Ionization Quadrupole Ion Trap Mass Research Articles

Sequence-independent acylation of the vaccinia virus A-type inclusion protein.

N-Terminal myristoylation of proteins typically occurs cotranslationally via an amide bond to the penultimate glycine residue within the canonical motif (M)GXXX(S/T/A) in a reaction catalyzed by N-myristoyltransferase. A second, less common myristoylation reaction occurs internally at dibasic amino acid doublets of proteins such as alpha-TNF. In this case, myristoylation occurs within a portion of the preprotein, which is subsequently removed by N-terminal proteolysis. The identity of the enzyme catalyzing internal myristoylation is unknown. Considering this information, the vaccinia virus (VV) A-type inclusion protein (ATI) presents a conundrum. Although this cytosolic protein is clearly myristoylated, the protein does not have the N-terminal myristoylation motif nor is it subject to proteolytic maturation. In the experiments reported here, we cleaved VV ATI with cyanogen bromide and determined that the myristoyl moiety was present in the C-terminal half of the protein. We also subjected a tryptic digest of VV ATI to liquid chromatography electrospray ionization quadrupole ion trap mass spectrometry analyses, which indicated that ATI is randomly myristoylated at six different lysines or arginines. Analysis of the modification sites reveals no obvious conserved acceptor motifs or dibasic doublets. Mutation of these residues alone or in combination does not abrogate myristoylation of the protein, suggesting utilization of alternative modification sites. This information implies that the VV ATI protein is myristoylated in a sequence-independent manner. Because viral acylproteins typically utilize the host cell modification apparatus, this result suggests there may be an alternative type of myristoylation pathway in mammalian cells.

Differentiation of isomeric C8-substituted alkylaniline adducts of guanine by electrospray ionization and tandem quadrupole ion trap mass spectrometry

Product ion spectra from thirteen C8-substituted alkylaniline adducts of guanine and deoxyguanosine were generated using electrospray ionization and quadrupole ion trap mass spectrometry and studied to investigate the possibility of differentiating isomeric adduct structures based upon the relative abundances of fragment ions derived from the alkylaniline-modified guanine bases (BH 2 + ions). The structural discrimination of the BH 2 + ions formed by attachment of isomeric alkylanilines to the C8 position of guanine is a challenging problem because the ions tend to yield product ion spectra that are qualitatively identical upon collisional activation. In this study, a statistical method, referred to as a similarity index, was used to compare the product ion spectra of isomeric BH 2 + ions and differentiate their structures. All the adducts investigated could be distinguished from SIs calculated using 5–6 product ions. These results suggest that a searchable database of product ion spectra may be created and used to characterize DNA adducts from aromatic amines whenever they are detected at levels amenable to mass spectral analysis.

Differentiation of isomeric C8-substituted alkylaniline adducts of guanine by electrospray ionization and tandem quadrupole ion trap mass spectrometry

Differentiation of isomeric C8-substituted alkylaniline adducts of guanine by electrospray ionization and tandem quadrupole ion trap mass spectrometry

Rapid analysis of aromatic contaminants in water samples by means of laser ionization mass spectrometry.

Three different approaches to laser ionization mass spectrometric analysis of aromatic compounds in water samples are described and their performances are compared. Whereas the first two methods are based on direct laser desorption and subsequent laser ionization of either frozen or adsorbed samples in a time-of-flight mass analyzer, the third performs laser ionization in a quadrupole ion-trap into which the sample is transferred from a GC injector via a short piece of capillary tubing. For the laser-desorption method a detection limit in the 100 microg L(-1) range was determined for fluorene in frozen samples. The easier to handle analysis of adsorbed samples yielded sensitivities which were lower by about two orders of magnitude. As both direct techniques do not reach the sensitivity required for ultra trace analysis in water a preconcentration step in form of solid-phase microextraction was added before measurement using the laser ionization quadrupole ion-trap mass spectrometer. Sensitivity in the desired ng L(-1) range was easily achieved.

Between-gel reproducibility of the human cerebrospinal fluid proteome.

This manuscript describes the between-gel reproducibility of the two-dimensional gel electrophoresis analysis of the human lumbar cerebrospinal fluid (CSF) proteome. This reproducibility study is a necessary component for our long-term research program that uses comparative proteomics to analyze lumbar CSF samples in a study of human idiopathic low back pain. A Protein-Plus Dodeca Cell electrophoresis apparatus and PDQuest software were used to measure the level of between-gel reproducibility of the CSF proteome. One pooled CSF sample was used to evaluate the level of within-sample, between-gel reproducibility, and a set of seven different CSF samples (CSF-1 to 7) was used to test the level of within-group and between-group variability. Differentially expressed proteins (six CSF samples versus the designated control, CSF-3) were characterized with mass spectrometry. The number of spots found in the pooled CSF sample was 490 +/- 30 (n = 10 gels); the percentage of protein spots found in those 10 gels was 92 +/- 6%, with a coefficient of variation of 6%; and a positive coefficient of correlation (r = 0.82) was found. In order to test the proof-of-principle, that set of seven CSF samples served as a test of our ability to perform reproducibility comparative proteomics, and to detect differentially expressed proteins within that set of test samples. One sample (CSF-3) served as the control for the other six to locate the differentially expressed proteins. A comparison of fifteen differentially expressed proteins found in that set of test CSF samples correlated with pathology. Matrix-assisted laser desorption/ionization-time-of-flight and electrospray ionization quadrupole ion trap mass spectrometry were used to characterize thirteen of those fifteen differentially expressed proteins. These results (reproducibility, protein characterization, set of test samples, and proof-of-principle) suggest that the analysis of human CSF two-dimensional gels can achieve a high level of within-sample and between-sample reproducibility, and that PDQuest software can measure the relative protein abundance in the human CSF proteome.

A new solution for an old problem: the regiochemical distribution of the acyl chains in galactolipids can be established by electrospray ionization tandem mass spectrometry.

Electrospray ionization-quadrupole ion trap mass spectrometry (ESI-QITMS), either in positive- or in negative-ion mode, has been used to establish the chemical structures (chain length, degree of unsaturation, positional distribution) of the fatty acids attached to the primary (sn-1) and secondary (sn-2) hydroxyl groups of the glycerol moiety of natural monogalactosyl- (MGDG) and digalactosyldiacylglycerols (DGDG), isolated from the freshwater dinoflagellate Glenodinium sanguineum and from a marine diatom belonging to the genus Chaetoceros. Fragmentation by collision-induced dissociation of a single component in MGDG and DGDG mixtures, separated by high-performance liquid chromatography (HPLC) and detected on-line by tandem positive-ion ESI-MS, leads to a clear-cut determination of the positional distribution of the sn-glycerol-bound fatty acyl chains. Reversed-phase liquid chromatography allowed a partial resolution of the component mixture before ESI-MS/MS analysis. These results were validated by comparison with ESI-MS data obtained for the sn-2 lysoglyceroglycolipids synthesized via regiospecific enzymatic hydrolysis of the corresponding diacylglycerols by Rhizopus arrhizus lipase.

Probing mass discriminations and mass shifts in the ITMS mass spectra of externally generated MALDI ions with synthetic polymers

Synthetic polymers are demonstrated to be very useful probes for the characterization of a newly constructed external ion source matrix-assisted laser desorption and ionization quadrupole ion trap mass spectrometer (MALDI-ITMS). Mass discrimination effects and space-charge-induced mass shifts can readily be identified and quantified with these synthetic polymer probes. Mass dependencies in the trapping efficiency were evaluated by comparison of measurements of poly(methyl methacrylate) (PMMA) standards in the mass range m/ z 400–3400 with MALDI-TOF-MS measurements of the same samples. This established the optimal experimental conditions for the analysis of mass ranges smaller than 1000 u with negligible mass discrimination. This is demonstrated on the basis of measurements on a tri-block copolymer of poly(ethylene oxide) poly(propylene oxide). As expected, mass measurements were highly influenced by the magnitude of the trapped ion population. Using poly(ethylene glycol) standards we were able to quantify these mass shifts as a function of the total ion load. Consequently, reproducible results can only be obtained by reproducing the total space-charge in the trap. In MALDI experiments, this can be achieved by tuning the laser power just above threshold. This approach allowed the mass determination of three different end groups in a complex Jeffamine D2000 sample with an accuracy of better than 0.1 u.

Multiple-stage mass spectrometric analysis of complex oligosaccharide antibiotics (everninomicins) in a quadrupole ion trap

Electrospray ionization (ESI) quadrupole ion-trap tandem mass spectrometry (MS/MS) was utilized to characterize a class of complex oligosaccharide antibiotics (everninomicins) that include SCH 27899, everninomicin-D, amino everninomicin (SCH 27900), and SCH 49088 (containing a hydroxylamino-ether sugar). The addition of sodium chloride (∼1 μg/mL) facilitates the formation of abundant metal complex ions, and this was used because protonation does not readily occur for most of these compounds. The multiple-stage mass analysis (MS n) of the sodiated species provides an important series of fragment ions that are specific for sugar sequence and for some sugar-ring opening. These data suggest a general charge-remote fragmentation pattern with the sodium cation residing in a specific, central location of the sugar chain and fragmentation occurring to trim the end of the molecule. For protonated everninomicin (SCH 27900), however, the proton appears to be mobile during the collisional activation process, opening different fragmentation pathways depending on the proton location. The use of water and acetonitrile with 0.1% acetic acid as the solvent in ESI-MS promotes rapid hydrolysis of the central ortho ester, resulting in the formation of abundant sodiated products that are hydrated. These product ions of the hydrated molecules are likely formed by the same charge-remote fragmentation processes as those that occur for the unhydrolyzed precursor.

HPLC/ESI-quadrupole ion trap mass spectrometry for characterization and direct quantification of amphoteric and nonionic surfactants in aqueous samples.

An amphoteric (cocamidopropylbetaine, CAPB) and a nonionic (alcohol polyethoxylate, AE) surfactant were characterized by electrospray ionization quadrupole ion trap mass spectrometry (ESI-MS) as to their homologue distribution and ionization/fragmentation chemistry. Quantitative methods involving reversed-phase gradient HPLC and (+)ESI-MSn were developed to directly determine these surfactants in hydroponic plant growth medium that received simulated graywater. The predominant homologues, 12 C alkyl CAPB and 9 EO AE, were monitored to represent the total amount of the respective surfactants. The methods demonstrated dynamic linear ranges of 0.5-250 ng (r2 > 0.996) for CAPB and 8-560 ng (r2 > 0.998) for AE homologue mixture, corresponding to minimum quantification limits of 25 ppb CAPB and 0.4 ppm AE with 20-microL injections. This translated into an even lower limit for individual components due to the polydispersive nature of the surfactants. The procedure was successfully employed for the assessment of CAPB and AE biodegradation in a hydroponic plant growth system used as a graywater bioreactor.

Characterization of methyl orange and its photocatalytic degradation products by HPLC/UV–VIS diode array and atmospheric pressure ionization quadrupole ion trap mass spectrometry

HPLC/UV–VIS diode array and HPLC/MS techniques were successfully applied to the analysis of sulfonated molecules present in samples coming from the photocatalytic degradation of the azo dye indicator, methyl orange. The substrate was chosen as a simple model for the study of reactions involving the more complex commercial products used for the dyeing of textile fibers. Unexpected MS fragmentation path was observed, due to the very stable methyl orange molecular structure. The chromatographic information were combined with the obtained MS, MS/MS data and the UV–VIS diode array spectra and allowed to rationalize the molecular structures attributable to the various degradation products.

Evaluation of complexation of metal-mediated DNA-binding drugs to oligonucleotides via electrospray ionization mass spectrometry.

The interactions of self-complementary oligonucleotides with a group of metal-mediated DNA-binding drugs, including chromomycin A(3), mithramycin and the novel compound UK-1, were examined via electrospray ionization quadrupole ion trap mass spectrometry. Both chromomycin and mithramycin were shown to bind preferentially to GC-rich oligonucleotide duplexes in a 2:1 drug:metal ratio, while UK-1 was shown to bind in a 1:1 drug:metal stoichiometric ratio without a strong sequence preference. These trends were observed in the presence of Co(2+), Ni(2+) and Zn(2+), with the exception that chromomycin-Zn(2+) complexes were not readily observed. The binding stoichiometries as well as the sequence specificities are in agreement with literature reports for solution studies. Binding selectivities and stabilities of the complexes were also probed using electrospray ionization mass spectrometry. Both of the GC-rich oligomers 5'-GCGCGC-3' and 5'-GCGCATGCGC-3' exhibited a binding preference for chromomycin over mithramycin in the presence of Co(2+) and Ni(2+). Energy-variable collisionally activated dissociation of the complexes was employed to determine the stabilities of the complexes. The relative metal-dependent binding energies were Ni(2+) > Zn(2+) > Co(2+) for UK-1-oligomer complexes and Ni(2+) > Co(2+) for both mithramycin and chromomycin complexes.

Site specific sequestering and stabilization of charge in peptides by supramolecular adduct formation with 18-crown-6 ether by way of electrospray ionization

The binding of 18-crown-6-ether (18C6) to biologically relevant groups is studied by way of electrospray ionization (ESI) quadrupole ion-trap mass spectrometry. Isolated lysine is a special case in which the 18C6 attaches to the N-terminus, deriving stability from the resulting salt-bridge structure. In a peptide, 18C6 forms a stable supramolecular adduct with lysine by way of specific hydrogen bonding with the primary protonated amine on the side chain. 18C6 will bind to adjacent lysines, forming multiply charged complexes. For example, the most intense peak in the spectrum of a mixture of 18C6 and tetralysine is the quadruply charged peptide with four crown ethers attached. Competitive binding of 18C6 by other basic sites such as the n-terminus, histidine, and arginine limits the utility of this technique for the molecular recognition of lysine. Adducts of 18C6 with proteins such as cytochrome-c (CytC) and bovine pancreatic trypsin inhibitor (BPTI) reveal structural information relevant to the accessibility of potential binding sites. Adduct formation with 18C6 appears to increase the surface activity of peptides in a charged droplet and inhibits deprotonation during ion desolvation in the ESI process. These effects increase both the primary charge state and ion abundance of the analyte. Sequestering and stabilization of charge provided by 18C6 allows for the detection of species that are normally difficult to observe using ESI, such as glycine. The relationship between the observed behaviors of 18C6 adducts and the proposed ion evaporation mechanism of ESI is discussed.

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.

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 alpha-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 MS(n) 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.

Determination of alkali metal binding selectivities of caged crown ligands by electrospray ionization quadrupole ion trap mass spectrometry

The alkali metal (Li +, Na +, K +, Rb +, and Cs +) binding selectivities of caged crown ether compounds are evaluated by way of electrospray ionization mass spectrometry and compared to the binding selectivities of three reference crown ethers, 15-crown-5, 1,7-diaza-15-crown-5, and 18-crown-6. The relative binding selectivities are estimated from the mass spectral intensities of the metal complexes and compared to calculated equilibrium distributions when possible. In general, the selectivities of the caged crown ethers parallel the selectivities for the noncaged reference crowns. However, as the cage moiety imparts a greater degree of rigidity in the polyether ring, the selectivities are shifted slightly toward larger cations. Further understanding of the metal binding preferences is obtained from molecular modeling calculations.

Identification of phosphorylation sites of proteins by high performance liquid chromatography-electrospray ionization-quadrupole ion trap mass spectrometry

The phosphorylation sites of two phosphorylated proteins, bovine beta-casein and myelin basic protein (MBP), were identified by high performance liquid chromatography-electrospray ionization-quadrupole ion trap mass spectrometry (HPLC-ESI-QITMS). The tryptic digest of each protein was separated by HPLC, the molecular weight of each peptide was determined by ESI-QITMS on line, and MS/MS spectrum of each peptide was simultaneously obtained by the combination of collision-induced desorption (CID) technique and tandem mass spectrometry (MS/MS) of QITMS. The phosphorylated peptide was identified by looking into whether the difference between the observed and predicted molecular weights of a peptide is 80 u or its integral multiple. Then the phosphorylation site was identified through manual interpretation of the MS/MS spectrum of the phosphorylated peptide or automatic SEQUEST data base-searching.

Methanol chemical ionization quadrupole ion trap mass spectrometry of O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothiolate (VX) and its degradation products

Mass spectrometric analysis of O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothiolate (VX) degradation products by electron ionization produces extensive fragmentation with little or no molecular ion information making product identification difficult. Milder chemical ionization (CI) is commonly used to provide molecular mass and structure confirmation. In this study, methanol was used as a CI reagent in combination with an ion trap detector for detection and identification of over 30 compounds present in a thermally degraded sample of VX. The use of methanol provides superior results for this class of compounds with less fragmentation than commonly observed with gas reagents and offers logistical advantages for on-site analysis by being easier to transport and safer to use than gas cylinders.

Analysis of 8-aminonaphthalene-1,3,6-trisulfonate-derivatized oligosaccharides by capillary electrophoresis–electrospray ionization quadrupole ion trap mass spectrometry

Dextran was partially hydrolyzed with 0.1 mol/l HCl and the hydrolysate was derivatized with 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) by reductive amination. The derivatized-oligosaccharide mixture was separated by capillary electrophoresis (CE) in a buffer of 1% HAc–NH 4OH, pH 3.4, and the separated components were detected on-line by electrospray ionization quadrupole ion trap mass spectrometry (ESI-QIT-MS) in the negative ion mode. A mass accuracy lower than 0.01% could be achieved and as low as 1.6 pmol of detxran octaose could be detected. ANTS-derivatized dextran oligosaccharide with a degree of polymerization (DP) lower than 6 produced both [M−H] − and [M−2H] 2− ions, whereas those with a DP of 6 or higher than 6 produced only [M−2H] 2− ion. As 1≤DP≤6, the percentage of [M−2H] 2− ion in the total ions of [M−H] − and [M−2H] 2− was found to be a linear function of the logarithmic DP. Molecular mass determination with ESI-QIT-MS strengthens the power of CE analysis of oligosaccharides.

Multicomponent quantification of diastereomeric hexosamine monosaccharides using ion trap tandem mass spectrometry.

A rapid means of stereochemical differentiation and quantification for the hexosamine monosaccharides was achieved using electrospray ionization quadrupole ion trap mass spectrometry. The hexosamine monosaccharides, glucosamine, galactosamine, and mannosamine, were derivatized with [Co(DAP)2Cl2]Cl, and the complex [Co(DAP)2(HexNH2)]Cl was generated. Subjecting this complex to collision-induced dissociation provided a unique product ion spectrum for each of the diastereomeric monosaccharide complexes, thus differentiating the stereoisomers. Furthermore, the stereoisomers were quantified. This was achieved by using the relative abundances of product ions from pure standards and using these values to determine the ratio of isomeric products in a mixture. The utility of this quantification method was demonstrated by successfully determining the composition of two- and three-component mixtures of the hexosamines.

Electrospray ionization-ion trap mass spectrometry for structural analysis of complex N-linked glycoprotein oligosaccharides.

Electrospray ionization-ion trap mass spectrometry, with its capacity to perform multiple stages of fragmentation (MSn), is demonstrated as an effective method for the structural characterization of permethylated N-linked complex glycoprotein oligosaccharides. Complex glycan structural features, such as N-acetyllactosamine antenane, neuraminic acids, and nonreducing terminal GlcNAc monosaccharides, commonly suppress cross-ring and core saccharide cleavages in traditional MS/MS experiments. Using ion trap mass spectrometry, removal of these substituents permits determination of branching patterns and intersaccharide linkages by MS3 and MS4. Both sequence and linkage data are obtained for N-acetyllactosamine and sialyl-N-acetyllactosamine oligosaccharide antennae from biantennary glycans using MS3, and the location of a bisecting GlcNAc residue is also established after exposing the core pentasaccharide. Higher-order experiments further illustrate the potential of electrospray ionization-quadrupole ion trap mass spectrometry for carbohydrate analysis, as MS8 is used to produce significant and otherwise unobtainable branching information for an oligosaccharide from chicken ovalbumin. These studies constitute further evidence of the unique role that ion trap mass spectrometry can assume in the area of oligosaccharide analysis.

Characterization of oligosaccharide composition and structure by quadrupole ion trap mass spectrometry.

The use of electrospray ionization-quadrupole ion trap mass spectrometry for the characterization of linear oligosaccharides and N-linked protein oligosaccharide mixtures is described. Tandem mass spectrometry (MS/MS) experiments with orders higher than two offer a number of ways to enhance MS/MS spectra and to derive information not present in MS and MS2 spectra. Three such methods are presented in this paper. (a) Collisional activation of permethylated oligosaccharide molecular ions (MS2) as illustrated by maltoheptaose, produces abundant fragments from glycosidic bond cleavages which indicate composition and sequence, and weak cross-ring cleavage products which denote specific linkages within the oligosaccharide. Through the trapping and further dissociation of these fragments (MSn), cross-ring cleavage products can be confirmed and their relative abundances increased to facilitate interpretation. (b) The mechanisms of formation of two isobaric ions or ions isobaric with another ion's isotope peaks, such as those present in the MS2 spectrum of the ribonuclease B oligosaccharide GlcNAc2-Man5 can be independently established by separate MS3 experiments. (c) Ions in the MS2 spectrum, specific for individual components of an isobaric mixture, can be isolated and characterized by further stages of fragmentation. This is illustrated by two isobaric oligosaccharides from chicken ovalbumin of the composition HexNAc5Hex5. These findings indicate the utility of ion trap mass spectrometry towards the facile determination of oligosaccharide composition, sequence, branching and linkage, providing a wealth of structural information not obtainable by other individual methods of carbohydrate mass spectrometric analysis.