Fragmentation studies on monensin A and B by accurate-mass electrospray tandem mass spectrometry

Monensin A and B were studied by electrospray ionisation tandem mass spectrometry (ESI-MS/MS) and the fragment ions were confirmed by accurate-mass measurements. Analyses were performed on both a quadrupole time-of-flight (QTOF) and a Fourier-transform ion cyclotron resonance (FTICR) mass spectrometer. The analysis revealed that fragment ions were produced by Grob-Wharton fragmentations and pericyclic rearrangements in addition to various simple neutral losses. A study of the protonated and sodiated sodium salt revealed different fragmentation pathways for these species, thus complementary structural information could be gained. A complete fragmentation pathway of monensin A and B protonated sodium salt [(M-H+Na)+H])+) and sodiated sodium salt [(M-H+Na)+Na](+) is proposed. MS(3) analysis confirmed the separate fragmentation pathways.     

Isomer differentiation and structural characterization of penem β-lactam antibiotics by mass-analyzed ion kinetic energy spectrometry

A number of clinically significant penem β-lactams, both free acids and sodium salts, were investigated by mass-analyzed ion kinetic energy spectrometry (MIKES) following fast atom bombardment (FAB) ionization. The collisionally activated dissociation (CAD) products of [M + H] ⁺ and [M + Na]⁺ ions are described. Carbon dioxide loss was observed for some of the free acids, whereas a daughter ion generated by β-lactam ring cleavage was characteristic of the sodiated species. Other fragments included successive cleavages and rearrangements of the substituent side-chain, permitting complete characterization of these chains. The fragmentation pattern for both protonated and sodiated species were more clearly established by CAD MIKES than by normal FAB mass spectral analyses. A notable feature of this technique was its ability to differentiate between pairs of regioisomeric penems on the basis of their fragmentation patterns. These compounds could not be differentiated in the usual mass spectra.     

Characterization of limonin glucoside metabolites from human prostate cell culture medium using high-performance liquid chromatography/electrospray ionization mass spectrometry and tandem mass spectrometry

The metabolism of limonin 17-beta-D-glucopyranoside (LG) by non-cancerous (RWPE-1) and cancerous (PC-3) human prostate epithelial cells was investigated using high-performance liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) with in-source fragmentation and tandem mass spectrometry (MS/MS). During positive ion LC/ESI-MS, LG formed an abundant sodiated species ([M+Na]+) while the protonated molecule was barely observable. [M+Na]+ further fragmented into the less abundant [LARL+H]+ and a predominantly protonated aglycone molecule (limonin) due to in-source fragmentation. The major metabolite, limonin A-ring lactone (LARL), formed an abundant protonated molecule that was fragmented into a protonated molecule of limonin by loss of one molecule of water. In MS/MS by collisionally activated dissociation (CAD), LG produced the sodiated aglycone, [aglycone+Na]+, while LARL fragmented into [M+H]+ of limonin and fragment ions resulted by further loss of water, carbon monoxide and carbon dioxide, indicating the presence of oxygenated-ring structures. The limits of detection of LG were 0.4 and 20 fmol in selected-ion monitoring (SIM) and selected-reaction monitoring (SRM) detection, respectively.     

Novel phosphoryl derivatization method for peptide sequencing by electrospray ionization mass spectrometry

A one-step phosphoryl derivatization method has been used in a peptide sequencing procedure for electrospray ionization tandem mass spectrometry (ESI-MS/MS). The sodiated derivatized peptides exhibit very simple dissociation patterns, in which two kinds of fragment ions, [b(n) + OH + Na]+ and [a(n) + Na]+, are formed. Since the amino acid residues are lost sequentially from the C-terminus, peptide sequences can be identified easily. The fragmentation efficiency of peptides increased as a result of the phosphorylation, and also provided peaks of useful intensity at lower m/z. A peptide with lysine at the C-terminus was derivatized and analyzed by ESI-MS/MS. Similar mass spectra, from which the sequence could be read out, were obtained. This is a novel derivatization method yielding neutral derivatives that should be suitable for peptide sequencing by LC/ESI-MS/MS.     

Structural analysis of phosphatidylcholines by post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The utility of post-source decay (PSD) matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was investigated for the structural analysis of phosphatidylcholine (PC). PC did not produce detectable negative molecular ion from MALDI, but positive ions were observed as both [PC+H](+) and [PC+Na](+). The PSD spectra of the protonated PC species contained only one fragment corresponding to the head group (m/z 184), while the sodiated precursors produced many fragment ions, including those derived from the loss of fatty acids. The loss of fatty acid from the C-1 position (sn-1) of the glycerol backbone was favored over the loss of fatty acid from the C-2 position (sn-2). Ions emanating from the fragmentation of the head group (phosphocholine) included [PC+Na-59](+), [PC+Na-183](+) and [PC+Na-205](+), which corresponded to the loss of trimethylamine (TMA), non-sodiated choline phosphate and sodiated choline phosphate, respectively. Other fragments reflecting the structure of the head group were observed at m/z 183, 146 and 86. The difference in the fragmentation patterns for the PSD of [PC+Na](+) compared to [PC+H](+) is attributed to difference in the binding of Na(+) and H(+). While the proton binds to a negatively charged oxygen of the phosphate group, the sodium ion can be associated with several regions of the PC molecule. Hence, in the sodiated PC, intermolecular interaction of the negatively charged oxygen of the phosphate group, along with sodium association at multiple sites, can lead to a complex and characteristic ion fragmentation pattern. The preferential loss of sn-1 fatty acid group could be explained by the formation of an energetically favorable six-member ring intermediate, as apposed to the five-member ring intermediate formed prior to the loss of sn-2 fatty acid group.     

Characterization of sodiated glycerol phosphatidylcholine phospholipids by mass spectrometry

Fast atom bombardment mass spectrometry in the positive mode was used for the characterization of sodiated glycerol phosphatidylcholines. The relative abundance (RA) of the protonated species is similar to the RA of the sodiated molecular species. The sodiated fragment ion, [M + Na - 59](+), corresponding to the loss of trimethylamine, and other sodiated fragment ions, were also observed. The decomposition of the sodiated molecule is very similar for all the studied glycerol phosphatidylcholines, in which the most abundant ion corresponds to a neutral loss of 59 Da. Upon collision-induced dissociation (CID) of the [M + Na](+) ion informative ions are formed by the losses of the fatty acids in the sn-1 and sn-2 positions. Other major fragment ions of the sodiated molecule result from loss of non-sodiated and sodiated choline phosphate, [M + Na - 183](+), [M + Na - 184](+.) and [M + Na - 205](+), respectively. The main CID fragmentation pathway of the [M + Na - 59](+) ion yields the [M + Na - 183](+) ion, also observed in the CID spectra of the [M + Na](+) molecular ion. Other major fragment ions are [M + Na - 205](+) and the fragment ion at m/z 147. Collisional activation of [M + Na - 205](+) results in charge site remote fragmentation of both fatty acid alkyl chains. The terminal ions of these series of charge remote fragmentations result from loss of part of the R(1) or R(2) alkyl chain. Other major informative ions correspond to acylium ions.     

Infrared spectroscopy of cationized arginine in the gas phase: direct evidence for the transition from nonzwitterionic to zwitterionic structure

The gas-phase structures of protonated and alkali metal cationized arginine (Arg) and arginine methyl ester (ArgOMe) are investigated with infrared spectroscopy and ab initio calculations. Infrared spectra, measured in the hydrogen-stretch region, provide compelling evidence that arginine changes from its nonzwitterionic to zwitterionic form with increasing metal ion size, with the transition in structure occurring between lithium and sodium. For sodiated arginine, evidence for both forms is obtained from spectral deconvolution, although the zwitterionic form is predominant. Comparisons of the photodissociation spectra with spectra calculated for low-energy candidate structures provide additional insights into the detailed structures of these ions. Arg*Li+, ArgOMe*Li+, and ArgOMe*Na+ exist in nonzwitterionic forms in which the metal ion is tricoordinated with the amino acid, whereas Arg*Na+ and Arg*K+ predominately exist in a zwitterionic form where the protonated side chain donates one hydrogen bond to the N terminus of the amino acid and the metal ion is bicoordinated with the carboxylate group. Arg*H+ and ArgOMe*H+ have protonated side chains that form the same interaction with the N terminus as zwitterionic, alkali metal cationized arginine, yet both are unambiguously determined to be nonzwitterionic. Calculations indicate that for clusters with protonated side chains, structures with two strong hydrogen bonds are lowest in energy, in disagreement with these experimental results. This study provides new detailed structural assignments and interpretations of previously observed fragmentation patterns for these ions.     

Electrospray ionization for analysis of platelet-activating factor.

Platelet-activating factor (PAF) was analyzed by electrospray-ionization mass spectrometry (ESI-MS) using a single quadrupole mass spectrometer. The positive-ion spectrum was dominated by an ion corresponding to a sodiated molecule when a low potential difference between the capillary exit (nozzle) and the skimmer was employed, but when the capillary exit voltage was increased, fragmentation of PAF was observed. Initial fragmentation involved the loss of the elements of trimethylamine from the sodiated molecule to yield [M+Na-59]+. An intense ion at m/z 147, generated by the loss of trimethylamine from the sodiated phosphocholine portion of the molecule was also detected, along with a lower intensity ion at m/z 184 which is representative of a protonated phosphocholine moiety. With negative-ion detection the major molecular species was [M+Cl]-. Interpretation of the mass spectral fragments was verified by ESI tandem mass spectrometry on a triple-quadrupole tandem mass spectrometer.     

Fragmentation studies on lasalocid acid by accurate mass electrospray mass spectrometry

Lasalocid acid is an important polyether ionophore veterinary drug. Polyether ionophores have been the subject of MS study for many years, but this is the first rigorous study of the complex fragmentation processes occurring in ESI MS/MS for lasalocid, underpinned by high-resolution accurate-mass measurement. Initial low-resolution analyses were performed on an ion-trap instrument. High-resolution analyses were performed on a Fourier-transform ion cyclotron resonance mass spectrometer. The MS/MS analysis of the pseudo-molecular ion shows that fragment ions are produced either by beta-elimination or by neutral losses of water. Additional ions were observed in the source dissociation analysis, indicating that additional fragmentation reactions occur in the source region. Some of these ions can then undergo additional ion-ion or ion-molecule reactions before being extracted from the source. The study of both the protonated and sodiated sodium salts shows the same fragmentation pathways, with fragment ions containing two sodiums at low intensity. A fragmentation pathway of the lasalocid acid protonated sodium salt [(M-H+Na)+H]+ (m/z 613) and sodiated sodium salt [(M-H+Na)+Na]+ (m/z 635) is presented. The increased understanding afforded by this study will help in the development of unequivocal analytical methods for lasalocid and related polyether ionophore veterinary drugs.     

Metal ion adducts in the structural analysis of ginsenosides by electrospray ionization with multi-stage mass spectrometry

The effect of metal (Li+, Na+, K+, Ag+) cationization on collision-induced dissociation of ginsenosides was investigated by electrospray ionization mass spectrometry combined with multi-stage mass spectrometry (ESI-MS(n)). The fragments of sodiated and lithiated molecules give valuable structural information regarding the nature of the aglycone and the sequence and linkage information of sugar moieties. However, the number and relative abundances of fragment ions from lithiated ginsenosides are significantly greater than for the sodiated species. The K+ adducts undergo glycosidic cleavages and very limited cross-ring reactions. The silver ion adducts fragment mainly through glycosidic cleavages.     

Charge state effect on the zwitterion influence on stability of non-covalent interaction of single-stranded DNA with peptides

Negative ion ESI mass spectrometry was used to study the gas-phase stability and dissociation pathways of peptide-DNA complexes. We show that bradykinin and three modified peptides containing the basic residue arginine or lysine form stable interactions with single-stranded oligonucleotides. ESI-MS/MS of complexes of T(8) with PPGFSPFRR resulted in a major dissociation pathway through cleavage of the peptide covalent bond. The stability of the complex is due to electrostatic interaction between the negatively charged phosphate group and the basic side chain of the arginine and lysine residues as demonstrated by Vertes et al. and Woods et al. In fact, the present work establishes the role played by zwitterions on complex stabilisation. The presence of protons in nucleobase and/or amino acid contributes in reinforcing the strength of the salt bridge (SB) interaction. The zwitterionic form of the most basic of amino acid residues, arginine, is assumed to form a strong SB interaction to the negatively charged phosphate groups of DNA. This non-covalent complex is stable enough to withstand disruption of the non-covalent interaction and to first break the covalent bond. Moreover, the dependence of fragmentation patterns upon the complex charge state is explained by the fact that the net number of negative charges modulates the number of zwitterionic sites, which stabilise the complexes. Finally, the weak influence of the nucleobase is assumed by the existence of competition for proton addition between the nucleobase and the R/K side chain leading to a decrease in the stabilisation of the SB interaction.     

甘露糖磷酰氨基酸缀合物的ESI-MS研究

一系列全乙酰保护甘露糖-1-磷酰氨基酸酯缀合物的α构型和β构型异构体的ESI-MSn裂解规律研究表明, β构型异构体会出现一系列特征的m/z 433, 391, 371的碎片离子, 且［M-CH2CHCH3+Na］+碎片丰度较大. ［M-糖基+Na］+和［糖基+Na］+碎片相对丰度较小. α构型分子反之. 同时对全乙酰保护的［糖基+Na］+ m/z 353碎片离子进行三级碎裂, 其主要特征是消除CH2CO和AcOH中性分子的碎片离子, 而且其它位羟基构型的差异对质谱中碎裂方式影响不大.     

Solid-phase synthesis of alpha-glucosamine sulfoforms with fragmentation analysis by tandem mass spectrometry

Sulfated epitopes of alpha-glucosamine (GlcN sulfoforms) were prepared by solid-phase synthesis as models of internal glucosamines within heparan sulfate. An orthogonally protected 2'-hydroxyethyl GlcN derivative was immobilized on a trityl resin support and subjected to regioselective deprotection and sulfonation conditions, which were optimized with the aid of on-resin infrared or Raman analysis. The sulfoforms were cleaved from the resin under mild Lewis acid conditions without affecting the O- or N-sulfate groups and purified by reversed-phase high-performance liquid chromatography (HPLC). The alpha-GlcN sulfoforms and their 4- O-benzyl ethers were examined by electrospray ionization tandem mass spectrometry (ESI-MS/MS), with product ion spectra produced by collision-induced dissociation (CID). ESI-MS/MS revealed significant differences in parent ion stabilities and fragmentation rates as a function of sulfate position. Ion fragmentation by CID resulted in characteristic mass losses with strong correlation to the positions of both free hydroxyl groups and sulfate ions. Most of these fragmentation patterns are consonant with elimination pathways, and suggest possible strategies for elucidating the structures of glucosamine-derived sulfoforms with identical m/ z ratios. In particular, fragmentation analysis can easily distinguish GlcN sulfoforms bearing the relatively rare 3- O-sulfate from isomers with the more common 6- O-sulfate.     

Sequencing cyclic peptide inhibitors of mammalian ribonucleotide reductase by electrospray ionization mass spectrometry.

Mammalian ribonucleotide reductase (mRR) is a potential target for cancer intervention. A series of lactam-bridged cyclic peptide inhibitors (1-9) of mRR have been synthesized and tested in previous work. These inhibitors consist of cyclic and linear regions, causing their mass spectral characterization to be a challenge. We determined the fragmentation mechanism of cyclic peptides 1-9 using an ion-trap mass spectrometer equipped with an ESI source. Low-energy collision-induced dissociation of sodiated cyclic peptides containing linear branches follows a general pathway. Fragmentation of the linear peptide region produced mainly a and b ions. The ring peptide region was more stable and ring opening required higher collision energy, mainly occurring at the amide bond adjacent to the lactam bridge. The sodium ion, which bound to the carbonyl oxygen of the lactam bridge, acted as a fixed charge site and directed a charge-remote, sequence-specific fragmentation of the ring-opened peptide. Amino acid residues were cleaved sequentially from the C-terminus to the N-terminus. Our findings have established a new way to sequence cyclic peptides containing a lactam bridge based on charge-remote fragmentation. This methodology will permit unambiguous identification of high-affinity ligands within cyclic peptide libraries.     

Gas-phase H/D exchange of sodiated glycine oligomers with ND3: exchange kinetics do not reflect parent ion structures

H/D exchange is a method commonly used to probe molecular structure. The majority of studies in the gas phase have involved protonated molecular ions. The present study gives attention to molecular ions formed by coordination with a sodium ion. In particular, ND(3) is reacted with sodiated glycine oligomers, Gly(n)(), where n = 1-5, and the results are interpreted using density functional calculations. Experimentally, Gly(1)Na(+), Gly(4)Na(+), and Gly(5)Na(+) all undergo three fast exchanges with ND(3), while Gly(2)Na(+) and Gly(3)Na(+) undergo one fast and two slow exchanges with ND(3). The methyl esters Gly(3)OMeNa(+) and Gly(5)OMeNa(+) do not exchange with ND(3). In agreement with earlier experimental studies, theoretical calculations show that the lowest-energy conformers of the sodiated glycine oligomers are charge-solvated structures. Calculations further indicate that, in the process of H/D exchange with ND(3), sodiated monoglycine and tetraglycine adopt zwitterionic structures, sodiated diglycine adopts a salt-bridge form, and sodiated triglycine takes on an ion-stabilized ion pair form. Sodiated monoglycine and diglycine exchange via an onium-ion mechanism. The proposed exchange mechanisms require a carboxylic acid hydrogen to complete the exchange, which is in agreement with the experimental results showing that no exchange occurs with methyl ester glycine oligomers. These studies clearly demonstrate that, in the process of H/D exchange, noncovalent complexation of the exchange reagent provides the energy required to access intermediates structurally distinct from the parent ions. H/D exchange is facile for these intermediates. Contrary to the assumption often expressed in earlier studies, H/D exchange kinetics may not directly reflect ion structures.     

Fragmentation study of salinomycin and monensin A antibiotics using electrospray quadrupole time-of-flight mass spectrometry

The fragmentation pathways of two selected ionophore antibiotics, salinomycin and monensin A, were studied using electrospray (ES) orthogonal acceleration quadrupole time-of-flight mass spectrometry in positive-ion mode. The identity of fragment ions was determined by accurate-mass measurements. In ES mass spectra, ion signals of relatively high intensity were observed for [M+Na](+) and [M-H+2Na](+) for each antibiotic. Each of the ion species [M+Na](+) and [M-H+2Na](+) for salinomycin and [M-H+2Na](+) for monensin A were isolated in turn and subjected to fragmentation. In the fragmentation of [M+Na](+) and [M-H+2Na](+) from salinomycin, only Cbond;C single bond cleavage and dehydration were observed. Product ion mass spectra obtained from [M-H+2Na](+) of monensin A showed that ether ring opening, Cbond;C single bond cleavage and dehydration fragmentations had occurred. Fragment ions containing two sodium atoms were observed in the product ion mass spectrum of [M-H+2Na](+) from salinomycin, but not from monensin A. Both type A (containing the terminal carboxyl group) and type F (containing the terminal hydroxyl group) fragment ions were observed in the product ion mass spectra of sodium adduct ions of salinomycin and monensin A.     

Structure and further fragmentation of significant [a3 + Na − H]+ ions from sodium‐cationized peptides

A good understanding of gas‐phase fragmentation chemistry of peptides is important for accurate protein identification. Additional product ions obtained by sodiated peptides can provide useful sequence information supplementary to protonated peptides and improve protein identification. In this work, we first demonstrate that the sodiated a₃ ions are abundant in the tandem mass spectra of sodium‐cationized peptides although observations of a₃ ions have rarely been reported in protonated peptides. Quantum chemical calculations combined with tandem mass spectrometry are used to investigate this phenomenon by using a model tetrapeptide GGAG. Our results reveal that the most stable [a₃ + Na ? H]⁺ ion is present as a bidentate linear structure in which the sodium cation coordinates to the two backbone carbonyl oxygen atoms. Due to structural inflexibility, further fragmentation of the [a₃ + Na ? H]⁺ ion needs to overcome several relatively high energetic barriers to form [b₂ + Na ? H]⁺ ion with a diketopiperazine structure. As a result, low abundance of [b₂ + Na ? H]⁺ ion is detected at relatively high collision energy. In addition, our computational data also indicate that the common oxazolone pathway to generate [b₂ + Na ? H]⁺ from the [a₃ + Na ? H]⁺ ion is unlikely. The present work provides a mechanistic insight into how a sodium ion affects the fragmentation behaviors of peptides. Copyright © 2015 John Wiley & Sons, Ltd.     

Structural determination of monoacylglycerols extracted from marine sponge by fast atom bombardment tandem mass spectrometry

Five new monoacylglycerols (MAGs) were isolated from the marine sponge Stelletta sp. by reversed-phase high-performance liquid chromatography and analyzed by positive ion fast atom bombardment mass spectrometry (FAB-MS). FAB mass spectra of these compounds produced abundant sodium-adducted molecules [M+Na]+ from a mixture of 3-nitrobenzyl alcohol and sodium iodide. The structural elucidation of these sponge MAGs was carried out by FAB tandem mass spectrometry (MS/MS). To find diagnostic ions for the characterization of the MAGs, authentic MAGs were initially analyzed by collision-induced dissociation (CID) MS/MS. The CID MS/MS of [M+Na]+ precursor ions resulted in the formation of numerous characteristic product ions via a series of dissociative processes. The product ions formed by charge-remote fragmentation (CRF) provided important information for the characterization of acyl chains substituted at the glycerol backbone, and product ions at m/z 84, 97, 113 and 139 were diagnostic for the sodiated glycerol backbone. On the basis of these fragmentation patterns, the structures of five MAGs extracted from marine sponge were elucidated. In addition, high-resolution mass measurement was performed to obtain the elemental compositions of the MAGs.     

Determination of linkage position and anomeric configuration in Hex-Fuc disaccharides using electrospray ionization tandem mass spectrometry

Fixed-energy sequential tandem mass spectrometry (MS(n)) capabilities offered by quadrupole ion trap instruments have been explored in a systematic study of six isomers of Gal-Fucalpha-OBenzyl disaccharides. Under collision-induced dissociation (CID), sodiated molecular species generated in the positive-ion electrospray ionization mode yield simple and predictable mass spectra. Information on interglycosidic linkages and configurations can be deduced from the relative intensities of the selected diagnostic fragments arising from the glycosidic bond cleavages and corroborated by the fragments arising from cross-ring cleavages. As the CID patterns are not dependent on the number of prior tandem mass spectrometric steps, structures can be unambiguously assigned by matching the spectra with a library. The rules governing the fragmentation behavior of this class of oligosaccharides were tested for a representative isomeric disaccharide, Glcbeta1,3Fucalpha-OAllyl. The findings establish a basis for using MS(n) with a quadrupole ion trap instrument to elucidate structures of hexose-fucose subunits from more complicated oligosaccharides. Energy-resolved mass spectra were also acquired by CID tandem triple-quadrupole mass spectrometry. The breakdown behavior of the molecular ions revealed patterns which could differentiate stereoisomers of Gal-Fuc disaccharides over a range of collision energy from 20 to 50 eV.