveries exceeded 90% for all seawater samples spiked with DA and approximated 98% of toxin standard added to cultured phytoplankton material. Acceptable reproducibility (ca. 5% or less) was obtained for all intra-day and inter-day samples. The detection limit was 30 pg/ml level with a 20 microl injection volume, which demonstrated the value of this method for not only confirming DA production by minimally toxic phytoplankton species, but also for investigating the potentially important role of dissolved DA in marine food webs.

  2. Metabolism profiles of nuciferine in rats using ultrafast liquid chromatography with tandem mass spectrometry.

    PubMed

    Ye, Lin-Hu; Xiao, Bing-Xin; Liao, Yong-Hong; Liu, Xin-Min; Pan, Rui-Le; Chang, Qi

    2016-08-01

    Nuciferine (NF) is one of the main aporphine alkaloids existing in the traditional Chinese medicine Folium Nelumbinis (lotus leaves). Modern pharmacological studies have demonstrated that NF has a broad spectrum of bioactivities, such as anti-HIV and anti-hyperlipidemic effects, and has been recommended as a leading compound for new drug development. However, the metabolites and biotransformation pathway of NF in vivo have not yet been comprehensively investigated. The present study was performed to identify the metabolites of NF for exploring in vivo fates. Rat plasma and urine samples were collected after oral administration and prepared by liquid-liquid extraction with ethyl acetate. A method based on ultrafast liquid chromatography with tandem mass spectrometry was applied to identify the metabolites. Q1 (first quadrupole) full scan combined with a multiple reaction monitoring (MRM) survey scan were used for the detection of metabolites. MRM-information-dependent acquisition of enhanced product ions was used for the structural identification of detected metabolites. A total of 10 metabolites were identified, including phase I (demethylation, oxidation and dehydrogenation) and phase II (glucuronidation, sulfation and glutathione) biotransformation products. Demethylation is the main metabolic pathway of NF in the body. These results can help in improving understanding of the disposition and pharmacological mechanism of NF in the body. Copyright © 2016 John Wiley & Sons, Ltd.

  3. Electrospray tandem mass spectrometry of alkali-cationized BocN-carbo-alpha,beta- and -beta,alpha-peptides: Differentiation of positional isomers.

    PubMed

    Reddy, P Nagi; Srikanth, R; Srinivas, R; Sharma, V U M; Sharma, G V M; Nagendar, P

    2006-01-01

    Dissociation pathways of a series of alkali-cationized hybrid peptides, viz., Boc-alpha,beta- and -beta,alpha-carbopeptides, synthesized from C-linked carbo-beta3-amino acids [Caa (S)] and alpha-alanine (L-Ala), have been investigated by electrospray ionization tandem mass spectrometry. The positional isomers (six pairs) of the cationized alpha,beta- and beta,alpha-peptides can be differentiated by the collision-induced dissociation (CID) spectra of their [M + Cat-Boc + H]+ ions which give characteristic series of alkali-cationized C- (x(n)+, y(n)+, z(n)+) and N-terminal (a(n)+, b(n)+, c(n)+) ions. Another noteworthy difference is cationized beta,alpha-peptides eliminate a molecule of ammonia whereas this pathway is absent for alpha,beta-peptides. This is useful for identifying the presence of a beta-amino acid at the N-terminus. The CID spectra of [M + Cat-Boc + H]+ ions of these peptide acids show abundant rearrangement [b(n) + 17 + Cat]+ (n = 1 to n-1) ions which is diagnostic for distinguishing between alpha- and beta-amino acid at the C-terminus. MS(n) experiments of [b(n) + Li-H]+ ions from these hybrid peptides showed the loss of CO and 72 u giving rise to [a(n) + Li-H]+ and cationized nitrile product ions which render support to earlier proposals that b(n)+ or [b(n) + Cat-H]+ ions have protonated or cationized oxazolinone structures, respectively.

  4. Analysis of Mammalian Sphingolipids by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) and Tissue Imaging Mass Spectrometry (TIMS)

    PubMed Central

    Sullards, M. Cameron; Liu, Ying; Chen, Yanfeng; Merrill, Alfred H.

    2011-01-01

    Sphingolipids are a highly diverse category of molecules that serve not only as components of biological structures but also as regulators of numerous cell functions. Because so many of the structural features of sphingolipids give rise to their biological activity, there is a need for comprehensive or “sphingolipidomic” methods for identification and quantitation of as many individual subspecies as possible. This review defines sphingolipids as a class, briefly discusses classical methods for their analysis, and focuses primarily on liquid chromatography tandem mass spectrometry (LC-MS/MS) and tissue imaging mass spectrometry (TIMS). Recently, a set of evolving and expanding methods have been developed and rigorously validated for the extraction, identification, separation, and quantitation of sphingolipids by LC-MS/MS. Quantitation of these biomolecules is made possible via the use of an internal standard cocktail. The compounds that can be readily analyzed are free long-chain (sphingoid) bases, sphingoid base 1-phosphates, and more complex species such as ceramides, ceramide 1-phosphates, sphingomyelins, mono- and di-hexosylceramides sulfatides, and novel compounds such as the 1-deoxy- and 1-(deoxymethyl)-sphingoid bases and their N-acyl-derivatives. These methods can be altered slightly to separate and quantitate isomeric species such as glucosyl/galactosylceramide. Because these techniques require the extraction of sphingolipids from their native environment, any information regarding their localization in histological slices is lost. Therefore, this review also describes methods for TIMS. This technique has been shown to be a powerful tool to determine the localization of individual molecular species of sphingolipids directly from tissue slices. PMID:21749933

  5. Evaluation of microdosing to assess pharmacokinetic linearity in rats using liquid chromatography-tandem mass spectrometry.

    PubMed

    Balani, Suresh K; Nagaraja, Nelamangala V; Qian, Mark G; Costa, Arnaldo O; Daniels, J Scott; Yang, Hua; Shimoga, Prakash R; Wu, Jing-Tao; Gan, Liang-Shang; Lee, Frank W; Miwa, Gerald T

    2006-03-01

    The microdosing strategy allows for early assessment of human pharmacokinetics of new chemical entities using more limited safety assessment requirements than those requisite for a conventional phase I program. The current choice for evaluating microdosing is accelerator mass spectrometry (AMS) due to its ultrasensitivity for detecting radiotracers. However, the AMS technique is still expensive to be used routinely and requires the preparation of radiolabeled compounds. This report describes a feasibility study with conventional liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology for oral microdosing assessment in rats, a commonly used preclinical species. The nonlabeled drugs fluconazole and tolbutamide were studied because of their similar pharmacokinetics characteristics in rats and humans. We demonstrate that pharmacokinetics can be readily characterized by LC-MS/MS at a microdose of 1 microg/kg for these molecules in rats, and, hence, LC-MS/MS should be adequate in human microdosing studies. The studies also exhibit linearity in exposure between the microdose and >or=1000-fold higher doses in rats for these drugs, which are known to show a linear dose-exposure relationship in the clinic, further substantiating the potential utility of LC-MS/MS in defining pharmacokinetics from the microdose of drugs. These data should increase confidence in the use of LC-MS/MS in microdose pharmacokinetics studies of new chemical entities in humans. Application of this approach is also described for an investigational compound, MLNX, in which the pharmacokinetics in rats were determined to be nonlinear, suggesting that MLNX pharmacokinetics at microdoses in humans also might not reflect those at the therapeutic doses. These preclinical studies demonstrate the potential applicability of using traditional LC-MS/MS for microdose pharmacokinetic assessment in humans.

  6. Determination of bedaquiline in human serum using liquid chromatography-tandem mass spectrometry.

    PubMed

    Alffenaar, Jan-Willem C; Bolhuis, Mathieu; van Hateren, Kai; Sturkenboom, Marieke; Akkerman, Onno; de Lange, Wiel; Greijdanus, Ben; van der Werf, Tjip; Touw, Daan

    2015-09-01

    Bedaquiline, a diarylquinoline for the treatment of multidrug-resistant tuberculosis (TB), relies on exposure-dependent killing. As data on drug exposure in specific populations are scarce, pharmacokinetic studies may be of interest. No simple and robust validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been reported to date. Therefore, a new method using a quadrupole mass spectrometer was developed for analysis of bedaquiline and N-monodesmethyl bedaquiline (M2) in human serum, using deuterated bedaquiline as the internal standard. The calibration curve was linear over a range of 0.05 (lower limit of quantification [LLOQ]) to 6.00 mg/liter for both bedaquiline and M2, with correlation coefficient values of 0.997 and 0.999, respectively. The calculated accuracy ranged from 1.9% to 13.6% for bedaquiline and 2.9% to 8.5% for M2. Within-run precision ranged from 3.0% to 7.2% for bedaquiline and 3.1% to 5.2% for M2, and between-run precision ranged from 0.0% to 4.3% for bedaquiline and 0.0% to 4.6% for M2. Evaluation of serum concentrations in a patient receiving bedaquiline showed high levels at the end of treatment, reflecting accumulation of the drug. More observational pharmacokinetic data are needed to relate altered drug concentrations to clinical outcome or adverse drug effects. A simple LC-MS/MS method to quantify bedaquiline and M2 levels in human serum using a deuterated internal standard has been validated. This method can be used in clinical studies and daily practice.

  7. Characterisation of polyacetylenes isolated from carrot (Daucus carota) extracts by negative ion tandem mass spectrometry.

    PubMed

    Rai, Dilip K; Brunton, Nigel P; Koidis, Anastasios; Rawson, Ashish; McLoughlin, Padraig; Griffiths, William J

    2011-08-15

    The potential use of negative electrospray ionisation mass spectrometry (ESI-MS) in the characterisation of the three polyacetylenes common in carrots (Daucus carota) has been assessed. The MS scans have demonstrated that the polyacetylenes undergo a modest degree of in-source decomposition in the negative ionisation mode while the positive ionisation mode has shown predominantly sodiated ions and no [M+H](+) ions. Tandem mass spectrometric (MS/MS) studies have shown that the polyacetylenes follow two distinct fragmentation pathways: one that involves cleavage of the C3-C4 bond and the other with cleavage of the C7-C8 bond. The cleavage of the C7-C8 bond generated product ions m/z 105.0 for falcarinol, m/z 105/107.0 for falcarindiol, m/z 147.0/149.1 for falcarindiol-3-acetate. In addition to these product ions, the transitions m/z 243.2 → 187.1 (falcarinol), m/z 259.2 → 203.1 (falcarindiol), m/z 301.2 → 255.2/203.1 (falcarindiol-3-acetate), mostly from the C3-C4 bond cleavage, can form the basis of multiple reaction monitoring (MRM)-quantitative methods which are poorly represented in the literature. The 'MS(3) ' experimental data confirmed a less pronounced homolytic cleavage site between the C11-C12 bond in the falcarinol-type polacetylenes. The optimised liquid chromatography (LC)/MS conditions have achieved a baseline chromatographic separation of the three polyacetylenes investigated within 40 min total run-time.

  8. A Machine Learning Based Approach to de novo Sequencing of Glycans from Tandem Mass Spectrometry Spectrum.

    PubMed

    Kumozaki, Shotaro; Sato, Kengo; Sakakibara, Yasubumi

    2015-01-01

    Recently, glycomics has been actively studied and various technologies for glycomics have been rapidly developed. Currently, tandem mass spectrometry (MS/MS) is one of the key experimental tools for identification of structures of oligosaccharides. MS/MS can observe MS/MS peaks of fragmented glycan ions including cross-ring ions resulting from internal cleavages, which provide valuable information to infer glycan structures. Thus, the aim of de novo sequencing of glycans is to find the most probable assignments of observed MS/MS peaks to glycan substructures without databases. However, there are few satisfiable algorithms for glycan de novo sequencing from MS/MS spectra. We present a machine learning based approach to de novo sequencing of glycans from MS/MS spectrum. First, we build a suitable model for the fragmentation of glycans including cross-ring ions, and implement a solver that employs Lagrangian relaxation with a dynamic programming technique. Then, to optimize scores for the algorithm, we introduce a machine learning technique called structured support vector machines that enable us to learn parameters including scores for cross-ring ions from training data, i.e., known glycan mass spectra. Furthermore, we implement additional constraints for core structures of well-known glycan types including N-linked glycans and O-linked glycans. This enables us to predict more accurate glycan structures if the glycan type of given spectra is known. Computational experiments show that our algorithm performs accurate de novo sequencing of glycans. The implementation of our algorithm and the datasets are available at http://glyfon.dna.bio.keio.ac.jp/.

  9. Identification and Quantification of Glucosinolates in Kimchi by Liquid Chromatography-Electrospray Tandem Mass Spectrometry

    PubMed Central

    Lee, Mi Jin; Jeong, Min Hee

    2017-01-01

    A novel and simple method for detecting five glucosinolates (glucoalyssin, gluconapin, glucobrassicanapin, glucobrassicin, and 4-methoxyglucobrassicin) in kimchi was developed using liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS). The chromatographic peaks of the five glucosinolates were successfully identified by comparing their retention times, mass spectra. The mobile phase was composed of A (acetonitrile) and B (water). As for glucosinolate, the relative quantities were found through sinigrin, and five different compounds that have not been previously discovered in kimchi were observed. Monitoring was carried out on the glucosinolate in 20 kimchis distributed in markets, and this study examined the various quality and quantity compositions of the five components. The glucoalyssin content ranged from 0.00 to 7.07 μmol/g of day weight (DW), with an average content of 0.86 μmol/g of DW, whereas the gluconapin content ranged from 0.00 to 5.85 μmol/g of DW, with an average of 1.17 μmol/g of DW. The content of glucobrassicanapin varied between 0.00 and 11.87 μmol/g of DW (average = 3.03 μmol/g of DW), whereas that of glucobrassicin varied between 0.00 and 0.42 μmol/g of DW (average = 0.06 μmol/g of DW). The 4-methoxyglucobrassicin content ranged from 0.12 to 9.36 μmol/g of DW (average = 3.52 μmol/g of DW). A comparison of the contents revealed that, in most cases, the content of 4-methoxyglucobrassicin was the highest. PMID:28298926

  10. Direct tandem mass spectrometric analysis of amino acids in plasma using fluorous derivatization and monolithic solid-phase purification.

    PubMed

    Tamashima, Erina; Hayama, Tadashi; Yoshida, Hideyuki; Imakyure, Osamu; Yamaguchi, Masatoshi; Nohta, Hitoshi

    2015-11-10

    In this study, we developed a novel direct tandem mass spectrometric method for rapid and accurate analysis of amino acids utilizing a fluorous derivatization and purification technique. Amino acids were perfluoroalkylated with 2H,2H,3H,3H-perfluoroundecan-1-al in the presence of 2-picoline borane via reductive amination. The derivatives were purified by perfluoroalkyl-modified silica-based monolithic solid-phase extraction (monolithic F-SPE), and directly analyzed by tandem mass spectrometry using electrospray ionization without liquid chromatographic separation. The perfluoroalkyl derivatives could be sufficiently distinguished from non-fluorous compounds, i.e. the biological matrix, due to their fluorous interaction. Thus, rapid and accurate determination of amino acids was accomplished. The method was validated with human plasma samples and applied to the analysis of amino acids in the plasma of mice with maple syrup urine disease or phenylketonuria.

  11. Development of a high sensitivity bioanalytical method for alprazolam using ultra-performance liquid chromatography/tandem mass spectrometry.

    PubMed

    Mather, Joanne; Rainville, Paul D; Potts, Warren B; Smith, Norman W; Plumb, Robert S

    2010-01-01

    A rapid, specific, assay was developed for the benzodiapine alprazolam in rat plasma using sub-2 µm particle liquid chromatography (LC) and tandem quadrupole mass spectrometry (MS/MS). The limit of quantification using protein precipitation was determined to 10 pg/mL, whereas the limit of quantification using solid-phase extraction (SPE) was determined to be 1.0 pg/mL. The assay was optimized for throughput and resolution of the analyte of interest from the hydroxy metabolite. During the method development process the plasma matrix signal was monitored, for lipids and other endogenous metabolites, to maximize signal response and minimize ion suppression. This was achieved by using a tandem quadrupole mass spectrometer equipped with a novel collision cell design which allowed for the simultaneous collection of full scan MS and multiple reaction monitoring (MRM) data. The lipid profile from the SPE process was significantly less than obtained with the protein precipitation approach.

  12. Structure of Lipid A from Pseudomonas corrugata by electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

    PubMed

    Corsaro, M M; Piaz, F Dal; Lanzetta, R; Naldi, T; Parrilli, M

    2004-01-01

    The use of the electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-QTOFMS) technique for the structural determination of Lipid A from Pseudomonas corrugata is described. This technique appears to be more sensitive with respect to other commonly used tandem mass spectrometric approaches, and was very valuable in the structural determination of the highly heterogeneous Lipid A fractions. The Lipid A fraction consists mainly of a pentaacyl component in which 3-hydroxydecanoyl [10:0(3-OH)] and 3-hydroxydodecanoyl [12:0(3-OH)] are linked as primary acyl substituents to the classical bisphosphorylated beta-(1' --> 6)-linked D-glucosamine disaccharide. Secondary substitution of N-acyl fatty acids with dodecanoyl residues [12:0] and/or its 2-OH derivatives was also observed.

  13. Free amino acids analysis by liquid chromatography with tandem mass spectrometry in several botanicals with antioxidant character.

    PubMed

    Moldoveanu, Serban C; Zhu, Jeff; Qian, Nancy

    2015-07-01

    A novel method based on liquid chromatography with tandem mass spectrometry for the analysis of 19 amino acids in plant materials is described. For the analysis, the plant material is extracted with 0.1 N hydrochloric acid with internal standards present in the extraction solution. The filtered extracts are injected using no clean-up into the liquid chromatographic system coupled with a triple-quadrupole tandem mass spectrometer with an electrospray ionization source. The analytes are separated using ion pair chromatography on a reversed-phase column. The detection is performed in multiple-reaction monitoring positive-ion mode. Quantitation is obtained using calibrations. The validated procedure has been applied for the analysis of amino acids in 18 samples of plant material including botanicals with antioxidant character. The analysis requires 16 min separation time, has excellent precision and accuracy allowing amino acid analysis in a wide range of concentrations.

  14. Enhancing capillary liquid chromatography/tandem mass spectrometry of biogenic amines by pre-column derivatization with 7-fluoro-4-nitrobenzoxadiazole.

    PubMed

    Song, Yaru; Quan, Zhe; Evans, Joseph L; Byrd, Edward A; Liu, Yi-Ming

    2004-01-01

    This paper describes a capillary liquid chromatography/tandem mass spectrometry (LC/MS/MS) determination of biogenic amines enhanced by pre-column derivatization with 7-fluoro-4-nitrobenzoxadiazole (NBD-F). Biogenic amines including tryptamine, N-methylsalsolinol, histamine, and agmatine were studied. The biogenic NBD-amine derivatives could be quantitatively enriched in-line on 20 x 0.25 mm capillary columns packed in-house with 5 microm C(8) silica particles. In an electrospray ionization (ESI) source these derivatives were ionized effectively, and collision-induced dissociation (CID) produced predominant characteristic ions allowing sensitive MS/MS detection. Agmatine, a potential neurotransmitter/modulator, was taken as a reference compound to study the analytical figures of merit of the procedure. The detection limit of agmatine was estimated to be 0.6 ng/mL (signal-to-noise (S/N) = 3). A linear calibration curve in the range 15-1000 ng/mL agmatine with an r value of 0.9997 was obtained. Tissue samples of rat brain, stomach, and intestine were analyzed. Minimum sample pre-treatment was needed. Each analysis was accomplished within ca. 12 min. The concentration of agmatine was found to be 0.246, 3.31, and 0.058 microg/g wet tissue in the brain, stomach, and intestine, respectively.

  15. Application of liquid chromatography-tandem mass spectrometry for the characterization of galactosylated and tagatosylated beta-lactoglobulin peptides derived from in vitro gastrointestinal digestion.

    PubMed

    Corzo-Martínez, Marta; Lebrón-Aguilar, Rosa; Villamiel, Mar; Quintanilla-López, Jesús Eduardo; Moreno, F Javier

    2009-10-23

    This article describes a comprehensive characterization of bovine beta-lactoglobulin peptides glycated with an aldohexose (galactose) or a ketohexose (tagatose), derived from in vitro gastrointestinal digestion, by liquid chromatography coupled to positive electrospray ion trap tandem mass spectrometry. In addition to the dissociation pathway previously described for aldohexoses-derived Amadori compounds, i.e. formation of the pyrylium ([M+H](+)-54) and furylium ions ([M+H](+)-84) via the oxonium ion ([M+H](+)-18), another and more direct fragmentation route involving the formation of the imminium ion ([M+H](+)-150) is also reported following extensive glycation rates of beta-lactoglobulin with both carbohydrates. These results indicated that the analysis of digested proteins by LC-ESI-MS(2) on a three-dimensional ion trap monitoring neutral losses is an efficient and direct method to identify peptides glycated not only through the Amadori rearrangement but also via the Heyns rearrangement. Nevertheless, as the predominating MS(2) fragmentation pattern of the glycated peptides is derived from the sugar moiety, the sequence-informative b- and y-ions resulting from peptide backbone cleavage were undetected. To overcome this drawback, and taking advantage of multi-stage fragmentation capabilities of ion traps, the indicative Amadori and Heyns-derived imminium ions were successfully used in MS(3) analyses to identify the peptide backbone, as well as the specific glycation site. In addition, further MS(4) analyses were needed to carry out the characterization of doubly glycated peptides.

  16. Multianalyte determination of different classes of pesticides (acidic, triazines, phenyl ureas, anilines, organophosphates, molinate and propanil) by liquid chromatography-electrospray-tandem mass spectrometry.

    PubMed

    Borba da Cunha, Ana C; López de Alda, Maria J; Barceló, Damià; Pizzolato, Tania M; dos Santos, Joao Henrique Z

    2004-02-01

    This work describes the optimization of a liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS-MS) method for the multianalyte determination of twenty pesticides, selected based on current regulations and extent of use. Chromatographic separation was carried out on a Purospher STAR RP-18e column using gradient acetonitrile-water as mobile phase. Triazines, phenylureas, organophosphates, anilines, and molinate were determined in the positive ionization mode, and acidic pesticides and propanil in the negative ion mode. Two different precursor ion-product ion transitions were selected for each analyte and monitored under time scheduled multiple reaction monitoring (MRM) conditions. The optimized method was shown to be linear in the range 1 to 1000 ng/mL with correlation coefficients higher than 0.99 for all but one (diazinon) of the analytes, very sensitive (with limits of detection between 0.010 and 4.528 ng/mL), and repeatable (with relative standard deviations, calculated from the replicate analysis of standard mixtures, lower than 14%). The present work was also devoted to the elucidation of the structures of the principal fragment ions obtained after collision-induced dissociation of the pesticides investigated, an aspect often overlooked in the literature.

  17. β-Lactoglobulin detected in human milk forms noncovalent complexes with maltooligosaccharides as revealed by chip-nanoelectrospray high-resolution tandem mass spectrometry.

    PubMed

    Capitan, Florina; Robu, Adrian C; Schiopu, Catalin; Ilie, Constantin; Chait, Brian T; Przybylski, Michael; Zamfir, Alina D

    2015-11-01

    Cow's milk protein allergy in exclusively breastfed infants, the main cause of food intolerance during the first 6 months of life, is triggered by the mother's diet. β-Lactoglobulin (BLG) present in cow's milk is one of the most potent allergens for newborns. Since no prophylactic treatment is available, finding ligands capable of binding BLG and reducing its allergenicity is currently the focus of research. In this work, an innovative methodology encompassing microfluidics based on fully automated chip-nanoelectrospray ionization (nanoESI), coupled with high-resolution mass spectrometry (MS) on a quadrupole time-of-flight (QTOF MS) instrument was developed. This platform was employed for the assessment of the noncovalent interactions between maltohexaose (Glc6) and β-lactoglobulin extracted from human milk upon deliberate intake of cow's milk. The experiments were carried out in (+) ESI mode, using ammonium acetate (pH 6.0) as the buffer and also in pure water. In both cases, the MS analysis revealed the formation of BLG-Glc6 complex, which was characterized by top-down fragmentation in tandem MS (MS/MS) using collision-induced dissociation (CID). Our findings have a significant biomedical impact, indicating that Glc6 binds BLG under conditions mimicking the in vivo environment and therefore might represent a ligand, able to reduce its allergenicity.

  18. Dissociative diffusion mechanism in vacancy-rich materials according to mass action kinetics

    NASA Astrophysics Data System (ADS)

    Biderman, N. J.; Sundaramoorthy, R.; Haldar, Pradeep; Lloyd, J. R.

    2016-05-01

    Two sets of diffusion-reaction numerical simulations using a finite difference method (FDM) were conducted to investigate fast impurity diffusion via interstitial sites in vacancy-rich materials such as Cu(In,Ga)Se2 (CIGS) and Cu2ZnSn(S, Se)4 (CZTSSe or CZTS) via the dissociative diffusion mechanism where the interstitial diffuser ultimately reacts with a vacancy to produce a substitutional. The first set of simulations extends the standard interstitial-limited dissociative diffusion theory to vacancy-rich material conditions where vacancies are annihilated in large amounts, introducing non-equilibrium vacancy concentration profiles. The second simulation set explores the vacancy-limited dissociative diffusion where impurity incorporation increases the equilibrium vacancy concentration. In addition to diffusion profiles of varying concentrations and shapes that were obtained in all simulations, some of the profiles can be fitted with the constant- and limited-source solutions of Fick's second law despite the non-equilibrium condition induced by the interstitial-vacancy reaction. The first set of simulations reveals that the dissociative diffusion coefficient in vacancy-rich materials is inversely proportional to the initial vacancy concentration. In the second set of numerical simulations, impurity-induced changes in the vacancy concentration lead to distinctive diffusion profile shapes. The simulation results are also compared with published data of impurity diffusion in CIGS. According to the characteristic properties of diffusion profiles from the two set of simulations, experimental detection of the dissociative diffusion mechanism in vacancy-rich materials may be possible.

  19. Rare disorders of metabolism with elevated butyryl- and isobutyryl-carnitine detected by tandem mass spectrometry newborn screening.

    PubMed

    Koeberl, Dwight D; Young, Sarah P; Gregersen, Niels S; Vockley, Jerry; Smith, Wendy E; Benjamin, Daniel Kelly; An, Yan; Weavil, Susan D; Chaing, Shu H; Bali, Deeksha; McDonald, Marie T; Kishnani, Priya S; Chen, Y-T; Millington, David S

    2003-08-01

    Tandem mass spectrometry was adopted for newborn screening by North Carolina in April 1999. Since then, three infants with short-chain acyl-CoA dehydrogenase (SCAD) and one with isobutyryl-CoA dehydrogenase deficiency were detected on the basis of elevated butyrylcarnitine/isobutyrylcarnitine (C4-carnitine) concentrations in newborn blood spots analyzed by tandem mass spectrometry. For three SCAD-deficient infants, biochemical evaluation included a plasma acylcarnitine profile with markedly elevated C4-carnitine, urine organic acid analysis with markedly elevated ethylmalonic and 2-methylsuccinic acids, and markedly elevated [U-13C]butyrylcarnitine concentrations in medium from fibroblasts incubated with [U-13C]palmitic acid and excess l-carnitine, consistent with classic SCAD deficiency. Two of three infants diagnosed with classic SCAD deficiency remained asymptomatic; however, the third infant presented with seizures and a cerebral infarct at 10 wk of age. All three infants had putatively inactivating mutations in both alleles of the SCAD gene. The highly elevated plasma C4-carnitine levels in the three infants detected by newborn screening tandem mass spectrometry differentiated them from infants and children who were homozygous or compound heterozygous for one of two SCAD gene susceptibility variations; for the latter group the C4-carnitine levels were normal. Isobutyryl-CoA dehydrogenase deficiency in a fourth infant was confirmed after isolated elevation of C4-carnitine in the acylcarnitine profile.

  20. Binomial probability distribution model-based protein identification algorithm for tandem mass spectrometry utilizing peak intensity information.

    PubMed

    Xiao, Chuan-Le; Chen, Xiao-Zhou; Du, Yang-Li; Sun, Xuesong; Zhang, Gong; He, Qing-Yu

    2013-01-04

    Mass spectrometry has become one of the most important technologies in proteomic analysis. Tandem mass spectrometry (LC-MS/MS) is a major tool for the analysis of peptide mixtures from protein samples. The key step of MS data processing is the identification of peptides from experimental spectra by searching public sequence databases. Although a number of algorithms to identify peptides from MS/MS data have been already proposed, e.g. Sequest, OMSSA, X!Tandem, Mascot, etc., they are mainly based on statistical models considering only peak-matches between experimental and theoretical spectra, but not peak intensity information. Moreover, different algorithms gave different results from the same MS data, implying their probable incompleteness and questionable reproducibility. We developed a novel peptide identification algorithm, ProVerB, based on a binomial probability distribution model of protein tandem mass spectrometry combined with a new scoring function, making full use of peak intensity information and, thus, enhancing the ability of identification. Compared with Mascot, Sequest, and SQID, ProVerB identified significantly more peptides from LC-MS/MS data sets than the current algorithms at 1% False Discovery Rate (FDR) and provided more confident peptide identifications. ProVerB is also compatible with various platforms and experimental data sets, showing its robustness and versatility. The open-source program ProVerB is available at http://bioinformatics.jnu.edu.cn/software/proverb/ .

  1. A Supercritical Fluid Chromatography/Tandem Mass Spectrometry Method for the Simultaneous Quantification of Metformin and Gliclazide in Human Plasma

    PubMed Central

    Agrawal, Y. K.; Gogoi, P. J.; Manna, K.; Bhatt, H. G.; Jain, V. K.

    2010-01-01

    Present study reports the development and validation of a simultaneous estimation of metformin and gliclazide in human plasma using supercritical fluid chromatography followed by tandem mass spectrometry. Acetonitrile:water (80:20) mixture was used as a mobile phase along with liquid CO2 in supercritical fluid chromatography and phenformin as an internal standard. The modified plasma samples were analyzed by electro-spray ionization method in selective reaction monitoring mode in tandem mass spectrometry. Supercritical fluid chromatographic separation was performed using nucleosil C18 containing column as a stationary phase. The separated products were identified by characteristic peaks and specific fragments peaks in tandem mass spectrometry as m/z 130 to 86 for metformin, m/z 324 to 110 for gliclazide and m/z 206 to 105 for phenformin. The present method was found linear in the concentration ranges of 6.0-3550 ng/ml and 7.5-7500 ng/ml for metformin and gliclazide, respectively. Pharmacokinetic study was performed after an oral administration of dispersible tablets containing 500 mg of metformin and 80 mg of gliclazide using same techniques. PMID:20582190

  2. Ultra-high-pressure liquid chromatography-tandem mass spectrometry method for the determination of alkylphenols in soil.

    PubMed

    Wang, Jing; Pan, Hefang; Liu, Zhengzheng; Ge, Fei

    2009-03-20

    A novel method has been developed for the determination of alkylphenols in soil by ultra-high-pressure liquid chromatography employing small particle sizes, combined with tandem mass spectrometry. Soil samples were extracted with pressurized liquid extraction (PLE) and then cleaned with solid-phase extraction (SPE). The extracts were separated on C18 column (1.7 microm, 50 mm x 2.1mm) with a gradient elution and a mobile phase consisting of water and acetonitrile, and then detected by an electrospray ionization tandem mass spectrometry in negative ion mode with multiple reaction monitoring (MRM). Compared with traditional liquid chromatography, it took ultra-high-pressure liquid chromatography much less time to analyze alkylphenols. Additionally, the ultra-high-pressure liquid chromatography/tandem mass spectrometry method produces satisfactory reliability, sensitivity, and accuracy. The average recoveries of the three target analytes were 74.0-103.4%, with the RSD<15%. The calibration curves for alkylphenols were linear within the range of 0.01-0.4 microg/ml, with the correlation coefficients greater than 0.99. When 10 g soil sample was used for analysis, the limits of quantification (LOQs) of the three alkylphenols were all 1.0 microg/kg.

  3. Simultaneous determination of four alkaloids in Lindera aggregata by ultra-high-pressure liquid chromatography-tandem mass spectrometry.

    PubMed

    Han, Zheng; Zheng, Yunliang; Chen, Na; Luan, Lianjun; Zhou, Changxin; Gan, Lishe; Wu, Yongjiang

    2008-11-28

    A new separation and quantification method using liquid chromatography under ultra-high-pressure in combination with tandem mass spectrometry (MS/MS) was developed for simultaneous determination of four alkaloids in Lindera aggregata. The analysis was performed on an Acquity UPLC BEH C(18) column (50mmx2.1mm, 1.7microm particle size; Waters, Milford, MA, USA) utilizing a gradient elution profile and a mobile phase consisting of (A) water containing 10mM ammonium acetate adjusted to pH 3 with acetic acid and (B) acetonitrile. An electrospray ionization (ESI)-tandem interface in the positive mode was employed prior to mass spectrometric detection. The calibration curve was linear over the range of 17.1-856ng for boldine, 42.4-2652ng for norboldine, 6.1-304ng for reticuline and 0.5-50ng for linderegatine, respectively. The average recoveries ranged from 99.2 to 101.4% with RSDs< or =2.7%. Then, four L. aggregata samples from different batches were analyzed using the established method. The results indicated that ultra-high-pressure liquid chromatography-tandem mass spectrometry provided improved chromatographic parameters resulting in significantly increased sample throughput including lower solvent consumption and lower limits of quantitation (LOQs) for most of target analytes compared to previous method employing conventional high-performance liquid chromatography (HPLC) separation. So, the established method was validated, sensitive and reliable for the determination of four alkaloids in L. aggregata.

  4. Comparison of tandem-in-space and tandem-in-time mass spectrometry in gas chromatography determination of pesticides: application to simple and complex food samples.

    PubMed

    Garrido Frenich, A; Plaza-Bolaños, P; Martínez Vidal, J L

    2008-09-05

    Gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) is one of the most powerful techniques in pesticide residue analysis. MS/MS can be conceived in two ways: tandem in space (e.g. triple quadrupole, QqQ) or in time (e.g. ion trap, IT). QqQ and IT are commonly interfaced to GC; however, there has not been any direct comparison between them in pesticide residue analysis so far. In the present work, the performance of GC coupled to these two analyzers (GC-QqQ-MS/MS and GC-IT-MS/MS) was studied and compared for pesticide residue analysis as well as its application in food analysis. The large volume injection (LVI) technique together with programmed-temperature vaporization (PTV) was applied. For this purpose, 19 pesticides, including organochlorine and organophophorus pesticides and pyrethroids, were analyzed in both systems. Mass spectrometric data, performance characteristics (linearity, intra-day and inter-day precision) and the influence of the matrix nature on the analysis of low concentrations were compared. The target compounds were analyzed in solvent and in two representative food matrices such as cucumber (high water content) and egg (high fat content). MS data and intra-day precision were similar in QqQ and IT, whereas inter-day precision was significantly worse in QqQ. Linearity (expressed as determination coefficient, R(2)) in the range 10-150 microg L(-1) was adequate in both systems; however, better R(2) values were obtained with the QqQ analyzer in high and low concentration ranges (1-50 and 1-750 microg L(-1), respectively). The influence of the matrix nature on the analysis of low concentrations of each analyzer was also evaluated. The QqQ and IT performance was similar in cucumber and solvent. However, QqQ provided better sensitivity in egg working in selected reaction monitoring (SRM).

  5. MEASUREMENT OF PYRETHROID RESIDUES IN ENVIRONMENTAL AND FOOD SAMPLES BY ENHANCED SOLVENT EXTRACTION/SUPERCRITICAL FLUID EXTRACTION COUPLED WITH GAS CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY

    EPA Science Inventory

    The abstract summarizes pyrethorid methods development research. It provides a summary of sample preparation and analytical techniques such as supercritical fluid extraction, enhance solvent extraction, gas chromatography and tandem mass spectrometry.

  6. EPA CRL MS014: Analysis of Aldicarb, Bromadiolone, Carbofuran, Oxamyl and Methomyl in Water by Multiple Reaction Monitoring Liquid Chromatography / Tandem Mass Spectrometry (LC/MS/MS)

    EPA Pesticide Factsheets

    Method MS014 describes procedures for solvent extraction of aldicarb, bromadiolone, carbofuran, oxamyl and methomyl from water samples, followed by analysis using liquid chromatography tandem mass spectrometry (LC-MS-MS).

  7. An antibiotic linked to peptides and proteins is released by electron capture dissociation fourier transform ion cyclotron resonance mass spectrometry.

    PubMed

    Fagerquist, Clifton K; Hudgins, Robert R; Emmett, Mark R; Håkansson, Kristina; Marshall, Alan G

    2003-04-01

    Desfuroylceftiofur (DFC) is a bioactive beta-lactam antibiotic metabolite that has a free thiol group. Previous experiments have shown release of DFC from plasma extracts after addition of a disulfide reducing agent, suggesting that DFC may be bound to plasma and tissue proteins through disulfide bonds. We have reacted DFC with [Arg(8)]-vasopressin (which has one disulfide bond) and bovine insulin (which has three disulfide bonds) and analyzed the reaction products by use of electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry (ECD FT-ICR MS), which has previously shown preferential cleavage of disulfide bonds. We observe cleavage of DFC from vasopressin and insulin during ECD, suggesting that DFC is indeed bound to peptides and proteins through disulfide bonds. Specifically, we observed dissociative loss of one, as well as two, DFC species during ECD of [vasopressin + 2(DFC-H) + 2H](2+) from a single electron capture event. Loss of two DFCs could arise from either consecutive or simultaneous loss, but in any case implies a gas phase disulfide exchange step. ECD of [insulin + DFC + 4H](4+) shows preferential dissociative loss of DFC. Combined with HPLC, ECD FT-ICR-MS may be an efficient screening method for detection of drug-biomolecule binding.

  8. Pilot study of newborn screening for six lysosomal storage diseases using Tandem Mass Spectrometry☆

    PubMed Central

    Elliott, Susan; Buroker, Norman; Cournoyer, Jason J.; Potier, Anna M.; Trometer, Joseph D.; Elbin, Carole; Schermer, Mack J.; Kantola, Jaana; Boyce, Aaron; Turecek, Frantisek; Gelb, Michael H.; Scott, C. Ronald

    2017-01-01

    Background There is current expansion of newborn screening (NBS) programs to include lysosomal storage disorders because of the availability of treatments that produce an optimal clinical outcome when started early in life. Objective To evaluate the performance of a multiplex-tandem mass spectrometry (MS/MS) enzymatic activity assay of 6 lysosomal enzymes in a NBS laboratory for the identification of newborns at risk for developing Pompe, Mucopolysaccharidosis-I (MPS-I), Fabry, Gaucher, Niemann Pick-A/B, and Krabbe diseases. Methods and Results Enzyme activities (acid α-glucosidase (GAA), galactocerebrosidase (GALC), glucocerebrosidase (GBA), α-galactosidase A (GLA), α-iduronidase (IDUA) and sphingomyeline phosphodiesterase-1 (SMPD-1)) were measured on ~43,000 de-identified dried blood spot (DBS) punches, and screen positive samples were submitted for DNA sequencing to obtain genotype confirmation of disease risk. The 6-plex assay was efficiently performed in the Washington state NBS laboratory by a single laboratory technician at the bench using a single MS/MS instrument. The number of screen positive samples per 100,000 newborns were as follows: GAA (4.5), IDUA (13.6), GLA (18.2), SMPD1 (11.4), GBA (6.8), and GALC (25.0). Discussion A 6-plex MS/MS assay for 6 lysosomal enzymes can be successfully performed in a NBS laboratory. The analytical ranges (enzyme-dependent assay response for the quality control HIGH sample divided by that for all enzyme-independent processes) for the 6-enzymes with the MS/MS is 5- to 15-fold higher than comparable fluorimetric assays using 4-methylumbelliferyl substrates. The rate of screen positive detection is consistently lower for the MS/MS assay compared to the fluorimetric assay using a digital microfluidics platform. PMID:27238910

  9. Determination of sodium cromoglycate in human plasma by liquid chromatography with tandem mass.

    PubMed

    Liu, Xiao-yan; Qu, Ting-ting; Wang, Ben-jie; Wei, Chun-min; Yuan, Gui-yan; Zhang, Rui; Guo, Rui-chen

    2008-09-01

    A sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the determination of sodium cromoglycate (SCG) in human plasma after a nasal dose of 10.4 mg sodium cromoglycate nasal spray, using pravastatin sodium as the internal standard. The method was validated over a linear range of 0.300-20.0 ng/mL. SCG and I.S. were extracted from 1.0 mL of heparinized plasma by C(18) solid-phase extraction cartridges using methanol as eluting solvent. The dried residue was reconstituted with 100 microL of mobile phase, and 10 microL was injected onto the LC-MS/MS system. Chromatographic separation was achieved on a C(18) column (250 x 4.6 mm i.d., 5 microm particle size) with a mobile phase of methanol-acetonitrile-water (containing 2 mmol/L ammonium acetate; 42.5:42.5:15, v/v/v) at a flow rate of 0.4 mL/min. The analytes were detected with a triple quad LC-MS/MS using ESI with positive ionization. Ions monitored in the multiple reaction monitoring mode were m/z 469.0 (precursor ion) to m/z 245.0 (product ion) for SCG and m/z 447.2 (precursor ion) to m/z327.1 (product ion) for pravastatin sodium (internal standard) The average recovery of SCG from human plasma was 94.88% and the lower limit of quantitation was 0.3 ng/mL. Results from a 3-day validation study demonstrated excellent precision and accuracy across the calibration range of 0.3-20 ng/mL. The method was successfully applied to the pharmacokinetic study of SCG in healthy Chinese volunteers.

  10. High Performance Liquid Chromatography Tandem Mass Spectrometry Assay for the Determination of Cobinamide in Pig Plasma

    PubMed Central

    McCracken, Brent A.; Brittain, Matthew K.

    2015-01-01

    Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been widely utilized for the analysis of compounds in biological matrices due to its selectivity and sensitivity. This study describes the application of an LC-MS/MS-based approach toward the analysis of cobinamide in Yorkshire pig plasma. The selectivity, accuracy, precision, recovery, linearity, range, carryover, sensitivity, matrix effect, interference, stability, reproducibility, and ruggedness of the method were investigated in pig plasma. The accuracy and precision of the method was determined to be within 10% over three different days over a range of concentrations (25–10,000 ng/mL) that spanned more than two orders of magnitude. The lower limit of quantitation (LLOQ) for dicyanocobinamide was determined to be 25 ng/mL in pig plasma. Carryover was acceptable, as the area response of the carryover blanks were ≤15% of the area response of the nearest LLOQ standard for the analyte, while it was nonexistent for the internal standard. Specificity was ensured using six different lots of pig plasma. While the matrix effects of dicyanocobinamide in plasma were enhanced, ginsenoside Rb1 experienced signal suppression under the described conditions. The absolute recovery results for both compounds were consistent, precise, and reproducibly lower than expected at ~60% for dicyanocobinamide and ~22% for ginsenoside Rb1, confirming that a matrix standard curve was required for accurate quantitation. Cobinamide was shown to be very stable in matrix at various storage conditions including room temperature, refrigerated, and frozen at time intervals of 20 hours, 4 days, and 60 days respectively. This method was demonstrated to be sensitive, reproducible, stable, and rugged, and it should be applicable to the analysis of cobinamide in other biological matrices and species. PMID:26540437

  11. Quantification of six cannabinoids and metabolites in oral fluid by liquid chromatography-tandem mass spectrometry.

    PubMed

    Desrosiers, Nathalie A; Scheidweiler, Karl B; Huestis, Marilyn A

    2015-08-01

    Δ(9) -Tetrahydrocannabinol (THC) is the most commonly analyzed cannabinoid in oral fluid (OF); however, its metabolite 11-nor-9-carboxy-THC (THCCOOH) offers the advantage of documenting active consumption, as it is not detected in cannabis smoke. Analytical challenges such as low (ng/L) THCCOOH OF concentrations hampered routine OF THCCOOH monitoring. Presence of minor cannabinoids like cannabidiol and cannabinol offer the advantage of identifying recent cannabis intake. Published OF cannabinoids methods have limitations, including few analytes and lengthy derivatization. We developed and validated a sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for THC, its metabolites, 11-hydroxy-THC and THCCOOH quantification, and other natural cannabinoids including tetrahydrocannabivarin (THCV), cannabidiol (CBD), and cannabigerol (CBG) in 1 mL OF collected with the Quantisal device. After solid-phase extraction, chromatography was performed on a Selectra PFPP column with a 0.15% formic acid in water and acetonitrile gradient with a 0.5 mL/min flow rate. All analytes were monitored in positive mode atmospheric pressure chemical ionization (APCI) with multiple reaction monitoring. Limits of quantification were 15 ng/L THCCOOH and 0.2 µg/L for all other analytes. Linear ranges extended to 3750 ng/L THCCOOH, 100 µg/L THC, and 50 µg/L for all other analytes. Inter-day analytical recoveries (bias) and imprecision at low, mid, and high quality control (QC) concentrations were 88.7-107.3% and 2.3-6.7%, respectively (n = 20). Mean extraction efficiencies and matrix effects evaluated at low and high QC were 75.9-86.1% and 8.4-99.4%, respectively. This method will be highly useful for workplace, criminal justice, drug treatment and driving under the influence of cannabis OF testing.

  12. Anion exchange SPE and liquid chromatography-tandem mass spectrometry in GHB analysis.

    PubMed

    Elian, Albert A; Hackett, Jeffery

    2011-12-01

    In this study, the extraction of γ-hydroxybutyrate (GHB) from urine using solid-phase extraction (SPE) is described. SPE was performed on anion exchange columns after samples of urine had been diluted with de-ionized water. After application of the diluted samples containing GHB-d(6) as an internal standard, the sorbent was washed with deionized water and methanol and dried. The GHB was eluted from the SPE column with a solvent consisting of methanol containing 6% glacial acetic acid. The eluent was collected, evaporated to dryness, and dissolved in mobile phase (100 μL) for analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in negative multiple reaction monitoring (MRM) mode. Liquid chromatography was performed in gradient mode employing a biphenyl column and a mobile phase consisting of acetontitrile (containing 0.1% formic acid) and 0.1% aqueous formic acid. The total run time for each analysis was less than 5 min. The limits of detection/quantification for this method were determined to be 50 and 100 ng/mL, respectively. The method was found to be linear from 500 ng/mL to 10,000 ng/mL (r(2)>0.995). The recovery of GHB was found to be greater than 75%. In this report, results of authentic urine samples analyzed for GHB by this method are presented. GHB concentrations in these samples were found to be range from less than 500 ng/mL to 5110 ng/mL.

  13. Screening for medium chain acyl-CoA dehydrogenase deficiency using electrospray ionisation tandem mass spectrometry

    PubMed Central

    Clayton, P.; Doig, M.; Ghafari, S.; Meaney, C.; Taylor, C.; Leonard, J.; Morris, M.; Johnson, A.

    1998-01-01

    OBJECTIVE—To establish criteria for the diagnosis of medium chain acyl-CoA dehydrogenase (MCAD) deficiency in the UK population using a method in which carnitine species eluted from blood spots are butylated and analysed by electrospray ionisation tandem mass spectrometry (ESI-MS/MS).
DESIGN—Four groups were studied: (1) 35 children, aged 4 days to 16.2 years, with proven MCAD deficiency (mostly homozygous for the A985G mutation, none receiving carnitine supplements); (2) 2168control children; (3) 482 neonates; and (4) 15 MCAD heterozygotes.
RESULTS—All patients with MCAD deficiency had an octanoylcarnitine concentration ([C8-Cn]) > 0.38 µM and no accumulation of carnitine species > C10 or < C6. Among the patients with MCAD deficiency, the [C8-Cn] was significantly lower in children > 10 weeks old and in children with carnitine depletion (free carnitine < 20 µM). Neonatal blood spots from patients with MCAD deficiency had a [C8-Cn] > 1.5 µM, whereas in heterozygotes and other normal neonates the [C8-Cn] was < 1.0 µM. In contrast, the blood spot [C8-Cn] in eight of 27 patients with MCAD deficiency > 10 weeks old fell within the same range as five of 15 MCAD heterozygotes (0.38-1.0 µM). However, the free carnitine concentrations were reduced (< 20 µM) in the patients with MCAD deficiency but normal in the heterozygotes.
CONCLUSIONS—Criteria for the diagnosis of MCAD deficiency using ESI-MS/MS must take account of age and carnitine depletion. If screening is undertaken at 7-10 days, the number of false positive and negative results should be negligible. Because there have been no instances of death or neurological damage following diagnosis of MCAD deficiency in our patient group, a strong case can be made for neonatal screening for MCAD deficiency in the UK.

 PMID:9797589

  14. Applicability of ultra-performance liquid chromatography-tandem mass spectrometry for heroin profiling.

    PubMed

    Lurie, Ira S; Toske, Steven G

    2008-04-25

    The applicability of ultra- performance liquid chromatography tandem-mass spectrometry (UPLC-MS/MS) for heroin profiling is described. The coupling of the high separation power of UPLC with the highly selective and sensitive detection of MS/MS is well suited for heroin profiling. An Acquity UPLC BEH C18 1.7 microm particle column (100 mm x 2.1mm) with binary gradients containing 1% formic acid (pH 2.0) or 10 mM ammonium bicarbonate (pH 10.0)/acetonitrile mixtures was investigated for the profiling. For MS/MS detection, an atmospheric pressure positive electrospray source was employed with multiple reaction monitoring (MRM). MRMs for individual basic impurities were generated for heroin profiling using low and high pH mobile phases, while MRMs for neutral impurities were generated using a high pH mobile phase. Compared to a pH 2.2 mobile phase, the use of a pH 10 mobile phase allowed for significantly greater sample loading, major selectivity differences, and lower MRM sensitivity. UPLC-MS/MS allowed for the highly selective and sensitive detection of many of the targeted solutes in seized heroin exhibits. Basic impurities detected included morphine, codeine, noscapine, papaverine and the previously unreported solutes reticuline, reticuline monoacetate (2 products), reticuline diacetate, narceine, codamine, laudanidine, cryptopine, laudanosine, and norlaudanosine. Neutral impurities found included N,3,6-triacetylnormorphine, N-acetylnorcodeine, N-acetylnornarcotine, 3,6-dimethoxy-4-acetyloxy-5-[2-(N-methylacetamido)]-ethylphenanthrene, and cis-n-acetylanhydronornarceine. The detection of these impurities, at levels as low as 10(-6)% w/w should allow for greatly enhanced heroin profiles.

  15. [Determination of aniline in water and fish by liquid chromatography-tandem mass spectrometry].

    PubMed

    He, Dechun; Zhao, Bo; Tang, Caiming; Xu, Zhencheng; Zhang, Sukun; Han, Jinglei

    2014-09-01

    A fast analytical method for the determination of aniline in water and fish meat by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed. The water sample was mixed with acetonitrile by 4:1 (v/v) and the fish sample was extracted by 2.00 mL acetonitrile for each gram of sample, and then the extracts of water and fish samples were centrifuged at 5,000 r/min for 5 min. The separation was performed on a reversed-phase C18 column using mobile phases of acetonitrile-0.5% (v/v) formic acid aqueous solution (85:15, v/v). Aniline was separated within 3 min. The calibration curve was linear in the range of 0.5-500 pg/L with R2 > 0.999. The limits of detection (LODs) were 0.50 μg/L and 1.00 μg/kg and the limits of quantification (LOQs) were 1.00 μg/L and 2.00 μg/kg for aniline in water and fish meat, respectively. The average recoveries of aniline in water were 93.7% at the spiked level of 40 ng and 86.7% at the spiked level of 400 ng (n = 5). The average recoveries of aniline in fish were 96.8%, 92.6% and 81.8% at the spiked levels of 5, 50 and 500 ng respectively (n = 5). The relative standard deviations were 1.5%-9.2%. Thirteen water samples and twelve fish samples were collected from a reservoir polluted by aniline and the maximum contents found were 1,943. 6 μg/L in water and 60.8 μg/kg in fish. The method is suitable for the determination of aniline residues in water and fish with the characteristics of easy operation, high accuracy and precision.

  16. Determination of Heterocyclic Amines and Acrylamide in Agricultural Products with Liquid Chromatography-Tandem Mass Spectrometry.

    PubMed

    Lee, Kyung-Jun; Lee, Gae-Ho; Kim, HaeSol; Oh, Min-Seok; Chu, Seok; Hwang, In Ju; Lee, Jee-Yeon; Choi, Ari; Kim, Cho-Il; Park, Hyun-Mee

    2015-09-01

    Heterocyclic amines (HCAs) and acrylamide are unintended hazardous substances generated by heating or processing of foods and are known as carcinogenic and mutagenic agents by the animal experiments. A simple method was established for a rapid and accurate determination of 12 types of HCAs (IQ, MeIQ, Glu-P-1, Glu-P-2, MeIQx, Trp-P-1, Trp-P-2, PhIP, AαC, MeAαC, Harman and Norharman) and acrylamide in three food matrices (non-fat liquid, non-fat solid and fat solid) by isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS). In every sample, a mixture of internal standards including IQ-d3, MeIQx-d3, PhIP-d3, Trp-P-2-(13)C2-(15)N and MeAαC-d3 was spiked for quantification of HCAs and (13)C3-acrylamide was also spiked for the analysis of acrylamide. HCAs and acrylamide in sample were extracted with acetonitrile and water, respectively, and then two solid-phase extraction cartridges, ChemElut: HLB for HCAs and Accucat: HLB for acrylamide, were used for efficiently removing interferences such as pigment, lipid, polar, nonpolar and ionic compounds. Established method was validated in terms of recovery, accuracy, precision, limit of detection, limit of quantitation, and linearity. This method showed good precision (RSD < 20%), accuracy (71.8~119.1%) and recovery (66.0~118.9%). The detection limits were < 3.1 ng/g for all analytes. The correlation coefficients for all the HCAs and acrylamide were > 0.995, showing excellent linearity. These methods for the detection of HCAs and acrylamide by LC-MS/MS were applied to real samples and were successfully used for quantitative monitoring in the total diet study and this can be applied to risk assessment in various food matrices.

  17. Analysis of Alkaloids in Areca Nut-Containing Products by Liquid Chromatography-Tandem Mass Spectrometry.

    PubMed

    Jain, Vipin; Garg, Apurva; Parascandola, Mark; Chaturvedi, Pankaj; Khariwala, Samir S; Stepanov, Irina

    2017-03-08

    Chewing of areca nut in different forms such as betel quid or commercially produced pan masala and gutkha is common practice in the Indian subcontinent and many parts of Asia and is associated with a variety of negative health outcomes, particularly oral and esophageal cancers. Areca nut-specific alkaloids arecoline, arecaidine, guvacoline, and guvacine have been implicated in both the abuse liability and the carcinogenicity of the areca nut. Therefore, variations in the levels of areca alkaloids could potentially contribute to variations in addictive and carcinogenic potential across areca nut-containing products. Here, we developed an accurate and robust liquid chromatography-tandem mass-spectrometry (LC-MS/MS) method for simultaneous quantitation of all four areca alkaloids and applied this method to the analysis of a range of products obtained from India, China, and the United States. The results of the analyses revealed substantial variations in the levels of alkaloids across the tested products, with guvacine being the most abundant (1.39-8.16 mg/g), followed by arecoline (0.64-2.22 mg/g), arecaidine (0.14-1.70 mg/g), and guvacoline (0.17-0.99 mg/g). Substantial differences in the relative contribution of individual alkaloids to the total alkaloid content were also observed among the different products. Our results highlight the need for systematic surveillance of constituent levels in areca nut-containing products and a better understanding of the relationship between the chemical profile and the harmful potential of these products.

  18. Reliable and sensitive determination of dutasteride in human plasma by liquid chromatography-tandem mass spectrometry.

    PubMed

    Contractor, Pritesh; Kurani, Hemal; Guttikar, Swati; Shrivastav, Pranav S

    2013-09-01

    An accurate and precise method was developed and validated using LC-MS/MS to quantify dutasteride in human plasma. The analyte and dutasteride-13C6 as internal standard (IS) were extracted from 300 μL plasma volume using methyl tert-butyl ether-n-hexane (80:20, v/v). Chromatographic analysis was performed on a Gemini C18 (150 × 4.6 mm, 5 µm) column using acetonitrile-5 mm ammonium formate, pH adjusted to 4.0 with formic acid (85:15, v/v) as the mobile phase. Tandem mass spectrometry in positive ionization mode was used to quantify dutasteride by multiple reaction monitoring. The entire data processing was done using Watson LIMS(TM) software, which provided excellent data integrity and high throughput with improved operational efficiency. The calibration curve was linear in the range of 0.1-25 ng/mL, with intra-and inter-batch values for accuracy and precision (coefficient of variation) ranging from 95.8 to 104.0 and from 0.7 to 5.3%, respectively. The mean overall recovery across quality controls was ≥95% for the analyte and IS, while the interference of matrix expressed as IS-normalized matrix factors ranged from 1.01 to 1.02. The method was successfully applied to support a bioequivalence study of 0.5 mg dutasteride capsules in 24 healthy subjects. Assay reproducibility was demonstrated by reanalysis of 103 incurred samples.

  19. Simultaneous Quantification of Multiple Urinary Naphthalene Metabolites by Liquid Chromatography Tandem Mass Spectrometry

    PubMed Central

    Ayala, Daniel C.; Morin, Dexter; Buckpitt, Alan R.

    2015-01-01

    Naphthalene is an environmental toxicant to which humans are exposed. Naphthalene causes dose-dependent cytotoxicity to murine airway epithelial cells but a link between exposure and human pulmonary disease has not been established. Naphthalene toxicity in rodents depends on P450 metabolism. Subsequent biotransformation results in urinary elimination of several conjugated metabolites. Glucuronide and sulfate conjugates of naphthols have been used as markers of naphthalene exposure but, as the current studies demonstrate, these assays provide a limited view of the range of metabolites generated from the parent hydrocarbon. Here, we present a liquid chromatography tandem mass spectrometry method for measurement of the glucuronide and sulfate conjugates of 1-naphthol as well as the mercapturic acids and N-acetyl glutathione conjugates from naphthalene epoxide. Standard curves were linear over 2 log orders. On column detection limits varied from 0.91 to 3.4 ng; limits of quantitation from 1.8 to 6.4 ng. The accuracy of measurement of spiked urine standards was -13.1 to + 5.2% of target and intra-day and inter-day variability averaged 7.2 (± 4.5) and 6.8 (± 5.0) %, respectively. Application of the method to urine collected from mice exposed to naphthalene at 15 ppm (4 hrs) showed that glutathione-derived metabolites accounted for 60-70% of the total measured metabolites and sulfate and glucuronide conjugates were eliminated in equal amounts. The method is robust and directly measures several major naphthalene metabolites including those derived from glutathione conjugation of naphthalene epoxide. The assays do not require enzymatic deconjugation, extraction or derivatization thus simplifying sample work up. PMID:25853821

  20. Validation of keratan sulfate level in mucopolysaccharidosis type IVA by liquid chromatography-tandem mass spectrometry.

    PubMed

    Tomatsu, Shunji; Montaño, Adriana M; Oguma, Toshihiro; Dung, Vu Chi; Oikawa, Hirotaka; de Carvalho, Talita Giacomet; Gutiérrez, María L; Yamaguchi, Seiji; Suzuki, Yasuyuki; Fukushi, Masaru; Kida, Kazuhiro; Kubota, Mitsuru; Barrera, Luis; Orii, Tadao

    2010-12-01

    Mucopolysaccharidosis type IVA (MPS IVA, Morquio A disease), a progressive lysosomal storage disease, causes skeletal chondrodysplasia through excessive storage of keratan sulfate (KS). KS is synthesized mainly in cartilage and released to the circulation. The excess storage of KS disrupts cartilage, consequently releasing more KS into circulation, which is a critical biomarker for MPS IVA. Thus, assessment of KS level provides a potential screening strategy and determines clinical course and efficacy of therapies. We have recently developed a tandem mass spectrometry liquid chromatography [LC/MS/MS] method to assay KS levels in blood. Forty-nine blood specimens from patients with MPS IVA [severe (n = 33), attenuated (n = 11) and undefined (n = 5)] were analyzed for comparison of blood KS concentration with that of healthy subjects and for correlation with clinical severity. Plasma samples were digested by keratanase II to obtain disaccharides of KS. Digested samples were assayed by LC/MS/MS. We found that blood KS levels (0.4-26 µg/ml) in MPS IVA patients were significantly higher than those in age-matched controls (0.67-4.6 µg/ml; P < 0.0001). It was found that blood KS level varied with age and clinical severity in the patients. Blood KS levels in MPS IVA peaked between 2 years and 5 years of age (mean 11.4 µg/ml). Blood KS levels in severe MPS IVA (mean 7.3 µg/ml) were higher than in the attenuated form (mean 2.1 µg/ml) (P = 0.012). We also found elevated blood KS levels in other types of MPS. These findings indicate that the new KS assay for blood is suitable for early diagnosis and longitudinal assessment of disease severity in MPS IVA.

  1. Enantiomeric fraction evaluation of pharmaceuticals in environmental matrices by liquid chromatography-tandem mass spectrometry.

    PubMed

    Ribeiro, Ana Rita; Santos, Lúcia H M L M; Maia, Alexandra S; Delerue-Matos, Cristina; Castro, Paula M L; Tiritan, Maria Elizabeth

    2014-10-10

    The interest for environmental fate assessment of chiral pharmaceuticals is increasing and enantioselective analytical methods are mandatory. This study presents an enantioselective analytical method for the quantification of seven pairs of enantiomers of pharmaceuticals and a pair of a metabolite. The selected chiral pharmaceuticals belong to three different therapeutic classes, namely selective serotonin reuptake inhibitors (venlafaxine, fluoxetine and its metabolite norfluoxetine), beta-blockers (alprenolol, bisoprolol, metoprolol, propranolol) and a beta2-adrenergic agonist (salbutamol). The analytical method was based on solid phase extraction followed by liquid chromatography tandem mass spectrometry with a triple quadrupole analyser. Briefly, Oasis MCX cartridges were used to preconcentrate 250 mL of water samples and the reconstituted extracts were analysed with a Chirobiotic V under reversed mode. The effluent of a laboratory-scale aerobic granular sludge sequencing batch reactor (AGS-SBR) was used to validate the method. Linearity (r(2)>0.99), selectivity and sensitivity were achieved in the range of 20-400 ngL(-1) for all enantiomers, except for norfluoxetine enantiomers which range covered 30-400 ngL(-1). The method detection limits were between 0.65 and 11.5 ngL(-1) and the method quantification limits were between 1.98 and 19.7 ngL(-1). The identity of all enantiomers was confirmed using two MS/MS transitions and its ion ratios, according to European Commission Decision 2002/657/EC. This method was successfully applied to evaluate effluents of wastewater treatment plants (WWTP) in Portugal. Venlafaxine and fluoxetine were quantified as non-racemic mixtures (enantiomeric fraction ≠ 0.5). The enantioselective validated method was able to monitor chiral pharmaceuticals in WWTP effluents and has potential to assess the enantioselective biodegradation in bioreactors. Further application in environmental matrices as surface and estuarine waters can be

  2. Determination of loperamide in human plasma and saliva by liquid chromatography-tandem mass spectrometry.

    PubMed

    Arafat, Tawfiq; Arafat, Basil; awad, Riad; awwad, Ahmad Abu

    2014-12-01

    A simple and sensitive liquid chromatography-tandem mass spectrometric method for quantification of loperamide in human plasma and saliva was developed and validated, and then successfully applied in pharmacokinetic clinical study to investigate and correlate bioavailability of Imodium(®) 2mg quartet tablet dose in both human plasma and saliva. Loperamide with labeled internal standard was extracted from its biological matrix by methanol as protein direct precipitant in single extraction step. Adequate chromatographic separation for analytes from plasma and saliva matrices was achieved using ACE C18 (50mm×2.1mm, 5μm) column, eluted by water/methanol/formic acid (30:70:0.1%, v/v), delivered isocratically at constant flow rate of 0.75ml/min. The method validation intends to investigate specificity, sensitivity, linearity, precision, accuracy, recovery, matrix effect and stability according to European guideline, and partial validation was applied on saliva, specificity, matrix effect, recovery, sensitivity, within and between day precision and accuracy. The calibration curve was linear through the range of 20-3000pg/ml in both plasma and saliva using a 50μl sample volume. The partial validation sections outcome in saliva was so close to those in plasma. The within- and between-day precisions were all below 8.7% for plasma and below 11.4% for saliva. Accuracies ranged from 94 to 105% for both matrices. In this study, 26 healthy volunteers participated in the clinical study, and 6 of gave their saliva samples in addition to plasma at the same time schedule. The pharmacokinetic parameters of Cmax, AUC0-t and AUC0-∞, Tmax and T1/2 in both plasma and saliva were calculated and correlated.

  3. Analysis of acylcarnitine levels by tandem mass spectrometry in epileptic children receiving valproate and oxcarbazepine.

    PubMed

    Cansu, Ali; Serdaroglu, Ayse; Biberoglu, Gursel; Tumer, Leyla; Hirfanoglu, Tugba Luleci; Ezgu, Fatih Suheyl; Hasanoglu, Alev

    2011-12-01

    This prospective study was designed to investigate whether or not monotherapy with sodium valproate (VPA) or oxcarbazepine (OXC) affects plasma levels of fatty acylcarnitine esters in children with epilepsy. A total of 56 children with idiopathic partial or generalised epilepsy were included in the study. Patients were assigned to receive either VPA or OXC monotherapy. Free carnitine (C0) and acylcarnitine profiles of the patients were investigated using tandem mass spectrometry at baseline and at six and 18 months after commencement of therapy. For patients receiving VPA or OXC monotherapy, there were no significant differences in plasma levels of C0, compared with baseline, at six and 18 months (p>0.05). Treatment with VPA for six and 18 months correlated with a significant increase in 3-hydroxy-isovalerylcarnitine (C5-OH) (six months: +23%; 18 months: +73%), and significant decreases in the following acylcarnitines: C6-acylcarnitine (six months: -60%; 18 months: -66%), C14-acylcarnitine (six months: -25%; 18 months: -38%), C16-acylcarnitine (six months: -73%; 18 months: -73%), and C18:1-OH-acylcarnitine (six months: -60%; 18 months: -70%), compared with baseline (p<0.05). In patients receiving OXC monotherapy, on the other hand, plasma concentrations (μmol/L) of acylcarnitines (from C2 to C18:1-OH) fell within the normal reference range. The results of this study indicate that there are significant biochemical changes in acylcarnitines in ambulatory children on VPA monotherapy but these are not clinically significant. OXC monotherapy had no effect on acylcarnitine metabolism in ambulatory children.

  4. Simultaneous quantification of Pacific ciguatoxins in fish blood using liquid chromatography-tandem mass spectrometry.

    PubMed

    Mak, Yim Ling; Wu, Jia Jun; Chan, Wing Hei; Murphy, Margaret B; Lam, James C W; Chan, Leo L; Lam, Paul K S

    2013-04-01

    Ciguatera fish poisoning (CFP) is a food intoxication caused by exposure to ciguatoxins (CTXs) in coral reef fish. Rapid analytical methods have been developed recently to quantify Pacific-CTX-1 (P-CTX-1) in fish muscle, but it is destructive and can cause harm to valuable live coral reef fish. Also fish muscle extract was complex making CTX quantification challenging. Not only P-CTX-1, but also P-CTX-2 and P-CTX-3 could be present in fish, contributing to ciguatoxicity. Therefore, an analytical method for simultaneous quantification of P-CTX-1, P-CTX-2, and P-CTX-3 in whole blood of marketed coral reef fish using sonication, solid-phase extraction (SPE), and liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed. The optimized method gave acceptable recoveries of P-CTXs (74-103 %) in fish blood. Matrix effects (6-26 %) in blood extracts were found to be significantly reduced compared with those in muscle extracts (suppressed by 34-75 % as reported in other studies), thereby minimizing potential for false negative results. The target P-CTXs were detectable in whole blood from four coral reef fish species collected in a CFP-endemic region. Similar trends in total P-CTX levels and patterns of P-CTX composition profiles in blood and muscle of these fish were observed, suggesting a relationship between blood and muscle levels of P-CTXs. This optimized method provides an essential tool for studies of P-CTX pharmacokinetics and pharmacodynamics in fish, which are needed for establishing the use of fish blood as a reliable sample for the assessment and control of CFP.

  5. Rapid determination of polyether marine toxins using liquid chromatography-multiple tandem mass spectrometry.

    PubMed

    Fernández Puente, Patricia; Fidalgo Sáez, María José; Hamilton, Brett; Lehane, Mary; Ramstad, Hanne; Furey, Ambrose; James, Kevin J

    2004-11-12

    The diarrhetic shellfish poisoning (DSP) toxins, okadaic acid (OA), dinophysistoxins (DTX); pectenotoxin-2 (PTX2) and pectenotoxin-2 seco acids, were determined in marine phytoplankton, Dinophysis acuta, and mussels (Mytilus edulis) collected along the southwest coast of Ireland. Liquid chromatography-multiple tandem mass spectrometry (LC-MS/MS) was employed for the simultaneous determination of a series of marine toxins with large polarity differences. Separation of five DSP toxins was achieved on a C18 column (Luna-2, 150 mm x 2.1 mm, 5 microm) using an acetonitrile-water gradient with ammonium acetate as an eluent modifier. Electrospray ionisation (ESI) in negative mode, was used to generate the molecule related ion, [M-H]-, for each toxin. To develop a multiple reaction monitoring (MRM) method, fragmentation studies were performed to determine the optimum precursor-product ion combinations: OA (803/255), DTX2 (803/255), DTX1 (817/255), PTX2SAs (875/137) and PTX2 (857/137). This highly sensitive method had detection limits better than 1 pg (on-column). Linear calibrations were obtained for shellfish extracts that were spiked with toxins, OA, 0.007-1.00 microg/ml (r2 = 0.9993, N = 3) and DTX2, 0.054-8.5 microg/ml (r2 = 0.9992, N = 3). Good reproducibility data were also achieved with %RSD values (N = 3) ranging from 3.15% (0.56 microg DTX2/ml) to 5.71% (0.14 microg DTX2/ml), for shellfish extracts. The method was sufficiently sensitive to permit the determination of DSP toxins in small numbers of picked phytoplankton cells (N = 12-40). In one sample of D. acuta the average toxin composition per cell was: OA (7.0 pg), DTX2 (11 pg) and PTX2 (7.2 pg).

  6. Quantitation of Cotinine in Nonsmoker Saliva Using Chip Based Nanoelectrospray Tandem Mass Spectrometry

    SciTech Connect

    Tomkins, Bruce A; Van Berkel, Gary J; Jenkins, Roger A; Counts, Richard Wayne

    2006-01-01

    A new analytical procedure was developed for the quantitation of nonsmoker salivary cotinine. Small volumes of saliva were diluted with water, fortified with cotinine-d{sub 3} (internal standard), then passed through small extraction columns. The analyte and internal standard were eluted with 0.1% (v/v) acetic acid/acetonitrile. Aliquots of each extract were analyzed directly, without chromatographic separation, using chip-based (NanoMate{sup TM}) nanospray tandem mass spectrometry. The calculated detection limit was 0.49 ng cotinine/mL saliva. This method was used to quantify salivary cotinine collected from nonsmoking human subjects living in one of three environmental tobacco smoke (ETS) exposure categories or 'cells': 1. smoking home/smoking workplace; 2. smoking home/nonsmoking workplace; and 3. nonsmoking home/smoking workplace. Samples were collected during five sequential days, including Saturday, as part of a larger study to evaluate potential variability in exposure to ETS. Salivary cotinine measurements were made for the purpose of excluding misclassified smokers and for comparison with known levels of exposure to airborne nicotine in each exposure category. The concentrations observed were consistent with those reported from other large studies reported elsewhere. A non-parametric statistical test was applied to the data within each cell. No statistically significant differences were found between the mean cotinine concentrations collected on a weekday as compared to those collected on a weekend day. When the non-parametric test was applied to the three cells, a statistically significant difference was observed between cell 1 compared to cells 2 and 3. The salivary cotinine concentrations were thus statistically invariant over a five-day exposure period, and they were greatest under the conditions of smoking home and smoking workplace.

  7. Analysis of mouse liver membrane proteins using multidimensional separations and tandem mass spectrometry.

    PubMed

    Wang, Zhuowei; Wang, Min; Tong, Wei

    2010-12-01

    In the field of proteomic investigation, the analysis of membrane proteins still faces many technical challenges. A fundamental question in this puzzle is how to maintain a proper solvent environment to allow the hydrophobic proteins to remain solubilized. We propose that the denaturation of membrane proteins in a highly concentrated urea solution enables them to be ionized such that ionic exchange chromatography can be employed to separate them. The membrane proteins prepared from the mouse liver were dissolved in 6M guanidine hydrochloride, 20mM Tris-HCl, pH 9.0, and loaded onto a tandem chromatography apparatus coupled with Q-Sepharose FF and Sephacryl S-200HR. These columns were able to adsorb 97.87% of the membrane protein preparations. Using a linear NaCl (0-1.0M) gradient, the bound proteins were eluted out at 0.1-1.0M NaCl, and examined by SDS-PAGE. Furthermore the protein bands underwent excision and digestion with trypsin, followed by reverse-phase chromatography for the separation of the digested peptides and ionic-trap mass spectrometry for the identification of the proteins. From the SDS-PAGE gels, the overlap between proteins from neighboring bands was only 21.34%, indicating that the anionic-size exclusion coupling chromatography efficiently separated these membrane proteins. Of a total of 392 proteins identified, 306 were membrane proteins or membrane-associated proteins. Based on the calculation of hydrophobicity, the GRAVY scores of 83 proteins are greater than, or equal to, 0.00. Taking all of this evidence together, our results revealed that this approach is satisfactory for studies on the membrane proteome from the mouse liver.

  8. Tetrahydrocannabinol and two of its metabolites in whole blood using liquid chromatography-tandem mass spectrometry.

    PubMed

    Coulter, Cynthia; Miller, Elizabeth; Crompton, Katherine; Moore, Christine

    2008-10-01

    An analytical procedure for the determination of Delta9-tetrahydrocannabinol (THC), 11-nor-9-carboxy-Delta9-tetrahydrocannabinol (THCA), and 11-hydroxy-Delta9-tetrahydrocannabinol (11-OH-THC) in whole blood has been developed and validated using liquid chromatography with tandem mass spectral detection (MS). Cannabinoids present in the blood samples were quantified using solid-phase extraction followed by MS detection in positive electrospray ionization mode. For confirmation, two transitions were monitored and one ratio determined. Samples being reported as positive were required to have both transitions present, the ratio of quantifying transition to qualifying transition being within 20% of that determined from known calibration standards. The monitoring of the qualifying transition and requirement for its presence within a specific ratio to the primary ion has the potential of limiting the sensitivity of the assay, however, the additional confidence in the final result as well as forensic defensibility were considered to be of greater importance. The limit of quantitation was 0.5 ng/mL for THC, 5 ng/mL for THCA, and 2 ng/mL for 11-OH-THC. The limit of detection was 0.5 ng/mL for THC, 4 ng/mL for THCA, and 1 ng/mL for 11-OH-THC. The percentage recovery of the cannabinoids from whole blood at a concentration of 5 ng/mL was 71.5% for THC, 64.5% for 11-OH-THC, and 61.2% for THCA (n = 3).

  9. Determination of triapine, a ribonucleotide reductase inhibitor, in human plasma by liquid chromatography tandem mass spectrometry.

    PubMed

    Feng, Ye; Kunos, Charles A; Xu, Yan

    2015-09-01

    Triapine is an inhibitor of ribonucleotide reductase (RNR). Studies have shown that triapine significantly decreases the activity of RNR and enhanced the radiation-mediated cytotoxicity in cervical and colon cancer. In this work, we have developed and validated a selective and sensitive LC-MS/MS method for the determination of triapine in human plasma. In this method, 2-[(3-fluoro-2-pyridinyl)methylene] hydrazinecarbothioamide (NSC 266749) was used as the internal standard (IS); plasma samples were prepared by deproteinization with acetonitrile; tripaine and the IS were separated on a Waters Xbridge Shield RP18 column (3.5 µm; 2.1 × 50 mm) using a mobile phase containing 25.0% methanol and 75.0% ammonium bicarbonate buffer (10.0 mM, pH 8.50; v/v); column eluate was monitored by positive turbo-ionspray tandem mass spectrometry; and quantitation of triapine was carried out in multiple-reaction-monitoring mode. The method developed had a linear calibration range of 0.250-50.0 ng/mL with correlation coefficient of 0.999 for triapine in human plasma. The IS-normalized recovery and the IS-normalized matrix factor of triapine were 101-104% and 0.89-1.05, respectively. The accuracy expressed as percentage error and precision expressed as coefficient of variation were ≤±6 and ≤8%, respectively. The validated LC-MS/MS method was applied to the measurement of triapine in patient samples from a phase I clinical trial.

  10. Differentiation of ring-substituted bromoamphetamine analogs by gas chromatography-tandem mass spectrometry.

    PubMed

    Inoue, Hiroyuki; Negishi, Shoko; Nakazono, Yukiko; Iwata, Yuko T; Tsujikawa, Kenji; Ohtsuru, Osamu; Miyamoto, Kazuna; Yamashita, Takuya; Kasuya, Fumiyo

    There has been a rapid increase over the last decade in the appearance of new non-controlled psychoactive substances. Minor changes in the chemical structures of these compounds, such as the extension of an alkyl residue or replacement of a single substituent, are regularly made to avoid regulatory control, leading to the manufacture of many new potentially dangerous drugs. Bromoamphetamine analogs (bromoamphetamine [Br-AP] and bromomethamphetamine (Br-MA]) are ring-substituted amphetamines that can behave as stimulants, as well as exhibiting inhibitory activity towards monoamine oxidases in the same way as amphetamines. Gas chromatography-tandem mass spectrometry (GC-MS-MS) was used in this study to differentiate ring-substituted bromoamphetamine analogs. Free bases, trifluoroacetyl derivatives, and trimethylsilyl (TMS) derivatives of six analytes were successfully separated using DB-1ms and DB-5ms columns. Electron ionization MS-MS analysis of the TMS derivatives allowed for the differentiation of three regioisomers. TMS derivatives of 2-positional isomers provided significant product ions. The spectral patterns of 3- and 4-positional isomers were different. Chemical ionization MS-MS analysis of free bases for [M+H-HBr](+) ions at m/z 134 and 148 allowed for differentiation of the regioisomers. The spectra of 2-positional isomers contained characteristic product ions formed by dehydrogenation at m/z 132 and m/z 146 for 2Br-AP and 2Br-MA, respectively. The spectra of 3-positional isomers contained α-cleaved iminium cations as the base peaks. The spectra of 4-positional isomers showed a tropylium cation at m/z 91 as the base peak. These results demonstrate that GC-MS-MS can be used for the differentiation of regioisomeric Br-AP analogs in forensic practice.

  11. Nitroproteins in Human Astrocytomas Discovered by Gel Electrophoresis and Tandem Mass Spectrometry.

    PubMed

    Peng, Fang; Li, Jianglin; Guo, Tianyao; Yang, Haiyan; Li, Maoyu; Sang, Shushan; Li, Xuejun; Desiderio, Dominic M; Zhan, Xianquan

    2015-12-01

    Protein tyrosine nitration is involved in the pathogenesis of highly fatal astrocytomas, a type of brain cancer. To understand the molecular mechanisms of astrocytomas and to discover new biomarkers/therapeutic targets, we sought to identify nitroproteins in human astrocytoma tissue. Anti-nitrotyrosine immunoreaction-positive proteins from a high-grade astrocytoma tissue were detected with two-dimensional gel electrophoresis (2DGE)-based nitrotyrosine immunoblots, and identified with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Fifty-seven nitrotyrosine immunopositive protein spots were detected. A total of 870 proteins (nitrated and non-nitrated) in nitrotyrosine-immunopositive 2D gel spots were identified, and 18 nitroproteins and their 20 nitrotyrosine sites were identified with MS/MS analysis. These nitroproteins participate in multiple processes, including drug-resistance, signal transduction, cytoskeleton, transcription and translation, cell proliferation and apoptosis, immune response, phenotypic dedifferentiation, cell migration, and metastasis. Among those nitroproteins that might play a role in astrocytomas was nitro-sorcin, which is involved in drug resistance and metastasis and might play a role in the spread and treatment of an astrocytoma. Semiquantitative immune-based measurements of different sorcin expressions were found among different grades of astrocytomas relative to controls, and a semiquantitative increased nitration level in high-grade astrocytoma relative to control. Nitro-β-tubulin functions in cytoskeleton and cell migration. Semiquantitative immunoreactivity of β-tubulin showed increased expression among different grades of astrocytomas relative to controls and semiquantitatively increased nitration level in high-grade astrocytoma relative to control. Each nitroprotein was rationalized and related to the corresponding functional system to provide new insights into tyrosine nitration and its potential role in the

  12. Nitroproteins in Human Astrocytomas Discovered by Gel Electrophoresis and Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Peng, Fang; Li, Jianglin; Guo, Tianyao; Yang, Haiyan; Li, Maoyu; Sang, Shushan; Li, Xuejun; Desiderio, Dominic M.; Zhan, Xianquan

    2015-12-01

    Protein tyrosine nitration is involved in the pathogenesis of highly fatal astrocytomas, a type of brain cancer. To understand the molecular mechanisms of astrocytomas and to discover new biomarkers/therapeutic targets, we sought to identify nitroproteins in human astrocytoma tissue. Anti-nitrotyrosine immunoreaction-positive proteins from a high-grade astrocytoma tissue were detected with two-dimensional gel electrophoresis (2DGE)-based nitrotyrosine immunoblots, and identified with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Fifty-seven nitrotyrosine immunopositive protein spots were detected. A total of 870 proteins (nitrated and non-nitrated) in nitrotyrosine-immunopositive 2D gel spots were identified, and 18 nitroproteins and their 20 nitrotyrosine sites were identified with MS/MS analysis. These nitroproteins participate in multiple processes, including drug-resistance, signal transduction, cytoskeleton, transcription and translation, cell proliferation and apoptosis, immune response, phenotypic dedifferentiation, cell migration, and metastasis. Among those nitroproteins that might play a role in astrocytomas was nitro-sorcin, which is involved in drug resistance and metastasis and might play a role in the spread and treatment of an astrocytoma. Semiquantitative immune-based measurements of different sorcin expressions were found among different grades of astrocytomas relative to controls, and a semiquantitative increased nitration level in high-grade astrocytoma relative to control. Nitro-β-tubulin functions in cytoskeleton and cell migration. Semiquantitative immunoreactivity of β-tubulin showed increased expression among different grades of astrocytomas relative to controls and semiquantitatively increased nitration level in high-grade astrocytoma relative to control. Each nitroprotein was rationalized and related to the corresponding functional system to provide new insights into tyrosine nitration and its potential role in the

  13. Analysis of free amino acids in natural waters by liquid chromatography-tandem mass spectrometry.

    PubMed

    How, Zuo Tong; Busetti, Francesco; Linge, Kathryn L; Kristiana, Ina; Joll, Cynthia A; Charrois, Jeffrey W A

    2014-11-28

    This paper reports a new analytical method for the analysis of 18 amino acids in natural waters using solid-phase extraction (SPE) followed by liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) operated in multiple reaction monitoring mode. Two different preconcentration methods, solid-phase extraction and concentration under reduced pressure, were tested in development of this method. Although concentration under reduced pressure provided better recoveries and method limits of detection for amino acids in ultrapure water, SPE was a more suitable extraction method for real samples due to the lower matrix effects for this method. Even though the strong cation exchange resin used in SPE method introduced exogenous matrix interferences into the sample extracts (inorganic salt originating from the acid-base reaction during the elution step), the SPE method still incorporates a broad sample clean-up and minimised endogenous matrix effects by reducing interferences originating from real water samples. The method limits of quantification (MLQ) for the SPE LC-MS/MS method in ultrapure water ranged from 0.1 to 100 μg L(-1) as N for the different amino acids. The MLQs of the early eluting amino acids were limited by the presence of matrix interfering species, such as inorganic salts in natural water samples. The SPE LC-MS/MS method was successfully applied to the analysis of amino acids in 3 different drinking water source waters: the average total free amino acid content in these waters was found to be 19 μg L(-1) as N, while among the 18 amino acids analysed, the most abundant amino acids were found to be tyrosine, leucine and isoleucine.

  14. Determination of Heterocyclic Amines and Acrylamide in Agricultural Products with Liquid Chromatography-Tandem Mass Spectrometry

    PubMed Central

    Lee, Kyung-Jun; Lee, Gae-Ho; Kim, HaeSol; Oh, Min-Seok; Chu, Seok; Hwang, In Ju; Lee, Jee-yeon; Choi, Ari; Kim, Cho-il

    2015-01-01

    Heterocyclic amines (HCAs) and acrylamide are unintended hazardous substances generated by heating or processing of foods and are known as carcinogenic and mutagenic agents by the animal experiments. A simple method was established for a rapid and accurate determination of 12 types of HCAs (IQ, MeIQ, Glu-P-1, Glu-P-2, MeIQx, Trp-P-1, Trp-P-2, PhIP, AαC, MeAαC, Harman and Norharman) and acrylamide in three food matrices (non-fat liquid, non-fat solid and fat solid) by isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS). In every sample, a mixture of internal standards including IQ-d3, MeIQx-d3, PhIP-d3, Trp-P-2-13C2-15N and MeAαC-d3 was spiked for quantification of HCAs and 13C3-acrylamide was also spiked for the analysis of acrylamide. HCAs and acrylamide in sample were extracted with acetonitrile and water, respectively, and then two solid-phase extraction cartridges, ChemElut: HLB for HCAs and Accucat: HLB for acrylamide, were used for efficiently removing interferences such as pigment, lipid, polar, nonpolar and ionic compounds. Established method was validated in terms of recovery, accuracy, precision, limit of detection, limit of quantitation, and linearity. This method showed good precision (RSD < 20%), accuracy (71.8~119.1%) and recovery (66.0~118.9%). The detection limits were < 3.1 ng/g for all analytes. The correlation coefficients for all the HCAs and acrylamide were > 0.995, showing excellent linearity. These methods for the detection of HCAs and acrylamide by LC-MS/MS were applied to real samples and were successfully used for quantitative monitoring in the total diet study and this can be applied to risk assessment in various food matrices. PMID:26483884

  15. Qualitative and quantitative analysis of polycyclic polyprenylated acylphloroglucinols from Garcinia species using ultra performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

    PubMed

    Zhou, Yan; Lee, Stephanie; Choi, Franky Fung Kei; Xu, Gang; Liu, Xin; Song, Jing-Zheng; Li, Song-Lin; Qiao, Chun-Feng; Xu, Hong-Xi

    2010-09-23

    Polycyclic polyprenylated acylphloroglucinols (PPAPs) are a group of natural products isolated from different Garcinia species with a wide range of important biological activities. In this study, an ultra performance liquid chromatography (UPLC) coupled to photodiode-array detection and quadrupole time-of-flight mass spectrometry (Q-TOF) method was developed to characterize 16 PPAPs in 10 Garcinia species. In source dissociation techniques based on cone voltage fragmentation were used to fragment the deprotonated molecules and multiple mass spectrometry (MS/MS) using ramping collision energy were used to further break down the resulting product ions. The resulting characteristic fragment ions were generated by cleavage of C1-C5 bond and C7-C8 bond through concerted pericyclic reaction, which is especially valuable for differentiating three types of PPAPs isomers. As such, two new PPAPs isomers present in minor amount in the extracts of Garcinia oblongifolia were tentatively characterized by comparing their tandem mass spectra to the known ones. In addition, an UPLC-Q-TOF-MS method was validated for the quantitative determination of PPAPs. The method exhibited limits of detection from 2.7 to 21.4 ng mL(-1) and intra-day and inter-day variations were less than 3.7% and the recovery was in the range of 89-107% with RSD less than 9.0%. This UPLC-Q-TOF-MS method has successfully been applied to quantify 16 PPAPs in 32 samples of 10 Garcinia species, which were found to be a rich source of PPAPs.

  16. Screening for multiple phosphodiesterase type 5 inhibitor drugs in dietary supplement materials by flow injection mass spectrometry and their quantification by liquid chromatography tandem mass spectrometry.

    PubMed

    Song, Fenhong; El-Demerdash, Aref; Lee, Shwn-Ji Susie H

    2012-11-01

    A flow injection tandem mass spectrometry method (FI-MS/MS) has been developed to detect enzyme phosphodiesterase type 5 inhibitors, including tadalafil, sildenafil, and vardenafil. Multiple reaction monitoring (MRM) was used to detect the drugs and product ion ratios were used for identification. FI-MS/MS was used for semi-quantification and liquid chromatography tandem mass spectrometry (LC-MS/MS) was used for further confirmation and quantification. One of 13 samples has been found to be adulterated with prescription levels of tadalafil and also low level of sildenafil. The method can be used for screening large numbers of herbal products for adulteration since it takes less than 1 min without chromatographic separation on a column.

  17. Morphine brain pharmacokinetics at very low concentrations studied with accelerator mass spectrometry and liquid chromatography-tandem mass spectrometry.

    PubMed

    Sadiq, Muhammad Waqas; Salehpour, Mehran; Forsgard, Niklas; Possnert, Göran; Hammarlund-Udenaes, Margareta

    2011-02-01

    Morphine has been predicted to show nonlinear blood-brain barrier transport at lower concentrations. In this study, we investigated the possibility of separating active influx of morphine from its efflux by using very low morphine concentrations and compared accelerator mass spectrometry (AMS) with liquid chromatography-tandem mass spectrometry (LC-MS/MS) as a method for analyzing microdialysis samples. A 10-min bolus infusion of morphine, followed by a constant-rate infusion, was given to male rats (n = 6) to achieve high (250 ng/ml), medium (50 ng/ml), and low (10 ng/ml) steady-state plasma concentrations. An additional rat received infusions to achieve low (10 ng/ml), very low (2 ng/ml), and ultralow (0.4 ng/ml) concentrations. Unbound morphine concentrations from brain extracellular fluid and blood were sampled by microdialysis and analyzed by LC-MS/MS and AMS. The average partition coefficient for unbound drug (K(p,uu)) values for the low and medium steady-state levels were 0.22 ± 0.08 and 0.21 ± 0.05, respectively, when measured by AMS [not significant (NS); p = 0.5]. For the medium and high steady-state levels, K(p,uu) values were 0.24 ± 0.05 and 0.26 ± 0.05, respectively, when measured by LC-MS/MS (NS; p = 0.2). For the low, very low, and ultralow steady-state levels, K(p,uu) values were 0.16 ± 0.01, 0.16 ± 0.02, and 0.18 ± 0.03, respectively, when measured by AMS. The medium-concentration K(p,uu) values were, on average, 16% lower when measured by AMS than by LC-MS/MS. There were no significant changes in K(p,uu) over a 625-fold concentration range (0.4-250 ng/ml). It was not possible to separate active uptake transport from active efflux using these low concentrations. The two analytical methods provided indistinguishable results for plasma concentrations but differed by up to 38% for microdialysis samples; however, this difference did not affect our conclusions.

  18. Electron photodetachment dissociation for structural characterization of synthetic and bio-polymer anions.

    PubMed

    Antoine, Rodolphe; Lemoine, Jérôme; Dugourd, Philippe

    2014-01-01

    Tandem mass spectrometry (MS-MS) is a generic term evoking techniques dedicated to structural analysis, detection or quantification of molecules based on dissociation of a precursor ion into fragments. Searching for the most informative fragmentation patterns has led to the development of a vast array of activation modes that offer complementary ion reactivity and dissociation pathways. Collisional activation of ions using atoms, molecules or surface resulting in unimolecular dissociation of activated ions still plays a key role in tandem mass spectrometry. The discovery of electron capture dissociation (ECD) and then the development of other electron-ion or ion/ion reaction methods, constituted a significant breakthrough, especially for structural analysis of large biomolecules. Similarly, photon activation opened promising new frontiers in ion fragmentation owing to the ability of tightly controlled internal energy deposition and easy implementation on commercial instruments. Ion activation by photons includes slow heating methods such as infrared multiple photon dissociation (IRMPD) and black-body infrared radiative dissociation (BIRD) and higher energy methods like ultra-violet photodissociation (UVPD) and electron photo detachment dissociation (EPD). EPD occurs after UV irradiation of multiply negatively charged ions resulting in the formation of oxidized radical anions. The present paper reviews the hypothesis regarding the mechanisms of electron photo-detachment, radical formation and direct or activated dissociation pathways that support the observation of odd and even electron product ions. Finally, the value of EPD as a complementary structural analysis tool is illustrated through selected examples of synthetic polymers, oligonucleotides, polypeptides, lipids, and polysaccharides.

  19. Liquid chromatography-tandem mass spectrometry method for the determination of anthelmintics in alfalfa plants.

    PubMed

    Islam, M Dabalus; Haberhauer, G; Gerzabek, M; Cannavan, A

    2012-01-01

    A simple and inexpensive liquid chromatography-tandem mass spectrometric method for the determination of anthelmintics in alfalfa plants (Medicago sativa L.) was developed and validated. Anthelmintics in plant leaves and stems (green chops) were extracted with methanol/acetonitrile (7:3, v/v) followed by a concentration and clean-up step using solid-phase extraction (Strata-X, 500 mg, 6 ml cartridge). After drying with nitrogen gas, the adsorbed analytes were eluted with methanol/acetonitrile (50:50, v/v) mixture followed by 100% acetonitrile. Chromatographic separation was achieved on an Atlantis T-3 (2.1 × 100 mm × 3 µm) analytical column with a Phenomenex guard cartridge (C8, 4 × 3 mm) attached to a Waters triple quadrupole mass spectrometer operated in positive electrospray ionisation mode with selected reaction monitoring. Samples were analysed using gradient elution at a flow rate of 0.35 ml min⁻¹. The mobile phase consisted of a 10 mM ammonium formate solution in (A) water/acetonitrile (90:10, v/v) and (B) methanol/acetonitrile (50:50, v/v). The method was validated for levamisole, fenbendazole, fenbendazole sulphoxide and fenbendazole sulphone at 10, 20 and 40 µg kg⁻¹ and for eprinomectin at 20, 40 and 80 µg kg⁻¹. Limits of quantification (LOQ) were 10 µg kg⁻¹ for all analytes except eprinomectin, which had an LOQ of 20 µg kg⁻¹. The overall mean recovery in green plants was between 74.2% and 81.4% with repeatabilities ranging from 2.2% to 19.1% and reproducibilities in the range 3.8-8.7%. The validated method was applied to plant samples in a study on the behaviour of anthelmintic drugs in a soil, plant and water system.

  20. Simultaneous determination of cocaine and opiates in dried blood spots by electrospray ionization tandem mass spectrometry.

    PubMed

    Antelo-Domínguez, Ángel; Cocho, José Ángel; Tabernero, María Jesús; Bermejo, Ana María; Bermejo-Barrera, Pilar; Moreda-Piñeiro, Antonio

    2013-12-15

    A sample pre-treatment method based on blood spot collection filter cards was optimized as a means of using small volume samples for the screening and confirmation of cocaine and opiates abuse. Dried blood spots (DBSs) were prepared by dispersing 20 µL of whole blood specimens previously mixed with the internal standards (deuterated analogs of each target), and subjecting the whole DBS to extraction with 5 mL of methanol under orbital-horizontal shaking (180 rpm) for 10 min. Determinations were based on direct electrospray ionization tandem mass spectrometry (ESI-MS/MS) by injecting the re-dissolved methanol extract with the delivery solution (acetonitrile-water-formic acid, 80:19.875:0.125) at a flow rate of 60 µL min(-1), and using multiple reaction monitoring (MRM) mode with the m/z (precursor ion)→m/z (product ion) transitions for acquisition. Matrix effect has been found to be statistically significant (Multiple Range Test) when assessing cocaine, BZE, codeine and morphine, and the use of the standard addition method (dispersion of whole blood previously mixed with standards onto the filter papers) was needed for accurate determinations. The developed DBS-ESI-MS/MS procedure offered good intra-day and inter-day precisions (lower than 10% and 12%, respectively), as well as good intra-day and inter-day accuracies (inter-day absolute recoveries, expressed as the mean analytical recovery over three target concentration levels, of 103%, 100%, 101%, 98% and 100% for cocaine, BZE, codeine, morphine and 6-MAM, respectively). The high sensitivity inherent to MS/MS determinations combined with the minimal dilution of sample allowed low limits of quantification for all targets, and the developed method results therefore adequate for cocaine and opiates screening and confirmation purposes. The procedure was finally applied to DBSs prepared from whole blood from polydrug abusers, and results were compared with those obtained after a conventional sample pretreatment

  1. Quantitation of sirolimus using liquid chromatography-tandem mass spectrometry (LC-MS-MS).

    PubMed

    Korecka, Magdalena; Shaw, Leslie M

    2010-01-01

    A multiple reaction monitoring positive ion HPLC method with tandem mass spectrometric detection (MS-MS) for determination of sirolimus in human blood samples is described. This method utilizes an online cleanup step that provides simple and rapid sample preparation with a switching valve technique. This procedure includes: instrumentation, API 3000 triple quadrupole with turbo-ion spray (Applied Biosystems, Foster City, CA); HPLC system (Agilent Technologies series 1100, Wilmington, DE); two position switching valve (Valco, Houston, TX); 10 mm guard cartridge (C(18)) used as an extraction column (Perkin Elmer, Norwalk, CT); analytical column (Nova-Pak C(18) column, 2.1 x 150 mm I.D., 4 microm, Waters Corp, Milford, MA) maintained at 65 degrees C; extraction solution, ammonium acetate (30 mM, pH 5.2), flow rate 1.0 mL/min; eluting solution, methanol:30 mM ammonium acetate buffer (pH 5.2, 97:3 v/v), flow rate 0.8 mL/min with 1/3 of the flow split post-column into the MS-MS; total run-time 3.5 min. Sample preparation is based on simple protein precipitation with a mixture of methanol and zinc sulfate (7:3, v/v) followed by online sample cleanup. This procedure provides a decreased sample preparation time by a factor of four compared to a method that uses an SPE column. The first and third quadrupoles were set to detect the ammonium adduct ion and a high mass fragment of sirolimus (m/z 931.8-->864.6), and of an internal standard (ascomycin) (m/z 809.5-->756.5). The lower limit of quantification of this method is 2.5 microg/L. The quantification of drug is made from standard curve using peak-area ratio of analyte vs. internal standard. Calibration curve is constructed using non-weighted linear through zero regression.

  2. Quantification of Galactose-1-Phosphate Uridyltransferase Enzyme Activity by Liquid Chromatography–Tandem Mass Spectrometry

    PubMed Central

    Li, Yijun; Ptolemy, Adam S.; Harmonay, Lauren; Kellogg, Mark; Berry, Gerard T.

    2013-01-01

    Background The diagnosis of galactosemia usually involves the measurement of galactose-1-phosphate uridyltransferase (GALT) activity. Traditional radioactive and fluorescent GALT assays are nonspecific, laborious, and/or lack sufficient analytical sensitivity. We developed a liquid chromatography–tandem mass spectrometry (LC-MS/MS)–based assay for GALT enzyme activity measurement. Method Our assay used stable isotope-labeled α-galactose-1-phosphate ([13C6]-Gal-1-P) as an enzyme substrate. Sample cleanup and separation were achieved by reversed-phase ion-pair chromatography, and the enzymatic product, isotope-labeled uridine diphosphate galactose ([13C6]-UDPGal), was detected by MS/MS at mass transition (571 > 323) and quantified by use of [13C6]-Glu-1-P (265 > 79) as an internal standard. Results The method yielded a mean (SD) GALT enzyme activity of 23.8 (3.8) µmol · (gHgb)−1 · h−1 in erythrocyte extracts from 71 controls. The limit of quantification was 0.04 µmol · (g Hgb)−1 · h−1 (0.2% of normal control value). Intraassay imprecision was determined at 4 different levels (100%, 25%, 5%, and 0.2% of the normal control values), and the CVs were calculated to be 2.1%, 2.5%, 4.6%, and 9.7%, respectively (n = 3). Interassay imprecision CVs were 4.5%, 6.7%, 8.2%, and 13.2% (n = 5), respectively. The assay recoveries at the 4 levels were higher than 90%. The apparent Km of the 2 substrates, Gal-1-P and UDPGlc, were determined to be 0.38 mmol/L and 0.071 mmol/L, respectively. The assay in erythrocytes of 33 patients with classical galactosemia revealed no detectable activity. Conclusions This LC-MS/MS–based assay for GALT enzyme activity will be useful for the diagnosis and study of biochemically heterogeneous patients with galactosemia, especially those with uncommon genotypes and detectable but low residual activities. PMID:20348403

  3. [Determination of congo red in beef by high performance liquid chromatography-tandem quadrupole time of flight mass spectrometry and ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry].

    PubMed

    Lin, Hui; Xu, Chunxiang; Yan, Chunrong; Zhang, Zheng; Wang, Suilou

    2013-09-01

    A method was developed for the determination of congo red in beef. The analyte was identified by high performance liquid chromatography-tandem quadrupole time of flight mass spectrometry (LC-QTOF MS) and quantitatively determined by ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry. After purified by liquid-liquid extraction, the congo red in the beef sample was separated on an Agilent ZORBAX Eclipse Plus C18 Rapid Resolution HD UPLC column (50 mm x 2.1 mm, 1.8 microm) HPLC , using 95% (volume percentage) methanol as the mobile phase at 0.2 mL/min. The detection was performed on an AB 4000 + triple quadrupole mass spectrometer utilizing electrospray ionization (ESI) interface operated in negative ion mode and multiple-reaction monitoring (MRM) mode. The results showed that the linear range of congo red mass concentration was 0.03 - 1 mg/L with the correlation coefficient of 0.999 8. The method had a good precision with the RSDs lower than 5% and the recoveries ranging from 88% to 91%. The limit of detection (LOD) of congo red was 0.01 mg/L. With good reproducibility, the method is simple, fast and effective for the determination of the illegally added congo red in beef and other meat products.

  4. [Determination of sulfonamides in livestock products and seafoods by liquid chromatography/tandem mass spectrometry using glass bead homogenization].

    PubMed

    Fujita, Mizuka; Taguchi, Shuzo; Obana, Hirotaka

    2008-01-01

    A simple and rapid method using liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) was developed for the determination of 25 kinds of sulfonamides in livestock products and seafoods. The sulfonamides were extracted with acetonitrile by glass bead homogenization and cleaned up with a tandem-connected ODS and basic alumina column. The quantification limits of 25 kinds of sulfonamides were 0.0025-0.005 microg/g. When two sulfonamides of specific samples were excluded, the recoveries and relative standard deviations were 70 to 120% and less than 15%. These results show that the developed method, which minimizes the matrix effect, offers high precision and should be useful for the determination of sulfonamides in livestock products and seafoods.

  5. Amide-I relaxation-induced hydrogen bond distortion: An intermediate in electron capture dissociation mass spectrometry of alpha-helical peptides?

    PubMed

    Pouthier, Vincent; Tsybin, Yury O

    2008-09-07

    Electron capture dissociation (ECD) of peptides and proteins in the gas phase is a powerful tool in tandem mass spectrometry whose current description is not sufficient to explain many experimental observations. Here, we attempt to bridge the current understanding of the vibrational dynamics in alpha-helices with the recent experimental results on ECD of alpha-helical peptides through consideration of amide-I relaxation-induced hydrogen bond distortion. Based on a single spine of H-bonded peptide units, we assume that charge neutralization upon electron capture by a charged alpha-helix excites a nearby amide-I mode, which relaxes over a few picoseconds due to Fermi resonances with intramolecular normal modes. The amide-I population plays the role of an external force, which drives the displacements of each peptide unit. It induces a large immobile contraction of the H bonds surrounding the excited site whose lifetime is about the amide-I lifetime. In addition, it creates two lattice deformations describing H bond stretchings, which propagate from the excited region toward both termini of the alpha-helix, get reflected at the termini and yield H bond contractions which move back to the excited region. Consequently, we show that H bonds experience rather large contractions whose amplitude depends on general features such as the position of the amide-I mode, the peptide length and the H bond force constants. When an H bond contraction is sufficiently large, it may promote a hydrogen atom transfer between two neighboring peptide units leading to the formation of a radical at charge site remote carbonyl carbon which is known to be a precursor to the rupture of the corresponding N[Single Bond]C(alpha) bond. The introduced here way of excitation energy generation and transfer may significantly advance ECD understanding and complement existing ECD mechanisms.

  6. Protein Identification Via Surface-Induced Dissociation in an FT-ICR Mass Spectrometer and a Patchwork Sequencing Approach

    SciTech Connect

    Fernandez, Facundo; Wysocki, Vicki H.; Futrell, Jean H.; Laskin, Julia

    2006-05-01

    Surface-induced dissociation (SID) and collision-induced dissociation (CID) are ion activation techniques based on energetic collisions with a surface or gas molecules, respectively. One noticeable difference between CID and SID is that SID does not require a collision gas for ion activation and is therefore directly compatible with the high vacuum requirement of Fourier Transform Ion Cyclotron Resonance mass spectrometers (FT-ICR MS). Eliminating the introduction of collision gas into the ICR cell for collisional activation dramatically shortens the acquisition time for MS/MS experiments, suggesting that SID could be utilized for high-throughput MS/MS studies in FT-ICR MS. We demonstrate for the first time the utility of SID combined with FT-ICR MS for protein identification. Tryptic digests of standard proteins were analyzed using a hybrid 6-Tesla FT-ICR MS with SID and CID capabilities. SID spectra of mass-selected singly and doubly charged peptides were obtained using a diamond-coated target mounted at the rear trapping plate of the ICR cell. The broad internal energy distribution deposited into the precursor ion following collision with the diamond surface allowed a variety of fragmentation channels to be accessed by SID. Composition and sequence qualifiers produced by SID of tryptic peptides were used to improve the statistical significance of database searches. Protein identification MASCOT scores obtained using SID were comparable or better than scores obtained using sustained off-resonance irradiation collision-induced dissociation (SORI-CID) –the conventional ion activation technique in FT-ICR MS.

  7. Threshold collision-induced dissociation measurements using a ring ion guide as the collision cell in a triple-quadrupole mass spectrometer.

    PubMed

    Romanov, Vladimir; Verkerk, Udo H; Siu, Chi-Kit; Hopkinson, Alan C; Siu, K W Michael

    2009-08-15

    A triple-quadrupole mass spectrometer has been modified for bond-dissociation energy measurements via threshold collision-induced dissociations (TCIDs) by replacing the conventional collision cell with a ring ion guide. Optimal operating conditions for the ring ion guide were determined or derived, and validated using a set of complexes for which bond dissociation energies are known. A comparison with reference data (within a range of 16-57 kcal/mol) indicates an accuracy approaching that of TCID determined on a guided ion-beam mass spectrometer. Complexes for which bond-dissociation energies were measured include metal ion complexes of simple ligands, amino acids and peptides, as well as of carbonic acid. There is excellent agreement between our experimental data and literature data, as well as theoretical data determined using a high-level computational method.

  8. Mapping a Noncovalent Protein-Peptide Interface by Top-Down FTICR Mass Spectrometry Using Electron Capture Dissociation

    NASA Astrophysics Data System (ADS)

    Clarke, David J.; Murray, Euan; Hupp, Ted; Mackay, C. Logan; Langridge-Smith, Pat R. R.

    2011-08-01

    Noncovalent protein-ligand and protein-protein complexes are readily detected using electrospray ionization mass spectrometry (ESI MS). Furthermore, recent reports have demonstrated that careful use of electron capture dissociation (ECD) fragmentation allows covalent backbone bonds of protein complexes to be dissociated without disruption of noncovalent protein-ligand interactions. In this way the site of protein-ligand interfaces can be identified. To date, protein-ligand complexes, which have proven tractable to this technique, have been mediated by ionic electrostatic interactions, i.e., ion pair interactions or salt bridging. Here we extend this methodology by applying ECD to study a protein-peptide complex that contains no electrostatics interactions. We analyzed the complex between the 21 kDa p53-inhibitor protein anterior gradient-2 and its hexapeptide binding ligand (PTTIYY). ECD fragmentation of the 1:1 complex occurs with retention of protein-peptide binding and analysis of the resulting fragments allows the binding interface to be localized to a C-terminal region between residues 109 and 175. These finding are supported by a solution-phase competition assay, which implicates the region between residues 108 and 122 within AGR2 as the PTTIYY binding interface. Our study expands previous findings by demonstrating that top-down ECD mass spectrometry can be used to determine directly the sites of peptide-protein interfaces. This highlights the growing potential of using ECD and related top-down fragmentation techniques for interrogation of protein-protein interfaces.

  9. Isotopologue Distributions of Peptide Product Ions by Tandem Mass Spectrometry: Quantitation of Low Levels of Deuterium Incorporation1

    PubMed Central

    Wang, Benlian; Sun, Gang; Anderson, David R.; Jia, Minghong; Previs, Stephen; Anderson, Vernon E.

    2007-01-01

    Protonated molecular peptide ions and their product ions generated by tandem mass spectrometry appear as isotopologue clusters due to the natural isotopic variations of carbon, hydrogen, nitrogen, oxygen and sulfur. Quantitation of the isotopic composition of peptides can be employed in experiments involving isotope effects, isotope exchange, isotopic labeling by chemical reactions, and studies of metabolism by stable isotope incorporation. Both ion trap and quadrupole-time of flight mass spectrometry are shown to be capable of determining the isotopic composition of peptide product ions obtained by tandem mass spectrometry with both precision and accuracy. Tandem mass spectra obtained in profile-mode of clusters of isotopologue ions are fit by non-linear least squares to a series of Gaussian peaks (described in the accompanying manuscript) which quantify the Mn/M0 values which define the isotopologue distribution (ID). To determine the isotopic composition of product ions from their ID, a new algorithm that predicts the Mn/M0 ratios is developed which obviates the need to determine the intensity of all of the ions of an ID. Consequently a precise and accurate determination of the isotopic composition a product ion may be obtained from only the initial values of the ID, however the entire isotopologue cluster must be isolated prior to fragmentation. Following optimization of the molecular ion isolation width, fragmentation energy and detector sensitivity, the presence of isotopic excess (2H, 13C, 15N, 18O) is readily determined within 1%. The ability to determine the isotopic composition of sequential product ions permits the isotopic composition of individual amino acid residues in the precursor ion to be determined. PMID:17559791

  10. Surface-Induced Dissociation of Protein Complexes in a Hybrid Fourier Transform Ion Cyclotron Resonance Mass Spectrometer.

    PubMed

    Yan, Jing; Zhou, Mowei; Gilbert, Joshua D; Wolff, Jeremy J; Somogyi, Árpád; Pedder, Randall E; Quintyn, Royston S; Morrison, Lindsay J; Easterling, Michael L; Paša-Tolić, Ljiljana; Wysocki, Vicki H

    2017-01-03

    Mass spectrometry continues to develop as a valuable tool in the analysis of proteins and protein complexes. In protein complex mass spectrometry studies, surface-induced dissociation (SID) has been successfully applied in quadrupole time-of-flight (Q-TOF) instruments. SID provides structural information on noncovalent protein complexes that is complementary to other techniques. However, the mass resolution of Q-TOF instruments can limit the information that can be obtained for protein complexes by SID. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provides ultrahigh resolution and ultrahigh mass accuracy measurements. In this study, an SID device was designed and successfully installed in a hybrid FT-ICR instrument in place of the standard gas collision cell. The SID-FT-ICR platform has been tested with several protein complex systems (homooligomers, a heterooligomer, and a protein-ligand complex, ranging from 53 to 85 kDa), and the results are consistent with data previously acquired on Q-TOF platforms, matching predictions from known protein interface information. SID fragments with the same m/z but different charge states are well-resolved based on distinct spacing between adjacent isotope peaks, and the addition of metal cations and ligands can also be isotopically resolved with the ultrahigh mass resolution available in FT-ICR.

  11. Identification of isomers and control of ionization and dissociation processes using dual-mass-spectrometer scheme and genetic algorithm optimization

    NASA Astrophysics Data System (ADS)

    Chen, Zhou; Tong, Qiu-Nan; Zhang, Cong-Cong; Hu, Zhan

    2015-04-01

    Identification of acetone and its two isomers, and the control of their ionization and dissociation processes are performed using a dual-mass-spectrometer scheme. The scheme employs two sets of time of flight mass spectrometers to simultaneously acquire the mass spectra of two different molecules under the irradiation of identically shaped femtosecond laser pulses. The optimal laser pulses are found using closed-loop learning method based on a genetic algorithm. Compared with the mass spectra of the two isomers that are obtained with the transform limited pulse, those obtained under the irradiation of the optimal laser pulse show large differences and the various reaction pathways of the two molecules are selectively controlled. The experimental results demonstrate that the scheme is quite effective and useful in studies of two molecules having common mass peaks, which makes a traditional single mass spectrometer unfeasible. Project supported by the National Basic Research Program of China (Grant No. 2013CB922200) and the National Natural Science Foundation of China (Grant No. 11374124).

  12. Fragmentation of mycosporine-like amino acids by hydrogen/deuterium exchange and electrospray ionisation tandem mass spectrometry.

    PubMed

    Cardozo, Karina H M; Carvalho, Valdemir M; Pinto, Ernani; Colepicolo, Pio

    2006-01-01

    The determin