Fourier-transform electrospray instrumentation for tandem high-resolution mass spectrometry of large molecules

Department of Chemistry, Baker Laboratory, Cornell University, Ithaca, New York, USA Mass spectrometry instrumentation providing unit resolution and lo-ppm mass accuracy for molecules larger than 10 kDa was first reported in 1991. This instrumentation has now been improved with a 6.2-T magnet replacing that of 2.8 T, a more efficient vacuum system, ion injection with controlled ion kinetic energies, accumulated ion trapping with an open-cylindrical ion cell, acquisition of 2M data points, and updated electrospray apparatus. The resulting capabilities include resolving power of 5 × 10⁵ for a 29-kDa protein, less than l-ppm mass measuring error, and dissociation of protein molecular ions to produce dozens of fragment ions whose exact masses can be identified from their mass-to-charge ratio values and isotopic peak spacing.     

Analysis of urinary aromatic acids by liquid chromatography tandem mass spectrometry

The separation and detection of 11 urinary aromatic acids was developed using HPLC-MS/MS. The method features a simple sample preparation involving a single-step dilution with internal standard and a rapid 8 min chromatographic separation. The accuracy was evaluated by the recovery of known spikes between 87 and 110%. Inter- and intra-assay precision (CV) was below 11% in all cases and the analytes were observed to be stable for up to 8 weeks when stored at -20 degrees C. The method was validated based upon linearity, accuracy, precision and stability and was used to establish reference intervals for children and adults.     

Analysis of polycyclic aromatic hydrocarbons by ion trap tandem mass spectrometry

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 determine the conditions for CID ionization of 18 PAHs. Unlike in electron impact (EI) analysis, the relative abundances of progeny ions of isomers were statistically different (using Student’s t-test) in CID analysis, thus making isomer identification by CID possible. For comparison, CID and EI were applied to the analysis of used motor oil. CID analysis was shown to be more sensitive than EI analysis of the used motor oil. Precision at the 10-ppb level for EI and CID showed relative standard deviations of 5. 2 and 7. 7%, respectively.     

Resonance enhanced laser ionisation mass spectrometry of four aromatic molecules

Multiphoton ionisation and fragmentation of aniline, benzene, N,N-dimethyl and 2,4-dimethyl aniline has been studied by laser ionisation mass spectrometry under collision free conditions. All four molecules undergo efficient resonance-enhanced two-photon ionisation (R2PI) with relatively low laser intensities at λ = 266 nm producing the parent ion almost exclusively. At higher intensities, high order processes compete producing extensive fragmentation. At 266 nm, all the major fragment ions are produced by R3PI. For aniline excited at 294 nm, energetic considerations suggest R4P1 formation of fragments with differences in fragmentation between 266 and 294 nm reflecting the differing orders and energies above threshold of the competing processes. Comparison of R2P1 efficiencies in aniline and benzene shows that the cross sections for ionisation of the resonant intermediate ₁B² excited state in both molecules are approximately equal and independent of wavelength in the range 250–300 nm.     

Hybrid mass spectrometers for tandem mass spectrometry

Mass spectrometers that use different types of analyzers for the first and second stages of mass analysis in tandem mass spectrometry (MS/MS) experiments are often referred to as "hybrid" mass spectrometers. The general goal in the design of a hybrid instrument is to combine different performance characteristics offered by various types of analyzers into one mass spectrometer. These performance characteristics may include mass resolving power, the ion kinetic energy for collision-induced dissociation, and speed of analysis. This paper provides a review of the development of hybrid instruments over the last 30 years for analytical applications.     

Improved mass accuracy for tandem mass spectrometry

With the emergence of top-down proteomics, the ability to achieve high mass measurement accuracy on tandem MS/MS data will be beneficial for protein identification and characterization. (FT-ICR) Fourier transform ion cyclotron resonance mass spectrometers are the ideal instruments to perform these experiments with their ability to provide high resolution and mass accuracy. A major limitation to mass measurement accuracy in FT-ICR instruments arises from the occurrence of space charge effects. These space charge effects shift the cyclotron frequency of the ions, which compromises the mass measurement accuracy. While several methods have been developed that correct these space charge effects, they have limitations when applied to MS/MS experiments. It has already been shown that additional information inherent in electrospray spectra can be used for improved mass measurement accuracy with the use of a computer algorithm called DeCAL (deconvolution of Coulombic affected linearity). This paper highlights a new application of the strategy for improved mass accuracy in tandem mass analysis. The results show a significant improvement in mass measurement accuracy on complex electron capture dissociation spectra of proteins. We also demonstrate how the improvement in mass accuracy can increase the confidence in protein identification from the fragment masses of proteins acquired in MS/MS experiments.     

Mechanism study of SO2 elimination from sulfonamides by negative electrospray ionization mass spectrometry

The elimination of SO2 from deprotonated sulfonamides in the negative ion mode was confirmed by Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) experiments. For a set of N-arylbenzenesulfonamides substituted at the para position of the arylamine, the ln([M-H-SO2](-)/[M-H]-) values were correlated with the sigmap(-) substituent constants but, instead of a linear relationship, a bent line was obtained. Analyses of the complex curve led to the identification of two competing routes, which were further investigated by Hartree-Fock theoretical calculations. Furthermore, collision-induced dissociation (CID) of deprotonated N-alkylbenzenesulfonamides containing the -CHCHNHSO2- structure yielded a [M-H-66](-) product ion This characteristic ion could help to distinguish the side-chain isomers.     

Electrospray mass spectrometry and tandem mass spectrometry of the natural mixture of cyclic peptides from linseed

The cyclic peptides from linseed are composed exclusively of the hydrophobic amino acids: Phe, Leu, Ile, Val, Met, Pro, and Trp. Because these compounds does not contain functional groups which undergo easily protonation or deprotonation. their ionization in solvents used usually for peptide analysis is not efficient. A rapid and sensitive procedure for detection and structure elucidation of the cyclic peptides based on ionization with Na+ and NH4+ ions. A cationisation of methionine containing peptides with methyl iodide has been also described. The extract of seeds of Linum utitatissimum was analyzed directly by ESI-MS and neutral loss ESI-MS/MS technique. The analysis confirms the presence of cyclolinopeptides reported previously: CLA (c(Pro-Pro-Phe-Phe-Leu-Ile-Ile-Leu-Val), and CLB (c(Pro-Pro-Phe-Phe-Val-Ile-Met-Ile-Leu)). Cyclolinopeptides CLC, CLD, CLE, and CLG, which contain methionine oxide, were detected in relatively small quantities. These peptides results likely from the oxidation of their not reported precursors: CLD' (c (Pro- Phe-Phe-Trp-Ile-Met-Leu-Leu)), CLE'(c (Pro-Leu-Phe-Ile-Met-Leu-Val-Phe)), CLF (c (Pro-Phe-Phe-Trp- Val-Met-Leu-Met), and CLG (c (Pro-Phe-Phe-Trp-Ile-Met-Leu-Met), present at higher concentrations in the extract protected from atmospheric oxygen. The sequences of the unreported cyclic peptides were proposed on the basis of CID experiments and homology with peptides described by Morita,1,2 and supported by the fragmentation of synthetic analogues of CLA of a known structure.     

Ion activation methods for tandem mass spectrometry

This tutorial presents the most common ion activation techniques employed in tandem mass spectrometry. In-source fragmentation and metastable ion decompositions, as well as the general theory of unimolecular dissociations of ions, are initially discussed. This is followed by tandem mass spectrometry, which implies that the activation of ions is distinct from the ionization step, and that the precursor and product ions are both characterized independently by their mass/charge ratios. In collision-induced dissociation (CID), activation of the selected ions occurs by collision(s) with neutral gas molecules in a collision cell. This experiment can be done at high (keV) collision energies, using tandem sector and time-of-flight instruments, or at low (eV range) energies, in tandem quadrupole and ion trapping instruments. It can be performed using either single or multiple collisions with a selected gas and each of these factors influences the distribution of internal energy that the activated ion will possess. While CID remains the most common ion activation technique employed in analytical laboratories today, several new methods have become increasingly useful for specific applications. More recent techniques are examined and their differences, advantages and disadvantages are described in comparison with CID. Collisional activation upon impact of precursor ions on solid surfaces, surface-induced dissociation (SID), is gaining importance as an alternative to gas targets and has been implemented in several different types of mass spectrometers. Furthermore, unique fragmentation mechanisms of multiply-charged species can be studied by electron-capture dissociation (ECD). The ECD technique has been recognized as an efficient means to study non-covalent interactions and to gain sequence information in proteomics applications. Trapping instruments, such as quadrupole ion traps and Fourier transform ion cyclotron resonance instruments, are particularly useful for the photoactivation of ions, specifically for fragmentation of precursor ions by infrared multiphoton dissociation (IRMPD). IRMPD is a non-selective activation method and usually yields rich fragmentation spectra. Lastly, blackbody infrared radiative dissociation is presented with a focus on determining activation energies and other important parameters for the characterization of fragmentation pathways. The individual methods are presented so as to facilitate the understanding of each mechanism of activation and their particular advantages and representative applications.     

Artifacts in four-sector tandem mass spectrometry

Several types of artifacts were shown to be present in 4-sector tandem collision-induced dissociation (CID) mass spectra. In CID spectra of protonated peptides produced by liquid secondary-ion mass spectrometry (LSIMS), peaks corresponding to successive losses of matrix molecules from the precursor ion were observed. In addition, peaks corresponding to MH+ ions of smaller peptides that were also present in the sample/matrix mixture in greater abundance than the selected precursor ion were observed. Both of these types of artifact peaks were shown to originate from the 'peak-at-every-mass' chemical noise at the same nominal mass as that selected by the first 2 sectors (MS1). These noise ions are transmitted through to the collision cell and produce fragments that are analysed and detected in the next 2 sectors (MS2). A second, unrelated, kind of artifact was found to be due to decompositions in the second field-free region of MS2 in an EBEB geometry machine. These artifacts, which are detectable over only a very limited mass range when using a conventional single-point detector, can be present over a much greater mass range when an array detector is used and when the collision cell is floated above ground potential. A clear understanding of the origins of all peaks in a CID spectrum is important in order to have a firm foundation for interpretation, manual or computer-aided, of the spectra of unknown compounds.     

高效液相色谱-串联质谱法测定纺织品中的致癌芳香胺

建立了高效液相色谱-串联质谱法(LC/MS/MS)测定纺织品中致癌芳香胺的检测方法.纺织品中的偶氮染料在柠檬酸缓冲液中用连二亚硫酸钠还原为芳香胺,用硅藻土提取还原液中的芳香胺,用乙醚洗脱,浓缩至近干后用甲醇定容.使用液相色谱-串联质谱进行测定,色谱柱为XTerra MS C18柱,流动相为甲醇和0.025 mol/L乙...     

Comparison of negative and positive ion electrospray tandem mass spectrometry for the liquid chromatography tandem mass spectrometry analysis of oxidized deoxynucleosides

Oxidized deoxynucleosides are widely used as biomarkers for DNA oxidation and oxidative stress assessment. Although gas chromatography mass spectrometry is widely used for the measurement of multiple DNA lesions, this approach requires complex sample preparation contributing to possible artifactual oxidation. To address these issues, a high performance liquid chromatography (HPLC)-tandem mass spectrometric (LC-MS/MS) method was developed to measure 8-hydroxy-2'-deoxyguanosine (8-OH-dG), 8-hydroxy-2'-deoxyadenosine (8-OH-dA), 2-hydroxy-2'-deoxyadenosine (2-OH-dA), thymidine glycol (TG), and 5-hydroxy-methyl-2'-deoxyuridine (HMDU) in DNA samples with fast sample preparation. In order to selectively monitor the product ions of these precursors with optimum sensitivity for use during quantitative LC-MS/MS analysis, unique and abundant fragment ions had to be identified during MS/MS with collision-induced dissociation (CID). Positive and negative ion electrospray tandem mass spectra with CID were compared for the analysis of these five oxidized deoxynucleosides. The most abundant fragment ions were usually formed by cleavage of the glycosidic bond in both positive and negative ion modes. However, in the negative ion electrospray tandem mass spectra of 8-OH-dG, 2-OH-dA, and 8-OH-dA, cleavage of two bonds within the sugar ring produced abundant S1 type ions with loss of a neutral molecule weighing 90 u, [M - H - 90]-. The signal-to-noise ratio was similar for negative and positive ion electrospray MS/MS except in the case of thymidine glycol where the signal-to-noise was 100 times greater in negative ionization mode. Therefore, negative ion electrospray tandem mass spectrometry with CID would be preferred to positive ion mode for the analysis of sets of oxidized deoxynucleosides that include thymidine glycol. Investigation of the fragmentation pathways indicated some new general rules for the fragmentation of negatively charged oxidized nucleosides. When purine nucleosides contain a hydroxyl group in the C8 position, an S1 type product ion will dominate the product ions due to a six-membered ring hydrogen transfer process. Finally, a new type of fragment ion formed by elimination of a neutral molecule weighing 48 (CO2H4) from the sugar moiety was observed for all three oxidized purine nucleosides.     

Comparison of flow injection analysis electrospray mass spectrometry and tandem mass spectrometry and electrospray high-field asymmetric waveform ion mobility mass spectrometry and tandem mass spectrometry for the determination of underivatized amino acids

Twenty proteinogenic amino acids (AAs) were determined without derivatization using flow injection analysis followed by electrospray ionization mass spectrometry and tandem mass spectrometry (ESI-MS and ESI-MS/MS) and electrospray ionization high-field asymmetric waveform ion mobility mass spectrometry and tandem mass spectrometry (ESI-FAIMS-MS and ESI-FAIMS-MS/MS), in positive and negative ionization modes. Three separate sets of ESI-FAIMS conditions were used for the separation and detection of the 20 AAs. Typically ESI-FAIMS-MS showed somewhat improved sensitivity and significantly better signal-to-noise ratios than ESI-MS mainly due to the elimination of background noise. However, the difference between ESI-FAIMS-MS and ESI-MS/MS was significantly less. ESI-FAIMS was able to partially or completely resolve all the isobaric amino acid overlaps such as leucine, isoleucine and hydroxyproline or lysine and glutamine. Detection limits for the amino acids in ESI-FAIMS-MS mode ranged from 2 ng/mL for proline to 200 ng/mL for aspartic acid. Overall, ESI-FAIMS-MS is the preferred method for the quantitative analysis of AAs in a hydrolyzed yeast matrix.     

Electron impact ionization mass spectrometry and tandem mass spectrometry of phenylalkylpyridazines

The mass spectrometric fragmentation behaviour of pyridazine and four monosubstituted derivatives containing a pbenylalkyl side-chain (3- and 4-benizylpyridazine, 3- and 4-(2-pbenylethyl)pyridazine) was investigated. In the electron impact ionization mess spectra of the 3-substituted compounds abundant [M – H]⁺ peaks are observed. This allows a clear distinction between 3- and 4-substituted pyridazines, as the spectra of the latter isomers show only very weak [M – H]⁺ signals. The stability of [M – H]⁺ ions derived from 3-alkylpyridazines (deduced from only the very low abundance of further fragment ions) gives strong evidence for a cyclic structure of these ions. One fragmentation pathway typical of the parent pyridazine, the [M - N₂] fragmentation, was not detectable with any of the phenylalkylpyridazines investigated. Instead, loss of HCN, H₃CN⁺ and N²H⁺ was observed. An interesting fragmentation, observed with 3-(2-phenylethyl)pyridazine, is the loss of ⁺CH₃ from the molecular ion and also from the [M – H]⁺ ion.     

Electrospray mass spectrometry and tandem mass spectrometry of bimetallic oxovanadium complexes

A series of six bimetallic oxovanadium complexes (1-6; only one was purified) were investigated by electrospray quadrupole time-of-flight tandem mass spectrometry (ESI-QTOF-MS/MS) in negative-ion mode. Radical molecular anions [M](.-) were observed in MS mode. Fragmentation patterns of [M](.-) were proposed, and elemental compositions of most of the product ions were confirmed on the basis of the high-resolution ESI-CID-MS/MS spectra. A complicated series of low-abundance product ions similar to electron impact (EI) ionization spectra indicated the radical character of the precursor ions. Fragment ions at m/z 214, 200, and 182 seem to be the characteristic ions of bimetallic oxovanadium complexes. These ions implied the presence of a V-O-V bridge bond, which might contribute to stabilization of the radical. To obtain more information for structural elucidation, three representative bimetallic oxovanadium complexes (1-3) were analyzed further by MS in positive-ion mode. Positive-ion ESI-MS produced adduct ions of [M + H](+), [M + Na](+), and [M + K](+). The fragmentation patterns of [M + Na](+) were different than those of radical molecular anions [M](.-). Relatively simple fragmentation occurred for [M + Na](+), possibly due to even-electron ion character. Negative-ion MS and MS/MS spectra of the hydrolysis product of Complex 1 supported these finding, in particular, the existence of a V-O-V bridge bond.     

Electrospray tandem mass spectrometry of epipolythiodioxopiperazines

Low-energy collision-induced electrospray ionization tandem mass spectrometry ESI-CID-MS/MS (in the positive ion mode) was used for the structural characterization of a series of five representative epioplythiodioxopipreazines: dethiotetra(methylthio)chemotin, chaetocochins A, B and C, and chemotin isolated from the fungus Chaetomium cochliodes. The fragmentation pathways were elucidated by ESI-IT-MS(n). The elemental compositions of most of the product ions were confirmed by low-energy ESI-CID-QTOF-MS/MS analyses. The loss of the S(2) molecule seems always to be the first when the S--S bond is present. The loss of 77 Da corresponding to the loss of the [CH(3)SCH(2)O]' radical was diagnostic for chaetocochins A and B, in which the two piperazines rings are linked by an acetal group. It was found that a McLafferty rearrangement plays a significant role in the skeleton fragmentation of theses series of studied complex multicyclic piperazine compounds. This MacLafferty rearrangement affords the product ions at m/z 416 and 400, containing the two piperazine rings belonging to the epipolythiodioxopipreazines. In addition, the pentacyclic rearrangement involving the loss of the SMe(.) radical seems to occur in the presence of the unfused ring. Finally the product ions at m/z 635 and 591 seem to be the characteristic ions for chaetocochin A.