High-energy Collision-induced Dissociation Spectra Research Articles

Identification and characterization of the chemical components of Iris tectorum Maxim. and evaluation of their nitric oxide inhibitory activity.

Iris tectorum Maxim. is a traditional medicinal herb that is commonly used to treat inflammatory conditions. The present study investigated the fragmentation patterns of isoflavone glycosides and their qualitative analysis. In addition, lipopolysaccharide (LPS)-induced RAW264.7 macrophages were used to evaluate the anti-inflammatory properties of I. tectorum Maxim. samples collected at different time points during the year. High-performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (HPLC/QTOF-MS/MS) and HPLC with diode-array detection were employed for qualitative and quantitative analysis. The fragmentation patterns of the isoflavones were observed in negative electrospray ionization mode with collision-induced dissociation (CID). Their anti-inflammatory activity was assessed via nitric oxide (NO) production in LPS-treated RAW264.7 macrophages. A total of 15 chemical components were observed and tentatively identified using HPLC/QTOF-MS/MS. At low collision energy, the relative abundances of the aglycone radical anions Y0 - , [Y0 - H]-• , [Y0 - CH3 ]-• and [Y0 - H- CH2 ]-• were used for the structural characterization of tectoridin and tectorigenin-4'-O-β-D-glucoside. The radical ions [Y0 - CH3 ]-• and [Y0 - H - 2CH3 ]-• were also employed to differentiate between iristectorin A and iristectorin B based upon their high-energy CID spectra. Levels of 9.02 mg/g of tectoridin and 1.04 mg/g of tectorigenin were found in samples collected in June, which exhibited 69.7% NO inhibitory activity. The characteristic fragmentation patterns enabled us to reliably identify isoflavone glycosides. The results of the quantitative determination and NO inhibitory activity offer insight into the optimal I. tectorum Maxim. harvesting time.

Axial spatial distribution focusing: improving MALDI-TOF/RTOF mass spectrometric performance for high-energy collision-induced dissociation of biomolecules.

RationaleFor the last two decades, curved field reflectron technology has been used in matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometers, assisting in the generation of post-source-decay (PSD) or collision-induced dissociation (CID) without decelerating precursor ions, producing true high-energy CID spectra. The result was the generation of product ion mass spectra with product ions typical of high-energy (10 keV and beyond) collision processes. The disadvantage of this approach was the lack of resolution in CID spectra resulting from the excess laser energy deposition used to generate those MS/MS spectra. The work presented in this study overcomes this limitation and includes comprehensive examples of high-energy and high-resolution CID MALDI-MS/MS spectra of biomolecules.MethodsThe devices used in this study are TOF/RTOF instruments equipped with a high-vacuum MALDI ion source. High-resolution and high-energy CID spectra result from the use of axial spatial distribution focusing (ASDF) in combination with curved field reflectron technology.ResultsA CID spectrum of the P14R1 peptide exhibits product ion resolution in excess of 10,000 (FWHM) but at the same time yields typical high-energy product ions such as w- and [y–2]-type ion series. High-energy CID spectra of lipids, exemplified by a glycerophospholipid and triglyceride, demonstrate C–C backbone fragmentation elucidating the presence of a hydroxyl group in addition to double-bond positioning. A complex high mannose carbohydrate (Man)8(GlcNAc)2 was also studied at 20 keV collision energy and revealed further high-energy product ions with very high resolution, allowing unambiguous detection and characterization of cross-ring cleavage-related ions.ConclusionsThis is the first comprehensive study using a MALDI-TOF/RTOF instrument equipped with a curved field reflectron and an ASDF device prior to the reflectron. © 2015 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.

Development of a tandem time‐of‐flight mass spectrometer with an electrospray ionization ion source

A new tandem time-of-flight mass spectrometer with an electrospray ionization ion source 'ESI-TOF/quadTOF' was designed and constructed to achieve the desired aim of structural elucidation via high-energy collision-induced dissociation (CID), and the simultaneous detection of all fragment ions. The instrument consists of an orthogonal acceleration-type ESI ion source, a linear TOF mass spectrometer, a collision cell, a quadratic-field ion mirror and a microchannel plate detector. High-energy CID spectra of doubly protonated angiotensin II and bradykinin were obtained. Several fragment ions such as a-, d-, v- and w-type ions, characteristic of high-energy CID, were clearly observed in these spectra. These high-energy CID fragment ions enabled confirmation of the complete sequence, including leucine-isoleucine determinations. It was demonstrated that high-energy CID of multiply protonated peptides could be achieved in the ESI-TOF/quadTOF.

Study of Endocrine Disruptor Octylphenol Isomers Using Collision-Induced Dissociation Mass Spectrometry

Fragmentation processes of molecular-related ions M+· and [M-H]- of phenolic endocrine disruptors, 4-n-octylphenol, 4-(1,1,3,3-tetramethyl-butyl)phenol, and 4-(1-ethyl-1-methyl-butyl)phenol, which are octylphenol isomers, have been studied using high- and low-energy collision-induced dissociation (CID) mass spectrometry. The product ion spectra revealed the isomeric structures of octyl group C8H17 found in CID studies of both precursor ions, M+· and [M-H]-. The high-energy CID spectra of the deprotonated molecules [M-H]- were compared with the corresponding low-energy CID spectra. Although the fragmentation pattern of low-energy CID differed markedly from that of high-energy CID, both product ion spectra were useful in identifying the isomeric structures of the octyl group. The negative-ion products from the precursor [M-H]- could be explained by two mechanisms with homolytic cleavage, called charge-remote fragmentation (CRF), while the positive-ion products from M+· were understood as homolytic radical-initiated fragmentation (RIF) and heterolytic charge-initiated fragmentation (CIF).

マルチターンタンデム飛行時間型質量分析計におけるリン脂質のチャージリモートフラグメンテーション

High-energy collision-induced dissociation (CID) provides characteristic structural information about interested molecules, which cannot be obtained by low energy CID. High-energy CID is available using magnetic sector or time-of-flight (TOF) type mass spectrometers. In our laboratory, a multi-turn tandem TOF mass spectrometer (MULTUM-TOF/TOF) has been developed that has a multi-turn TOF mass spectrometer (MULTUM) as the first stage and a quadratic-field ion mirror as the second stage. MULTUM-TOF/TOF enables obtaining high-energy CID spectra with high resolving power for precursor ion selection. Furthermore, the signal-to-noise (S/N) ratio of the product ion mass spectrum is dramatically improved by increasing the number of cycles, because fragment ions caused by post-source decay are eliminated. The improved S/N ratio facilitates the recognition of signal peaks that have low intensity. In this paper, we performed the structural analysis of lysophosphatidylcholine (LPC) (18 : 1) using MULTUM-TOF/TOF. We obtained ions derived not only from a dissociation of the polar headgroup but also from charge-remote fragmentation generated by a high-energy CID process. Here, charge-remote fragmentation is the cleavage of a long-chain alkyl in a fatty acid moiety. Therefore, the position of the double bonds was successfully determined. The characteristics of product ion mass spectra obtained from sodiated and protonated LPC were also investigated.

Determination of the glycosylation site in flavonoid mono‐O‐glycosides by collision‐induced dissociation of electrospray‐generated deprotonated and sodiated molecules

The influence of the glycosylation site on the fragmentation behavior of 18 flavonoid glycoside standards was studied using positive and negative electrospray ionization mass spectrometry in combination with collision-induced dissociation and tandem mass spectrometry. The glycosylation position is shown to affect the relative abundance of the radical aglycone ions that can be observed in the [M-H]- collision-induced dissociation spectra. In particular, the radical aglycone ions are very abundant for deprotonated flavonol 3-O-glycosides. Collisional activation of the radical aglycone ions produced from positional isomers revealed minor differences: m,nB0- product ions are pronounced for 7-O-glycosides, whereas m,nA0- product ions are relatively more abundant for 4'-O-glycosides. In addition, the ratio between the radical aglycone and the regular aglycone ions in the [M+Na]+ high-energy collision-induced dissociation spectra gives an indication about the glycosylation site. This ion ratio allows the differentiation between flavonoid 3-O- and 7-O-glycosides or can be useful in the comparison of unknown compounds with standards. Unambiguous differentiation between O-glycosylation at the common positions of flavonoid O-glycosides, i.e. the 3-, 4'- and 7-positions, is achieved by collisional activation of sodiated molecules at high collision energy. The presence of a B-ring product ion containing the sugar residue indicates 4'-O-glycosylation, whereas the loss of the B-ring part from the aglycone product ion is characteristic of 3-O-glycosylation and the loss of the B-ring part from both the [M+Na]+ precursor ion and the aglycone product ion points to 7-O-glycosylation.

High-energy collision-induced dissociation of oxosteroids derivatised to Girard hydrazones.

Neutral oxosteroids have been derivatised with Girard T and P hydrazine reagents to give the corresponding Girard hydrazone quaternary ammonium salts. Both Girard T (GT) and Girard P (GP) hydrazones of oxosteroids give very intense [M](+) ion signals in electrospray (ES) mass spectra and fragment within the ES interface and collision cell to give characteristic fragment ions. GT and GP derivatives give informative high-energy collision-induced dissociation spectra, from which the structure of the precursor oxosteroid can be determined. Both charge-remote and charge-mediated mechanisms are responsible for the formation of the fragment ions at high collision-energy.

Characterisation of anthocyanidins by electrospray ionisation and collision-induced dissociation tandem mass spectrometry.

The present study describes the use of electrospray ionisation mass spectrometry, in combination with collision-induced dissociation (CID) and tandem mass spectrometry, for the structural characterisation of anthocyanidins and their O-glycosides. The high-energy CID spectra of [M-Cl](+) ions of the free aglycones show characteristic fragmentation pathways, which provide useful information about the substitution pattern in the A- and B-rings of each compound. The major fragmentation observed in the high-energy CID spectra of [M-Cl](+) ions of anthocyanins involves loss of the mono- or disaccharide units resulting in ions containing only the aglycone moiety. From the spectral data, the identity of the aglycone can be established as well as the number and the class of monosaccharide units in the O-glycosides.

Structural analysis of polymer end groups by electrospray ionization high-energy collision-induced dissociation tandem mass spectrometry

Chemical structures of polymer end groups play an important role in determining the functional properties of a polymeric system. We present a mass spectrometric method for determining end group structures. Polymeric ions are produced by electrospray ionization (ESI), and they are subject to source fragmentation in the ESI interface region to produce low-mass fragment ions. A series of source-fragment ions containing various numbers of monomer units are selected for high-energy collision-induced dissociation (CID) in a sector/time-of-flight tandem mass spectrometer. It is shown that high-energy CID spectra of source-induced fragment ions are very informative for end group structure characterization. By comparing the CID spectra of fragment ions with those of known chemicals, it is possible to unambiguously identify the end group structures. The utility of this technique is illustrated for the analysis of two poly(ethylene glycol)-based slow-releasing drugs where detailed structural characterization is of significance for drug formulation, quality control, and regulatory approval. Practical issues related to the application of this method are discussed.

High-energy collision-induced dissociation of small polycyclic aromatic hydrocarbons.

High-energy collision-induced dissociation (CID) experiments on polycyclic aromatic hydrocarbons (PAHs) having 2-6 rings, naphthalene, anthracene, phenanthrene, fluoranthene, pyrene and coronene, were performed, and the relative abundances of their fragment ions were investigated as a function of collision energy. The results revealed that the PAHs except naphthalene showed a bimodal-type distribution of positive fragmentation ions, which is closely similar to the fragment-ion distribution reported for the CID of three-dimensional fullerene, C(60)(+) and C(70)(+). The three-ring isomers of anthracene and phenanthrene and the four-ring isomers of fluoranthene and pyrene can be distinguishable in their spectra under an electron ionization energy of 70 eV, but the high-energy CID spectra of the three- and four-ring isomers were almost identical. The fragmentation corresponding to fragment ions in the low-mass region of the bimodal CID spectra could be interpreted by the simple statistical model that fragment ions are formed by random evaporation from the molecular ions after a considerable structural rearrangement, 'phase transition', occurring at some high-energy state.

Automated interpretation of high-energy collision-induced dissociation spectra of singly protonated peptides by 'SeqMS', a software aid for de novo sequencing by tandem mass spectrometry.

SeqMS, a software program designed for the automated interpretation of high-energy collision-induced dissociation (CID) mass spectra of singly protonated peptides ionized by fast atom bombardment, has been developed. The software is capable of probing the sequence of an unknown peptide, and even of certain modified peptides. The program, compiled for WINDOWS95 or NT, also permits the retrieval of raw data and the reconstruction of the spectra on a user-friendly graphical interface with the aid of several tools for processing the spectra, which include setting multiple threshold levels and automatic peak detection. SeqMS is capable of generating candidate sequences, based on the detected peaks, and of displaying the resulting assignments for each candidate in a spectrum or in tabular form. The software has the following capabilities: 1) the ions derived from backbone and side-chain fragmentations, internal and immonium ions, and side-chain loss ions can be used for calculation; 2) 18O-labeling of a peptide at the C terminus, a methodology which was developed to differentiate N-terminal from C-terminal ions, is applicable as an optional setting; 3) modified amino acids and N- or C-terminal blocking groups are taken into account for calculation according to the user's setting in a library; 4) amino acid composition and partial or complete amino acid sequence of a peptide can be used as input for calculation; 5) the assignments of signal output in a spectrum can be graphically edited, and then re-calculated based on the edited peaks. The efficacy of the program is demonstrated by testing 74 high-energy CID spectra, obtained using a four-sector instrument, of synthetic, proteolytic, and biologically active peptides, some of which contain modified groups.

Mechanistic aspects of high-energy collision-induced dissociation proximate to the charge in saturated fatty acidN-butyl esters cationized with lithium. Evidence for hydrogen radical removal

The high-energy collision-induced dissociation spectra of [M + Li]+ ions of n-butyl palmitate or heptadecanoate and their labelled isotopomers containing a 2H2-label at the C-2, C-3 and C-4 positions are reported. As is the case for positions remote from the charge, the removal of a hydrogen radical at proximate positions has been found to be an initial and decisive fragmentation reaction when [M + Li]+ ions are subjected to high-energy collision-induced dissociation. It is also demonstrated that direct C–C cleavage involving γ bonds relative to the carbonyl bond or bonds more remote from the charge may be regarded as a secondary fragmentation route. Direct C–C cleavage of the β bond should, however, be considered as a reaction competitive with C–H cleavage in the formation of the corresponding distonic ions. © 1997 John Wiley & Sons, Ltd.

Linkage determination of alditol disaccharides using high-energy collision-induced dissociation spectra of sodium cationized molecules

Oligosaccharide alditols containing a deuterium atom at the alditol C-1 position can be used to obtain information about the masses and linkages of the various residues. The high-energy collision-induced dissociation (CID) spectra of the protonated compounds show mainly glycosidic bond cleavage with charge retention on the alditol containing Y-type ions. The CID spectra of the sodium-cationized compounds show additional fragment ions resulting from cross-ring cleavages. Moreover the elimination of methanol, formaldehyde and ethylene glycol molecules, as deuterium labelled or unlabelled species, appeared to provide a reliable criterion to determine the linkage positions at the alditol unit. © 1997 John Wiley & Sons, Ltd.

Comparison of Low- and High-energy Collision-induced Dissociation Tandem Mass Spectrometry in the Analysis of Ricinoleic and Ricinelaidic Acid

Ricinoleic acid and itstransisomer ricinelaidic acid, and their methyl esters, were analyzed by low- and high-energy collision-induced dissociation (CID) tandem mass spectrometry. It is shown that a stable charge centre is required to observe the rearrangement, which occurs in the β-hydroxyalkene part and results in the loss of heptaldehyde, and that this rearrangement corresponds to a low-energy CID reaction. Differences between the CID behaviour of ricinoleic acid and that of itstransisomer are related to the loss of water, which can also be regarded as a low-energy rearrangement reaction. Comparison of low- and high-energy CID spectra further revealed that high-energy CID gives rise to loss of a H•radical and H2 together with low-energy fragmentation. Examination of different molecule ions, including [M-H]-, [M+Li]+ and [M-H+2Li]+ ions of free fatty acids and [M+Li]+ ions of the methyl esters shows that charge-remote homolytic fragmentation is most pronounced for the [M+Li]+ ions of the methyl esters. © 1997 by John Wiley & Sons, Ltd.

Low- and high-energy collision-induced dissociation of pyridinoline and deoxypyridinoline ions using Fourier transform ion cyclotron resonance electrospray and liquid secondary-ion magnetic sector mass spectrometry

The trifunctional collagen cross-link molecules pyridinoline and deoxypyridinoline have been structurally characterised by (1) low-energy collision-induced dissociation (CID) ( E COM ≈ 15 eV) using Fourier transform ion cyclotron resonance electrospray ionisation mass spectrometry (FT-ICR-ESI-MS), and (2) high-energy collision-induced dissociation ( E COM ≈ 400 eV) using liquid secondary-ion mass spectrometry (LSIMS) on a four-sector mass spectrometer. Both ESI and LSIMS ionisation readily produce the naturally occurring parent cations, although the LSIMS ionisation method is somewhat hampered by background ions from the matrix. Low-energy or high-energy collision-induced dissociation produces structurally informative fragmentations. The observed fragmentation patterns in the low- and high-energy CID spectra are, however, strikingly different. Both low- and high-energy CID are structurally informative and appear to be complementary in the characterisation of collagen cross-link molecules.

Search of sequence databases with uninterpreted high-energy collision-induced dissociation spectra of peptides

We have broadened the utility of the SEQUEST computer algorithms to permit correlation of uninterpreted high-energy collision-induced dissociation spectra of peptides with all sequences in a database. SEQUEST now allows for the additional fragment ion types observed under high-energy conditions. We analyzed spectra from peptides isolated following trypsin digestion of 13 proteins. SEQUEST ranked the correct sequence first for 90% (18/20) of the spectra in searches of the OWL database, without constraint by enzyme cleavage specificity or species of origin. All false-positives were flagged by the scoring system. SEQUEST searches databases for sequences that correspond to the precursor ion mass ±0.5 u. Preliminary ranking of the top 500 candidates is done by calculation of fragment ion masses for each sequence, and comparison to the measured ion masses on the basis of ion series continuity, summed ion intensity, and immonium ion presence. Final ranking is done by construction of model spectra for the 500 candidates and constructing/performing of a cross-correlation analysis with the actual spectrum. Given the need to relate mounting genome sequence information with corresponding suites of proteins that comprise the cellular molecular machinery, tandem mass spectrometry appears destined to play the leading role in accelerating protein identification on the large scale required.

Analysis of a Five-component Mixture of Polymer Additives by Means of High Energy Mass Spectrometry and Tandem Mass Spectrometry

High energy mass spectrometry and tandem mass spectrometry have been employed to analyse a five-component mixture of common organic polymer additives (300−1200 daltons). The experiments were performed by means of a four-sector instrument and a time-of-flight mass spectrometer. Field desorption was a good ionization method for the generation of molecular weight information from the polymer additives. High energy collision induced dissociation (CID) was found to be more applicable than low energy CID to the structural determination of polymer additives, as characteristic ions were observed in the spectra by means of the former method. Mechanisms have been proposed for the generation of some of the fragment ions observed in the high energy CID spectra

Matrix-assisted laser desorption /ionization tandem reflectron time-of-flight mass spectrometry of fullerenes

Atandem reflectron time-of-flight mass spectrometer developed in our laboratory provides a unique opportunity to investigate the collision-induced dissociation of fullerene ions formed by matrix-assisted laser desorption/ionization (MALDI). Specifically, this opportunity arises from the ability to utilize high energy collisional activation (normally available only on tandem sector instruments by using continuous ionization techniques) for ions formed by pulsed laser desorption, whereas most MALDI time-of-flight instruments record product ion mass spectra of ions formed by metastable or postsource decay. In this study we investigate the products of mass-selected and collisionally activated C 60 (+) and C 70 (+) ions by using different target gases over a range of target gas pressures. In general, heavier target gases produce more extensive fragmentation and improve the mass resolution of lower mass ionic products because a greater portion of these ions are formed by single collisions. Additionally, the tandem time-of-flight instrument utilizes a nonlinear (curved-field) reflectron in the second mass analyzer that enables high energy collision-induced dissociation spectra to be recorded without scanning or stepping the reflectron voltage.

Effect of the position of a basic amino acid on C-terminal rearrangement of protonated peptides upon collision-induced dissociation.

Internal rearrangement involving the loss of the C-terminal amino acid residue upon collision-induced dissociation (CID) or metastable decomposition was studied for protonated peptides. To investigate the structural characteristics of peptides responsible for this rearrangement, a series of synthetic peptides were prepared and subjected to B/E-linked scan or tandem mass spectrometric analyses using a four-sector instrument. The results showed that the position of a basic amino acid in the peptide sequence and its basicity have a significant influence on the rearrangement. Arginine (Arg) located at the n-1 position facilitates the rearrangement with about twice as many rearrangement ions as is observed for the other Arg-containing peptides. This can be attributed to the interaction of a positively charged guanidino group of Arg with its own carbonyl group via a salt bridge which is tightly formed in vacuo between a guanidino and carboxylate groups, the mechanism of which is analogous to that previously proposed for the formation of similar rearrangement ions observed in the spectra of metal-cationized peptides. This association would result in the facile attack of the C-terminal hydroxyl group on the penultimate carbonyl group, leading to the rearrangement. In addition, the rearrangement ion was observed both in metastable decomposition and high-energy CID spectra obtained by B/E-linked scan analyses without or with gas, respectively, but in a sequence dependent manner.

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

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