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Mass Accuracy (mass + accuracy)

Distribution by Scientific Domains
Chemistry91%

Kinds of Mass Accuracy

  • high mass accuracy
    		•

    Selected Abstracts

    Using a triple-quadrupole mass spectrometer in accurate mass mode and an ion correlation program to identify compounds,

    RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 18 2005
    Andrew H. Grange
    Atomic masses and isotopic abundances are independent and complementary properties for discriminating among ion compositions. The number of possible ion compositions is greatly reduced by accurately measuring exact masses of monoisotopic ions and the relative isotopic abundances (RIAs) of the ions greater in mass by +1,Da and +2,Da. When both properties are measured, a mass error limit of 6,10,mDa (<,31,ppm at 320,Da) and an RIA error limit of 10% are generally adequate for determining unique ion compositions for precursor and fragment ions produced from small molecules (less than 320,Da in this study). ,Inherent interferences', i.e., mass peaks seen in the product ion mass spectrum of the monoisotopic [M+H]+ ion of an analyte that are ,2, ,1, +1, or +2,Da different in mass from monoisotopic fragment ion masses, distort measured RIAs. This problem is overcome using an ion correlation program to compare the numbers of atoms of each element in a precursor ion to the sum of those in each fragment ion and its corresponding neutral loss. Synergy occurs when accurate measurement of only one pair of +1,Da and +2,Da RIAs for the precursor ion or a fragment ion rejects all but one possible ion composition for that ion, thereby indirectly rejecting all but one fragment ion-neutral loss combination for other exact masses. A triple-quadrupole mass spectrometer with accurate mass capability, using atmospheric pressure chemical ionization (APCI), was used to measure masses and RIAs of precursor and fragment ions. Nine chemicals were investigated as simulated unknowns. Mass accuracy and RIA accuracy were sufficient to determine unique compositions for all precursor ions and all but two of 40 fragment ions, and the two corresponding neutral losses. Interrogation of the chemical literature provided between one and three possible compounds for each of the nine analytes. This approach for identifying compounds compensates for the lack of commercial ESI and APCI mass spectral libraries, which precludes making tentative identifications based on spectral matches. Published in 2005 by John Wiley & Sons, Ltd. [source]

    Analysis of S -adenosylmethionine and related sulfur metabolites in bacterial isolates of Pseudomonas aeruginosa (BAA-47) by liquid chromatography/electrospray ionization coupled to a hybrid linear quadrupole ion trap and Fourier transform ion cyclotron resonance mass spectrometry

    RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 21 2009
    Tommaso R. I. Cataldi
    A comprehensive and highly selective method for detecting in bacterial supernatants a modified sulfur nucleoside, S -adenosyl-L-methionine (SAM), and its metabolites, i.e., S -adenosylhomocysteine (SAH), adenosine (Ado), 5,-deoxy-5,-methylthioadenosine (MTA), adenine (Ade), S -adenosyl-methioninamine (dcSAM), homocysteine (Hcy) and methionine (Met), was developed. The method is based on reversed-phase liquid chromatography with positive electrospray ionization (ESI+) coupled to a hybrid linear quadrupole ion trap (LTQ) and 7-T Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS). A gradient elution was employed with a binary solvent of 0.05,M ammonium formate at pH 4 and acetonitrile. The assay involves a simultaneous cleanup of cell-free bacterial broths by solid-phase extraction and trace enrichment of metabolites with a 50-fold concentration factor by using immobilized phenylboronic and anion-exchange cartridges. While the quantitative determination of SAM was performed using stable-isotope-labeled SAM-d3 as an internal standard, in the case of Met and Ade, Met- 13C and Ade- 15N2 were employed as isotope-labeled internal standards, respectively. This method enabled the identification of SAM and its metabolites in cell-free culture of Pseudomonasaeruginosa grown in Davis minimal broth (formulation without sulphur organic compounds), with routine sub-ppm mass accuracies (,0.27,±,0.68,ppm). The resulting contents of SCSS -SAM, SS -dcSAM, MTA, Ado and Met in the free-cell supernatant of P. aeruginosa was 56.4,±,2.1,nM, 32.2,±,2.2,nM, 0.91,±,0.10,nM, 19.6,±,1.2,nM and 1.93,±,0.02,µM (mean,±,SD, n,=,4 extractions), respectively. We report also the baseline separation (Rs ,1.5) of both diastereoisomeric forms of SAM (SCSS and SCRS) and dcSAM (SS and RS), which can be very useful to establish the relationship between the biologically active versus the inactive species, SCSS/SCRS and SS/RS of SAM and dcSAM, respectively. An additional confirmation of SAM-related metabolites was accomplished by a systematic study of their MS/MS spectra. Copyright © 2009 John Wiley & Sons, Ltd. [source]

    Electron transfer dissociation in the hexapole collision cell of a hybrid quadrupole-hexapole Fourier transform ion cyclotron resonance mass spectrometer

    RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 3 2008
    Desmond A. Kaplan
    Electron transfer dissociation (ETD) of proteins is demonstrated in a hybrid quadrupole-hexapole Fourier transform ion cyclotron resonance mass spectrometer (Qh-FTICRMS). Analyte ions are selected in the mass analyzing quadrupole, accumulated in the hexapole linear ion trap, reacted with fluoranthene reagent anions, and then analyzed via an FTICR mass analyzer. The hexapole trap allows for a broad fragment ion mass range and a high ion storage capacity. Using a 3,T FTICRMS, resolutions of 60,000 were achieved with mass accuracies averaging below 1.4,ppm. The high resolution, high mass accuracy ETD spectra provided by FTICR obviates the need for proton transfer reaction (PTR) charge state reduction of ETD product ions when analyzing proteins or large peptides. This is demonstrated with the ETD of ubiquitin and apomyoglobin yielding sequence coverages of 37 and 20%, respectively. We believe this represents the first reported successful combination of ETD and a FTICRMS. Copyright © 2008 John Wiley & Sons, Ltd. [source]

    Mass spectral characterization of phloroglucinol derivatives hyperforin and adhyperforin

    RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 18 2006
    Lekha Sleno
    Active phloroglucinol constituents of Hypericum perforatum (St. John's wort) extracts, hyperforin and adhyperforin, have been studied following ion activation using tandem mass spectrometry (MS/MS) and complemented by accurate mass measurements. These two compounds were readily analyzed as protonated and deprotonated molecules with electrospray ionization. MS/MS and MS3 data from a quadrupole-linear ion trap tandem mass spectrometer were employed to elucidate fragmentation pathways. Fourier transform ion cyclotron resonance measurements afforded excellent mass accuracies for the confirmation of elemental formulae of product ions formed via infrared multiphoton dissociation and sustained off-resonance irradiation collision-induced dissociation. Fragmentation schemes have been devised for the dissociation of hyperforin and adhyperforin in negative and positive ion modes. This information is expected to be especially valuable for the characterization of related compounds, such as degradation products, metabolites and novel synthetic analogs of hyperforin. Copyright © 2006 John Wiley & Sons, Ltd. [source]

    Accurate mass measurement in nano-electrospray ionization mass spectrometry by alternate switching of high voltage between sample and reference sprayers

    RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 4 2005
    Yoshinori Satomi
    An electrospray dual sprayer, which generates separate sample and reference sprays by alternately switching the high voltage between the two sprayers, is described. The technique permits accurate mass measurements in nano-electrospray ionization mass spectrometry (ESI-MS) to be obtained using a quadrupole/orthogonal acceleration time-of-flight mass spectrometer (Q-TOF). Similar to the method employed with a dual ESI source (Wolff JC et al., Anal. Chem. 2001; 73: 2605), the two sprays are orthogonal with respect to each other, but can be independently sampled without any baffle between these sprays. The reference sprayer is used in the original configuration of the ESI source and was optimized for a 1,2,,L/min flow, whereas the sample sprayer can be either a conventional glass capillary or a borosilicate tip of the type used for nano-ESI. Both sprayers can be positioned close to the cone so as to give maximum ion currents. The sample and reference sprays are independently generated by raising the potentials on the sample and reference sprayers to 1.4 and 3.0,kV, respectively; the high voltages can be rapidly turned on and off in ca. 1,ms. A nano-ESI-MS or nano-flow LC/ESI-MS experiment using a Q-TOF coupled with the above system gave mass accuracies within 3,ppm for measurements of ions up to m/z 1000 using subpicomole samples. Copyright © 2005 John Wiley & Sons, Ltd. [source]

    Differentiation of structural isomers in a target drug database by LC/Q-TOFMS using fragmentation prediction

    DRUG TESTING AND ANALYSIS, Issue 6 2010
    Elli Tyrkkö
    Abstract Isomers cannot be differentiated from each other solely based on accurate mass measurement of the compound. A liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/Q-TOFMS) method was used to systematically fragment a large group of different isomers. Two software programs were used to characterize in silico mass fragmentation of compounds in order to identify characteristic fragments. The software programs employed were ACD/MS Fragmenter (ACD Labs Toronto, Canada), which uses general fragmentation rules to generate fragments based on the structure of a compound, and SmartFormula3D (Bruker Daltonics), which assigns fragments from a mass spectra and calculates the molecular formulae for the ions using accurate mass data. From an in-house toxicology database of 874 drug substances, 48 isomer groups comprising 111 compounds, for which a reference standard was available, were found. The product ion spectra were processed with the two software programs and 1,3 fragments were identified for each compound. In 82% of the cases, the fragment could be identified with both software programs. Only 10 isomer pairs could not be differentiated from each other based on their fragments. These compounds were either diastereomers or position isomers undergoing identical fragmentation. Accurate mass data could be utilized with both software programs for structural elucidation of the fragments. Mean mass accuracy and isotopic pattern match values (SigmaFit; Bruker Daltonics Bremen, Germany) were 0.9 mDa and 24.6 mSigma, respectively. The study introduces a practical approach for preliminary compound identification in a large target database by LC/Q-TOFMS without necessarily possessing reference standards. Copyright © 2010 John Wiley & Sons, Ltd. [source]

    CE- and HPLC-TOF-MS for the characterization of phenolic compounds in olive oil

    ELECTROPHORESIS, Issue 5 2007
    Alegría Carrasco-Pancorbo
    Abstract We present an easy and rapid method for the analysis of phenolic compounds in extra-virgin olive oil by CZE coupled with ESI-TOF-MS. Optimum electrophoretic separation was obtained using a basic carbonate electrolyte. We thus achieved the determination of several important families (phenyl alcohols, phenyl acids, lignans, flavonoids, and secoiridoids) of the polar fraction of the olive oil. Furthermore, other "unknown" compounds were also identified. In addition to the CZE method, HPLC analyses were made, separating compounds belonging to the main families present in this polyphenolic fraction, as well as other new compounds. We compared the results obtained with both techniques and found it was possible to determine more than 45 compounds with both methods. The sensitivity, together with mass accuracy and true isotopic pattern of the TOF-MS, allowed the identification of a broad series of known and so far not described phenolic compounds present in extra-virgin olive oil. [source]

    Glycoform characterization of erythropoietin combining glycan and intact protein analysis by capillary electrophoresis , electrospray , time-of-flight mass spectrometry

    ELECTROPHORESIS, Issue 13 2006
    Elvira Balaguer
    Abstract Glycosylation of recombinant human erythropoietin (rHuEPO) is a post-translational process that alters biological activity, solubility and lifetime of the glycoprotein in blood, and strongly depends on the type of cell and the cell culture conditions. A fast and simple method providing extensive carbohydrate information about the glycans present in rHuEPO and other glycoproteins is needed in order to improve current methods in drug development or product quality control. Here, an improved method for intact rHuEPO glycoform characterization by CZE-ESI-TOF MS has been developed using a novel capillary coating and compared to a previous study. Both methods allow a fast separation in combination with accurate mass characterization of the single protein isoforms. The novel dynamic coating provides a separation at an EOF close to zero, enabling better separation. This results in an improved mass spectrometric resolution and the detection of minor isoforms. In order to assign an unequivocal carbohydrate composition to every intact glycoform, a CZE-ESI-MS separation method for enzymatically released underivatized N -glycans has been developed. The TOF,MS allows the correct identification of the glycans due to its high mass accuracy and resolution. Therefore, glycan modifications such as acetylation, oxidation, sulfation and even the exchange of OH by NH2 are successfully characterized. Information of the protein-backbone molecular mass has been combined with results from peptide analysis (revealing information about O -glycosylation) and from the glycan analysis, including the detection of as yet undescribed glycans containing four antennae and five sialic acids. This allows an unequivocal assignment of an overall glycosylation composition to the molecular masses obtained for the intact rHuEPO glycoforms. [source]

    Analysis of the glucosinolate pattern of Arabidopsis thaliana seeds by capillary zone electrophoresis coupled to electrospray ionization-mass spectrometry

    ELECTROPHORESIS, Issue 7-8 2005
    Gerhard Bringmann
    Abstract An easy and rapid method for the analysis of intact, non-desulfated glucosinolates by capillary zone electrophoresis (CZE) coupled to electrospray ionization-time of flight-mass spectrometry (ESI-TOF-MS) is described. Surprisingly, an electrolyte and a sheath liquid based on formic acid provided the best results. In this strongly acidic system, the glucosinolates were separated and detected as anions, resulting in an excellent selectivity. Thus, crude plant extracts could be analyzed without any interference of matrix constituents. The sensitivity together with mass accuracy and true isotopic pattern of the TOF-MS allowed identification of a broad series of glucosinolates in Arabidopsis thaliana seeds. [source]

    A fast, reproducible and low-cost method for sequence deconvolution of ,on-bead' peptides via ,on-target' maldi-TOF/TOF mass spectrometry

    JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 3 2010
    Giulio A. Amadei
    Abstract A novel approach to high-throughput sequence deconvolution of on-bead small peptides (MW < 2000 Da) using on-target MALDI-TOF/TOF instrumentation is presented. Short peptides of pentamer and octamer length, covalently attached to TentaGel polystyrene beads through a photolabile linker, were placed onto the MALDI target, apportioned with suitable matrix (2,5-dihydroxybenzoic acid) and then hit with the instrument laser (Nd : YAG, 355 nm). This induced easy and highly reproducible photochemical cleavage, desorption (MS mode) and fragmentation (MS/MS mode). Peptide fragments were identified with a mass accuracy of 0.1 Da of the expected values. This technique significantly accelerates the sequence determination of positive peptide hits obtained from random combinatorial libraries when screening against biological targets, paving the way for a rapid and efficient method to identify molecular imaging ligands specific to pathological targets in cancer and other diseases. Copyright © 2009 John Wiley & Sons, Ltd. [source]

    Modern MALDI time-of-flight mass spectrometry

    JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 3 2009
    Marvin L. Vestal
    Abstract This paper focuses on development of time-of-flight (TOF) mass spectrometry in response to the invention of matrix-assisted laser desorption/ionization (MALDI). Before this breakthrough ionization technique for nonvolatile molecules, TOF was generally considered as a useful tool for exotic studies of ion properties but was not widely applied to analytical problems. Improved TOF instruments and software that allow the full potential power of MALDI to be applied to difficult biological applications are described. A theoretical approach to the design and optimization of MALDI-TOF instruments for particular applications is presented. Experimental data are provided that are in excellent agreement with theoretical predictions of resolving power and mass accuracy. Data on sensitivity and dynamic range using kilohertz laser rates are also summarized. These results indicate that combinations of high-performance MALDI-TOF and TOF-TOF with off-line high-capacity separations may ultimately provide throughput and dynamic range several orders of magnitude greater than those currently available with electrospray LC-MS and MS-MS. Copyright © 2009 John Wiley & Sons, Ltd. [source]

    Matrix effects on accurate mass measurements of low-molecular weight compounds using liquid chromatography-electrospray-quadrupole time-of-flight mass spectrometry,

    JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 3 2006
    F. Calbiani
    Abstract Liquid chromatography (LC) with high-resolution mass spectrometry (HRMS) represents a powerful technique for the identification and/or confirmation of small molecules, i.e. drugs, metabolites or contaminants, in different matrices. However, reliability of analyte identification by HRMS is being challenged by the uncertainty that affects the exact mass measurement. This parameter, characterized by accuracy and precision, is influenced by sample matrix and interferent compounds so that questions about how to develop and validate reliable LC-HRMS-based methods are being raised. Experimental approaches for studying the effects of various key factors influencing mass accuracy on low-molecular weight compounds (MW < 150 Da) when using a quadrupole-time-of-flight (QTOF) mass analyzer were described. Biogenic amines in human plasma were considered for the purpose and the effects of peak shape, ion abundance, resolution and data processing on accurate mass measurements of the analytes were evaluated. In addition, the influence of the matrix on the uncertainty associated with their identification and quantitation is discussed. A critical evaluation on the calculation of the limits of detection was carried out, considering the uncertainty associated with exact mass measurement of HRMS-based methods. The minimum concentration level of the analytes that was able to provide a statistical error lower than 5 ppm in terms of precision was 10 times higher than those calculated with S/N = 3, thus suggesting the importance of considering both components of exact mass measurement uncertainty in the evaluation of the limit of detection. Copyright © 2006 John Wiley & Sons, Ltd. [source]

    Site-specific detection of S -nitrosylated PKB ,/Akt1 from rat soleus muscle using CapLC-Q-TOFmicro mass spectrometry

    JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 9 2005
    Xiao-Ming Lu
    Abstract Protein Kinase B,(PKB,, or Akt1) is believed to play a crucial role in programmed cell death, cancer progression and the insulin-signaling cascade. The protein is activated by phosphorylation at multiple sites and subsequently phosphorylates and activates eNOS. Free cysteine residues of the protein may capture reactive, endogenously produced nitric oxide (NO) as S -nitrosothiols. Site-specific detection of S -nitrosylated cysteine residues, usually at low stoichiometry, has been a major challenge in proteomic research largely due to the lack of mass marker for S -nitrosothiols that are very labile under physiologic conditions. In this report we describe a sensitive and specific MS method for detection of S -nitrosothiols in PKB ,/Akt1 in rat soleus muscle. PKB ,/Akt1 was isolated by immunoprecipitation and 2D-gel electrophoresis, subjected to in-gel tryptic digestion, and cysteinyl nitrosothiols were reacted with iodoacetic acids [2-C12/C13 = 50/50] under ascorbate reduction conditions. This resulted in the production of relatively stable carboxymethylcysteine (CMC) immonium ions (m/z 134.019 and m/z 135.019) within a narrow argon collision energy (CE = 30 ± 5 V) in the high MS noise region. In addition, free and disulfide-linked cysteine residues were converted to carboxyamidomethylcysteines (CAM). Tryptic S -nitrosylated parent ion was detected with a mass accuracy of 50 mDa for the two CMC immonium ions at the triggered elution time during capillary liquid chromatography (LC) separation. A peptide containing Cys296 was discriminated from four co-eluting tryptic peptides under lock mass conditions (m/z 785.8426). S -nitrosothiol in the tryptic peptide, ITDFGLBKEGIK (B: CAM, [M + 2H]2+ = 690.86, Found: 690.83), is believed to be present at a very low level, since the threshold for the CMC immonium trigger ions was set at 3 counts/s in the MS survey. The high levels of NO that are produced under stress conditions may result in increased S -nitrosylation of Cys296 which blocks disulfide bond formation between Cys296 and Cys310 and suppresses the biological effects of PKB ,/Akt1. With the procedures developed here, this process can be studied under physiological and pathological conditions. Copyright © 2005 John Wiley & Sons, Ltd. [source]

    The Orbitrap: a new mass spectrometer

    JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 4 2005
    Qizhi Hu
    Abstract Research areas such as proteomics and metabolomics are driving the demand for mass spectrometers that have high performance but modest power requirements, size, and cost. This paper describes such an instrument, the Orbitrap, based on a new type of mass analyzer invented by Makarov. The Orbitrap operates by radially trapping ions about a central spindle electrode. An outer barrel-like electrode is coaxial with the inner spindlelike electrode and mass/charge values are measured from the frequency of harmonic ion oscillations, along the axis of the electric field, undergone by the orbitally trapped ions. This axial frequency is independent of the energy and spatial spread of the ions. Ion frequencies are measured non-destructively by acquisition of time-domain image current transients, with subsequent fast Fourier transforms (FFTs) being used to obtain the mass spectra. In addition to describing the Orbitrap mass analyzer, this paper also describes a complete Orbitrap-based mass spectrometer, equipped with an electrospray ionization source (ESI). Ions are transferred from the ESI source through three stages of differential pumping using RF guide quadrupoles. The third quadrupole, pressurized to less than 10,3 Torr with collision gas, acts as an ion accumulator; ion/neutral collisions slow the ions and cause them to pool in an axial potential well at the end of the quadrupole. Ion bunches are injected from this pool into the Orbitrap analyzer for mass analysis. The ion injection process is described in a simplified way, including a description of electrodynamic squeezing, field compensation for the effects of the ion injection slit, and criteria for orbital stability. Features of the Orbitrap at its present stage of development include high mass resolution (up to 150 000), large space charge capacity, high mass accuracy (2,5 ppm), a mass/charge range of at least 6000, and dynamic range greater than 10.3 Applications based on electrospray ionization are described, including characterization of transition-metal complexes, oligosaccharides, peptides, and proteins. Use is also made of the high-resolution capabilities of the Orbitrap to confirm the presence of metaclusters of serine octamers in ESI mass spectra and to perform H/D exchange experiments on these ions in the storage quadrupole. Copyright © 2005 John Wiley & Sons, Ltd. [source]

    Negative and positive ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and positive ion nano-electrospray ionization quadrupole ion trap mass spectrometry of peptidoglycan fragments isolated from various Bacillus species

    JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 2 2001
    Gerold Bacher
    Abstract A general approach for the detailed characterization of sodium borohydride-reduced peptidoglycan fragments (syn. muropeptides), produced by muramidase digestion of the purified sacculus isolated from Bacillus subtilis (vegetative cell form of the wild type and a dacA mutant) and Bacillus megaterium (endospore form), is outlined based on UV matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and nano-electrospray ionization (nESI) quadrupole ion trap (QIT) mass spectrometry (MS). After enzymatic digestion and reduction of the resulting muropeptides, the complex glycopeptide mixture was separated and fractionated by reversed-phase high-performance liquid chromatography. Prior to mass spectrometric analysis, the muropeptide samples were subjected to a desalting step and an aliquot was taken for amino acid analysis. Initial molecular mass determination of these peptidoglycan fragments (ranging from monomeric to tetrameric muropeptides) was performed by positive and negative ion MALDI-MS using the thin-layer technique with the matrix ,-cyano-4-hydroxycinnamic acid. The results demonstrated that for the fast molecular mass determination of large sample numbers in the 0.8,10 pmol range and with a mass accuracy of ±0.07%, negative ion MALDI-MS in the linear TOF mode is the method of choice. After this kind of muropeptide screening often a detailed primary structural analysis is required owing to ambiguous data. Structural data could be obtained from peptidoglycan monomers by post-source decay (PSD) fragment ion analysis, but not from dimers or higher oligomers and not with the necessary sensitivity. Multistage collision-induced dissociation (CID) experiments performed on an nESI-QIT instrument were found to be the superior method for structural characterization of not only monomeric but also of dimeric and trimeric muropeptides. Up to MS4 experiments were sometimes necessary to obtain unambiguous structural information. Three examples are presented: (a) CID MSn (n = 2,4) of a peptidoglycan monomer (disaccharide-tripeptide) isolated from B. subtilis (wild type, vegetative cell form), (b) CID MSn (n = 2,4) of a peptidoglycan dimer (bis-disaccharide-tetrapentapeptide) obtained from a B. subtilis mutant (vegetative cell form) and (c) CID MS2 of a peptidoglycan trimer (a linear hexasaccharide with two peptide side chains) isolated from the spore cortex of B. megaterium. All MSn experiments were performed on singly charged precursor ions and the MS2 spectra were dominated by fragments derived from interglycosidic bond cleavages. MS3 and MS4 spectra exhibited mainly peptide moiety fragment ions. In case of the bis-disaccharide-tetrapentapeptide, the peptide branching point could be determined based on MS3 and MS4 spectra. The results demonstrate the utility of nESI-QIT-MS towards the facile determination of the glycan sequence, the peptide linkage and the peptide sequence and branching of purified muropeptides (monomeric up to trimeric forms). The wealth of structural information generated by nESI-QIT-MSn is unsurpassed by any other individual technique. Copyright © 2001 John Wiley & Sons, Ltd. [source]

    The minotaur proteome: Avoiding cross-species identifications deriving from bovine serum in cell culture models

    PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 16 2010
    Jakob Bunkenborg
    Abstract Cell culture is a fundamental tool in proteomics where mammalian cells are cultured in vitro using a growth medium often supplemented with 5,15% FBS. Contamination by bovine proteins is difficult to avoid because of adherence to the plastic vessel and the cultured cells. We have generated peptides from bovine serum using four sample preparation methods and analyzed the peptides by high mass accuracy LC-MS/MS. Distinguishing between bovine and human peptides is difficult because of a considerable overlap of identical tryptic peptide sequences. Pitfalls in interpretation, different database search strategies to minimize erroneous identifications and an augmented contaminant database are presented. [source]

    High-resolution imaging and proteomics of peptide fragments by TOF-SIMS

    PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 8 2010
    Håkan Nygren
    Abstract Thyroglobulin is an iodinated glycoprotein (m.w. 660,kD) required for the storage and formation of thyroid hormone. Thyroglobulin was digested by trypsin in distilled water and the resulting peptides were identified by TOF-secondary ion mass spectrometry, using TFA as a matrix to catalyze the ionization of the peptides. Cryostate sections of pig thyroid glands were incubated with trypsin in distilled water, followed by deposition of TFA. The sections were analyzed with TOF-secondary ion mass spectrometry, and the peptides formed were identified through comparison with the peptides of the thyroglobulin reference sample. The thyroglobulin fragments were localized in the thyroid follicle cells with a spatial resolution of 3 microns, a mass resolution m/,m of >6000 and a mass accuracy of <60,ppm. The thyroglobulin was found localized heterogeneously in the follicle cells. The heterogeneity may be due to thyroglobulin synthesis, uptake and degradation or globules representing insoluble polymers of thyroglobulin considered to be a mechanism for storing hormone at high concentrations. [source]

    De novo sequencing of peptides by MS/MS

    PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 4 2010
    Joerg Seidler
    Abstract The current status of de novo sequencing of peptides by MS/MS is reviewed with focus on collision cell MS/MS spectra. The relation between peptide structure and observed fragment ion series is discussed and the exhaustive extraction of sequence information from CID spectra of protonated peptide ions is described. The partial redundancy of the extracted sequence information and a high mass accuracy are recognized as key parameters for dependable de novo sequencing by MS. In addition, the benefits of special techniques enhancing the generation of long uninterrupted fragment ion series for de novo peptide sequencing are highlighted. Among these are terminal 18O labeling, MSn of sodiated peptide ions, N-terminal derivatization, the use of special proteases, and time-delayed fragmentation. The emerging electron transfer dissociation technique and the recent progress of MALDI techniques for intact protein sequencing are covered. Finally, the integration of bioinformatic tools into peptide de novo sequencing is demonstrated. [source]

    Increasing the mass accuracy of high-resolution LC-MS data using background ions , a case study on the LTQ-Orbitrap

    PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 22 2008
    Richard A. Scheltema
    Abstract With the advent of a new generation of high-resolution mass spectrometers, the fields of proteomics and metabolomics have gained powerful new tools. In this paper, we demonstrate a novel computational method that improves the mass accuracy of the LTQ-Orbitrap mass spectrometer from an initial ±1,2,ppm, obtained by the standard software, to an absolute median of 0.21,ppm (SD 0.21,ppm). With the increased mass accuracy it becomes much easier to match mass chromatograms in replicates and different sample types, even if compounds are detected at very low intensities. The proposed method exploits the ubiquitous presence of background ions in LC-MS profiles for accurate alignment and internal mass calibration, making it applicable for all types of MS equipment. The accuracy of this approach will facilitate many downstream systems biology applications, including mass-based molecule identification, ab initio metabolic network reconstruction, and untargeted metabolomics in general. [source]

    A strategy for high-resolution protein identification in surface-enhanced laser desorption/ionization mass spectrometry: Calgranulin A and chaperonin 10 as protein markers for endometrial carcinoma

    PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 7 2005
    Jingzhong Guo
    Abstract Surface-enhanced laser desorption/ionization-mass spectrometry (SELDI-MS) has conventionally been practiced on linear time of flight (TOF) which has low mass accuracy and resolution. Here we demonstrate in an examination of both malignant and nonmalignant endometrial tissue homogenates that high mass accuracy and resolution in the MS stage are crucial. Using a commercially available quadrupole/TOF (QqTOF), we were able to resolve two potential cancer markers, subsequently identified off-line as chaperonin 10 and calgranulin A, that differ by 8 Da in mass. Two off-line protein identification protocols were developed: the first was based on size-exclusion chromatography (SEC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein extraction, trypsin digestion, and matrix-assisted laser desorption/ionization-tandem MS (MALDI-MS/MS); the second on SEC and shotgun nano-liquid chromatography (nanoLC)-MS/MS. Analyses on a cohort of 44 endometrial homogenates showed 22 out of 23 nonmalignant samples had nondetectable to very low abundance of chaperonin 10 and calgranulin A; 17 of the 21 malignant samples had detectable to abundant levels of both proteins. Immunohistochemical staining of a tissue microarray of 32 samples showed that approximately half of malignant endometrial tissues exhibited positive staining for calgranulin A in the malignant epithelium, while 9 out of 10 benign tissues exhibited negative epithelial staining. In addition, macrophages/granulocytes in malignant as well as nonmalignant tissues showed positive staining. No immunostaining occurred in stroma or myometrium. Calgranulin A, in combination with chaperonin 10 and other proteins, may eventually constitute a panel of markers to permit diagnosis and screening of endometrial cancer. [source]

    High-resolution extracted ion chromatography, a new tool for metabolomics and lipidomics using a second-generation orbitrap mass spectrometer

    RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 10 2009
    Albert Koulman
    Most analytical methods in metabolomics are based on one of two strategies. The first strategy is aimed at specifically analysing a limited number of known metabolites or compound classes. Alternatively, an unbiased approach can be used for profiling as many features as possible in a given metabolome without prior knowledge of the identity of these features. Using high-resolution mass spectrometry with instruments capable of measuring m/z ratios with sufficiently low mass measurement uncertainties and simultaneous high scan speeds, it is possible to combine these two strategies, allowing unbiased profiling of biological samples and targeted analysis of specific compounds at the same time without compromises. Such high mass accuracy and mass resolving power reduces the number of candidate metabolites occupying the same retention time and m/z ratio space to a minimum. In this study, we demonstrate how targeted analysis of phospholipids as well as unbiased profiling is achievable using a benchtop orbitrap instrument after high-speed reversed-phase chromatography. The ability to apply both strategies in one experiment is an important step forward in comprehensive analysis of the metabolome. Copyright © 2009 John Wiley & Sons, Ltd. [source]

    A new approach to aid the characterisation and identification of metabolites of a model drug; partial isotope enrichment combined with novel formula elucidation software

    RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 2 2009
    Kirsten Hobby
    This work describes the identification of ,isotopically enriched' metabolites of 4-cyanoaniline using the unique features of the software package ,Spectral Simplicity'. The software is capable of creating the theoretical mass spectra for partially isotope-enriched compounds, and subsequently performing an elemental composition analysis to give the elemental formula for the ,isotopically enriched' metabolite. A novel mass spectral correlation method, called ,FuzzyFit', was employed. ,FuzzyFit' utilises the expected experimental distribution of errors in both mass accuracy and isotope pattern and enables discrimination between statistically probable and improbable candidate formulae. The software correctly determined the molecular formulae of ten previously described metabolites of 4-cyanoaniline confirming the technique of partial isotope enrichment can produce results analogous to standard methodologies. Six previously unknown species were also identified, based on the presence of the unique ,designer' isotope ratio. Three of the unknowns were tentatively identified as N-acetylglutamine, O-methyl-N acetylglucuronide and a putative fatty acid conjugate. The discovery of a significant number of unknown species of a model drug with a comprehensive history of investigation highlights the potential for enhancement to the analytical process by the use of ,designer' isotope ratio compounds. The ,FuzzyFit' methodology significantly aided the elucidation of candidate formulae, by provision of a vastly simplified candidate formula data set. Copyright © 2008 John Wiley & Sons, Ltd. [source]

    Metabolism of olaquindox in rat liver microsomes: structural elucidation of metabolites by high-performance liquid chromatography combined with ion trap/time-of-flight mass spectrometry,

    RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 7 2008
    Zhaoying Liu
    Olaquindox (N -(2-hydroxyethyl)-3-methyl-2-quinoxalincarboxamide-1,4-dioxide) is a growth-promoting feed additive for food-producing animals. Its toxicity is closely related to the metabolism. The complete metabolic pathways of olaquindox are not revealed. To improve studies of the metabolism and toxicity of olaquindox, its biotransformation in rat liver microsomes and the structure of its metabolites using high-performance liquid chromatography combined with ion trap/time-of-flight mass spectrometry (LC/MS-ITTOF) were investigated. When olaquindox was incubated with an NADPH-generating system and rat liver microsomes, ten metabolites (M1,M10) were detected. The structures of these metabolites were identified from mass spectra and comparison of their changes in their accurate molecular masses and fragment ions with those of the parent drug. With the high resolution and good mass accuracy achieved by this technique, the elemental compositions of the metabolites and their fragment ions were exactly determined. The results indicate that the N,,,O group reduction is the main metabolic pathway of olaquindox metabolism in rat liver microsomes, because abundant 1-desolaquindox (M2), 4-desolaquindox (M1) and bisdesoxyolaquindox (M9) were produced during the incubation step. Seven other minor metabolites were revealed which were considered to be hydroxylation metabolites, based on the position of the quinoxaline ring or 3-methyl group and a carboxylic acid derivative on the side chain at position 2 of the quinoxaline ring. Among the identified metabolites, five new hydroxylated metabolites (M3,M7) were found for the first time in rat liver microsomes. This work will conduce to complete clarification of olaquindox metabolism, and improve the in vivo metabolism of olaquindox in food animals. Copyright © 2008 John Wiley & Sons, Ltd. [source]

    A new hybrid electrospray Fourier transform mass spectrometer: design and performance characteristics

    RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 2 2006
    Peter B. O'Connor
    A new hybrid electrospray quadrupole Fourier transform mass spectrometry (FTMS) instrument design is shown and characterized. This instrument involves coupling an electrospray source and mass-resolving quadrupole, ion accumulation, and collision cell linear ion trap system developed by MDS Sciex with a home-built ion guide and ion cyclotron resonance (ICR) cell. The iterative progression of this design is shown. The final design involves a set of hexapole ion guides to transfer the ions from the accumulation/collision trap through the magnetic field gradient and into the cell. These hexapole ion guides are separated by a thin gate valve and two conduction limits to maintain the required <10,9,mbar vacuum for FTICR. Low-attomole detection limits for a pure peptide are shown, 220,000 resolving power in broadband mode and 820,000 resolving power in narrow-band mode are demonstrated, and mass accuracy in the <2,ppm range is routinely available provided the signal is abundant, cleanly resolved, and internally calibrated. This instrument design provides high experimental flexibility, allowing Q2 CAD, SORI-CAD, IRMPD, and ECD experiments with selected ion accumulation as well as experiments such as nozzle skimmer dissociation. Initial top-down mass spectrometry experiments on a protein is shown using ECD. Copyright © 2005 John Wiley & Sons, Ltd. [source]

    ,All-in-One' analysis for metabolite identification using liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry with collision energy switching

    RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 18 2005
    Mark Wrona
    The removal of bottlenecks in discovery stage metabolite identification studies is an ongoing challenge for the pharmaceutical industry. We describe the use of an ,All-in-One' approach to metabolite characterization that leverages the fast scanning and high mass accuracy of hybrid quadrupole time-of-flight mass spectrometry (QqToFMS) instruments. Full-scan MS and MS/MS data is acquired using collision energy switching without the preselection, either manually or in a data-dependent manner, of precursor ions. The acquisition of ,clean' MS/MS data is assisted by the use of ultrahigh-performance chromatography. Data acquired using this method can then be mined post-acquisition in a number of ways. These include using narrow window extracted ion chromatograms (nwXICs) for expected biotransformations, XICs for the product ions of the parent compound and/or expected modification of these product ions, and neutral loss chromatograms. This approach has the potential to be truly comprehensive for the determination of in vitro biotransformations in a drug discovery environment. Copyright © 2005 John Wiley & Sons, Ltd. [source]

    Mass spectrometry for the detection of differentially expressed proteins: a comparison of surface-enhanced laser desorption/ionization and capillary electrophoresis/mass spectrometry

    RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 2 2004
    Nils v. Neuhoff
    The discovery of biomarkers is currently attracting much interest as it harbors great potential for the diagnosis and monitoring of human diseases. Here we have used two advanced mass spectroscopy based technologies, surface enhanced laser desorption ionization (SELDI-MS) and capillary electrophoresis/mass spectrometry (CE/MS), to obtain proteomic patterns of urine samples from patients suffering from membranous glomerulonephritis (MGN) and healthy volunteers. The results indicate that CE/MS analysis is able to display a rich and complex pattern of polypeptides with high resolution and high mass accuracy. In order to analyze these patterns, the MosaiqueVisu software was developed for peak identification, deconvolution and the display of refined maps in a three-dimensional format. The polypeptide profiles obtained with SELDI-MS from the same samples are much sparser and show lower resolution and mass accuracy. The SELDI-MS profiles are further heavily dependent on analyte concentration. SELDI-MS analysis identified three differentially expressed polypeptides, which are potential biomarkers that can distinguish healthy donors from patients with MGN. In contrast, approximately 200 potential biomarkers could be identified by CE/MS. Thus, while SELDI-MS is easy to use and requires very little sample, CE/MS generates much richer data sets that enable an in-depth analysis. Copyright © 2003 John Wiley & Sons, Ltd. [source]

    Closely spaced external standard: a universal method of achieving 5 ppm mass accuracy over the entire MALDI plate in axial matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

    RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 19 2003
    Eugene Moskovets
    Close deposition of the sample and external standard was used in axial matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to achieve mass accuracy equivalent to that obtained with an internal standard across the entire MALDI plate. In this work, the sample and external standard were deposited by continuous deposition in separate traces, each approximately 200,,m wide. The dependence of the mass accuracy on the distance between the sample and standard traces was determined across a MALDI target plate with dimensions of 57.5,mm,×,57.0,mm by varying the gap between the traces from 100,,m to 4,mm. During acquisition, two adjacent traces were alternately irradiated with a 200-Hz laser, such that the peaks in the resulting mass spectra combined the sample and external standard. Ion suppression was not observed even when the peptide concentrations in the two traces differed by more than two orders of magnitude. The five peaks from the external standard trace were used in a four-term mass calibration of the masses of the sample trace. The average accuracy across the whole plate with this method was 5,ppm when peaks of the sample trace had signal-to-noise ratios of at least 30 and the gap between the traces was approximately 100,,m. This approach was applied to determining peptide masses of a reversed-phase liquid chromatographic (LC) separation of a tryptic digest of , -galactosidase deposited as a long serpentine trace across the MALDI plate, with accuracy comparable to that obtainable using internal calibration. In addition, the eluent from reversed-phase LC separation of a strong cation-exchange fraction containing tryptic peptides from a yeast lysate along with the closely placed external standard was deposited on the MALDI plate. The data obtained in the MS and MS/MS modes on a MALDI-TOF/TOF mass spectrometer were combined and used in database searching with MASCOT. Since the significant score is a function of mass accuracy in the MS mode, database searching with high mass accuracy reduced the number of false positives and also added peptides which otherwise would have been eliminated at lower mass accuracy (false negatives). Copyright © 2003 John Wiley & Sons, Ltd. [source]

    Primary structural determination of N-terminally blocked peptides from the bark of Eucommia ulmoides Oliv by mass spectrometric analysis

    RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 9 2003
    Ren-Huai Huang
    Sequencing of N-terminally blocked proteins/peptides is a challenge since these molecules inhibit processing by Edman degradation. By using high accuracy Fourier transform ion cyclotron resonance (FTICR) tandem mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), the primary structures of two novel N-terminally blocked antifungal peptides (EAFP1 and EAFP2), purified from the bark of Eucommia ulmoides Oliv, have been determined. The results show that the high mass accuracy provided by FTICR mass spectrometry is effective to determine the N-terminally blocking group, and can simplify the task of spectral interpretation and improve the precision of sequence determination. The combination of MALDI-TOFMS with carboxyl peptidase Y digestion was used to determine the C-terminal 36- and 27-residue sequences of EAFP1 and EAFP2, respectively, to provide the sequence linkage information for tryptic fragments. Compared with traditional peptide chemistry the advantage of mass spectrometric techniques is their simplicity, speed and sensitivity. Copyright © 2003 John Wiley & Sons, Ltd. [source]

    Analysis of native and chemically modified oligonucleotides by tandem ion-pair reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry

    RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 7 2003
    Kenneth J. Fountain
    Ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) was utilized in tandem with negative-ion electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS) for the analysis of native and chemically modified oligonucleotides. Separation was performed on a 1.0,×,50,mm column packed with porous C18 sorbent with a particle size of 2.5,,m and an average pore diameter of 140 Å. A method was developed which maximizes both chromatographic separation and mass spectrometric sensitivity using an optimized buffer system containing triethylamine and 1,1,1,3,3,3-hexafluoro-2-propanol with a methanol gradient. The ESI-TOFMS tuning parameters were also optimized in order to minimize in-source fragmentation and achieve the best sensitivity. Analyses of native, phosphorothioate, and guanine-rich oligonucleotides were performed by LC/MS. Detection limits were at sub-picomole levels with an average mass accuracy of 125,ppm. The described method allowed for the LC/MS analysis of oligonucleotides up to 110mer in length with little alkali cation adduction. Since sensitive detection of oligonucleotides was achieved with ultraviolet (UV) detection, we utilized a combination of UV-MS for quantitation (UV) and characterization (MS) of oligonucleotides and their failure sequence fragments/metabolites. Copyright © 2003 John Wiley & Sons, Ltd. [source]

    Micro-high-performance liquid chromatography/Fourier transform mass spectrometry with electron-capture dissociation for the analysis of protein enzymatic digests

    RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 10 2002
    Walter Davidson
    Electron-capture dissociation (ECD) Fourier transform mass spectrometry (FTMS) employed to generate comprehensive sequence information for the chromatographic analysis of enzymatic protein digests is described. A pepsin digest of cytochrome c was separated by reversed-phase micro-high-performance liquid chromatography (µHPLC) and ionized ,on-line' by electrospray ionization (ESI). The ions thus formed were transferred to and trapped in the FTMS analyzer cell. Typically, no precursor ion isolation was performed. The trapped ions were subjected to a pulse of electrons to induce fragmentation. Mass spectra were acquired continuously to produce a three-dimensional LC/MS data set. The spectra were dominated by c and, to a lesser degree, z ions, which provided near complete sequence coverage. External calibration provided good mass accuracy and resolution, typical of FTMS. Thus,µHPLC/ECD,,,FTMS is shown to be a highly informative method for the analysis of enzymatic protein digests. Copyright © 2002 John Wiley & Sons, Ltd. [source]