Structural characterization by infrared multiple photon dissociation spectroscopy of protonated gas-phase ions obtained by electrospray ionization of cysteine and dopamine

Structural characterization of protonated gas-phase ions of cysteine and dopamine by infrared multiple photon dissociation (IRMPD) spectroscopy using a free electron laser in combination with theory based on DFT calculations reveals the presence of two types of protonated dimer ions in the electrospray mass spectra of the metabolites. In addition to the proton-bound dimer of each species, the covalently bound dimer of cysteine (bound by a disulfide linkage) has been identified. The dimer ion of m/z 241 observed in the electrospray mass spectra of cysteine has been identified as protonated cystine by comparison of the experimental IRMPD spectrum to the IR absorption spectra predicted by theory and the IRMPD spectrum of a standard. Formation of the protonated covalently bound disulfide-linked dimer ions (i.e. protonated cystine) from electrospray of cysteine solution is consistent with the redox properties of cysteine. Both the IRMPD spectra and theory indicate that in protonated cystine the covalent disulfide bond is retained and the proton is involved in intramolecular hydrogen bonding between the amine groups of the two cysteine amino acid units. For cysteine, the protonated covalently bound dimer (m/z 241) dominated the mass spectrum relative to the proton-bound dimer (m/z 243), but this was not the case for dopamine, where the protonated monomer and the proton-bound dimer were both observed as major ions. An extended conformation of the ethylammonium side chain of gas-phase protonated dopamine monomer was verified from the correlation between the predicted IR absorption spectra and the experimental IRMPD spectrum. Dopamine has the same extended ethylamine side chain conformation in the proton-bound dopamine dimer identified in the mass spectra of electrosprayed dopamine. The structure of the proton-bound dimer of dopamine is confirmed by calculations and the presence of an IR band due to the shared proton. The presence of the shared proton in the protonated cystine ion can be inferred from the IRMPD spectrum.

Structure Elucidation of the Diagnostic Product Ion at <Emphasis Type="Italic">m/z</Emphasis> 97 Derived from Androst-4-en-3-One-Based Steroids by ESI-CID and IRMPD Spectroscopy

Structure elucidation of steroids by mass spectrometry has been of great importance to various analytical arenas and numerous studies were conducted to provide evidence for the composition and origin of (tandem) mass spectrometry-derived product ions used to characterize and identify steroidal substances. The common product ion at m/z 97 generated from androst-4-ene-3-one analogs has been subject of various studies, including stable isotope-labeling and (high resolution/high accuracy) tandem mass spectrometry, but its gas-phase structure has never been confirmed. Using high resolution/high accuracy mass spectrometry and low resolution tandem mass spectrometry, density functional theory (DFT) calculation, and infrared multiple photon dissociation (IRMPD) spectroscopy employing a free electron laser, the structure of m/z 97 derived from testosterone was assigned to protonated 3-methyl-2-cyclopenten-1-one. This ion was identified in a set of six cyclic C₆H₉O⁺ isomers as computed at the B3LYP/6-311++G(2d,2p) level of theory (protonated 3-methyl-2-cyclopenten-1-one, 2-methyl-2-cyclopenten-1-one and 2-cyclohexen-1-one). Product ions of m/z 97 obtained from MS² and MS³ experiments of protonated 3-methyl-2-cyclopenten-1-one, 2-methyl-2-cyclopenten-1-one, 2-cyclohexen-1-one, and testosterone corroborated the suggested gas-phase ion structure, which was eventually substantiated by IRMPD spectroscopy yielding a spectrum that convincingly matched the predicted counterpart. Finally, the dissociation pathway of the protonated molecule of testosterone to m/z 97 was revisited and an alternative pathway was suggested that considers the exclusion of C-10 along with the inclusion of C-5, which was experimentally demonstrated with stable isotope labeling.     

Ion-Molecule reactions in methylamine and dimethylamine and trimethylamine systems

Fourier transform ion cyclotron resonance mass spectrometry has been used to measure the reaction rates for ions derived from methylamine with dimethylamine or trimethylamine. The use of the selective ion ejection technique greatly simplifies the elucidation of the ion-molecule reaction channels. The rate constants for proton transfer from protonated metwlamine, CH₃NH ₃ ⁺ (m/z 32), to dimethylamine and trimethylamine are 16.1 ± 1.6 × 10^?10 and 9.3 ± 0.9 × 10^?10 cm³ molec^?1s^?1, respectively. The rate constants for charge transfer from methylamine molecular ion, CH₃NH ₂ ⁺ (m/z 31), to dimethylamine and trimethylamine are 9.3 ± 1.8 x 10^?10 and 15.0 ± 5 × 10^?10 cm³molec^?1s^?1, respectively.     

Fragmentation of the metastable molecular ion of methyl lactate: The formation of oxygen-protonated methanol [CH3OH2]+ involving double hydrogen atom transfers

The spontaneous unimolecular dissociation reaction of methyl lactate (1) ionized by electron impact was investigated by a combination of mass-analyzed ion kinetic energy spectrometry and deuterium labeling. The metastable ions 1^+· decompose in a variety of ways: four fragment peaks are observed at m/z 89, 76, 61, and 45, which correspond to the losses of ?H₃, CO, CH₃?O, and ?OOCH₃, respectively. Double hydrogen atom transfer occurs in the third reaction. The source-generated m/z 61 ions decompose into oxygen-protonated methanols at m/z 33 ([CH₃OH ₂ ⁺ ]) by the loss of CO with double hydrogen atom migration. Both hydroxyl and methyne hydrogen atoms in 1 ^+· are present in the resultant protonated methanols.     

Spectroscopic evidence for mobilization of amide position protons during CID of model peptide ions

Infrared multiple photon dissociation (IRMPD) spectroscopy was used to study formation of b ₂⁺ from nicotinyl-glycine-glycine-methyl ester (NicGGOMe). IRMPD shows that NicGGOMe is protonated at the pyridine ring of the nicotinyl group, and more importantly, that b ₂⁺ from NicGGOMe is not protonated at the oxazolone ring, as would be expected if the species were generated on the conventional b _n ⁺ /y _n ⁺ oxazolone pathway, but at the pyridine ring instead. IRMPD data support a hypothesis that formation of b ₂⁺ from NicGGOMe involves mobilization and transfer of an amide position proton during the fragmentation reaction.     

Gas Phase Decarbonylation and Cyclization Reactions of Protonated N-Methyl-N-Phenylmethacrylamide and Its Derivatives Via an Amide Claisen Rearrangement

Gas phase decarbonylation and cyclization reactions of protonated N-methyl-N-phenylmethacrylamide and its derivatives (M·H⁺) were studied by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). MS/MS experiments of M·H⁺ showed product ions were formed by loss of CO, which could only occur with an amide Claisen rearrangement. Mechanisms for the gas phase decarbonylation and cyclization reactions were proposed based on the accurate m/z measurements and MS/MS experiments with deuterated compounds. Theoretical computations showed the gas phase Claisen rearrangement was a major driving force for initiating gas phase decarbonylation and cyclization reactions of M·H⁺. Finally, the influence of different phenyl substituents on the gas phase Claisen rearrangement was evaluated. Electron-donating groups at the para-position of the phenyl moiety promoted the gas phase Claisen rearrangement to give a high abundance of fragment ions [M ? CO + H]⁺. By contrast, electron-withdrawing groups on the phenyl moiety retarded the Claisen rearrangement, but gave a fragment ion at m/z 175 by loss of neutral radicals of substituents on the phenyl, and a fragment ion at m/z 160 by further loss of a methyl radical.     

LC-DAD-ESI-MS Characterization of Carbohydrates Using a New Labeling Reagent

A novel labeling reagent 1-(2-naphthyl)-3-methyl-5-pyrazolone (NMP) coupling to liquid chromatography with electrospray ionization mass spectrometry for the detection of carbohydrates from the derivatized rape bee pollen samples is reported. Carbohydrates are derivatized to their bis-NMP-labeled derivatives. Derivatives showed an intense protonated molecular ion at m/z [M+H]⁺ in positive-ion detection mode. The mass-to-charge ratios of characteristic fragment ions at m/z 473.0 could be used for the accurately qualitative analysis of carbohydrates. This characteristic fragment ion is from the cleavage of C2–C3 bond in carbohydrate chain giving the specific fragment ions at m/z [MH-C _m H_2m+1O _m -H₂O]⁺ for pentose, hexose and glyceraldehydes and at m/z [MH-C _m H_2m-1O _m+1-H₂O]⁺ for alduronic acids such as galacturonic acid and glucuronic acid (m = n ? 2, n is carbon number of carbohydrate). No interferences for all aliphatic and aromatic aldehydes presented in natural environmental samples were observed due to the highly specific parent mass-to-charge ratio and the characteristic fragment ions. The method, in conjunction with a gradient elution, offered a baseline resolution of carbohydrate derivatives on a reversed-phase Hypersil ODS-2 column. The carbohydrates such as mannose, galacturonic acid, glucuronic acid, rhamnose, glucose, galactose, xylose, arabinose and fucose can successfully be detected.     

Diagnostic NH and OH Vibrations for Oxazolone and Diketopiperazine Structures: b<Subscript>2</Subscript> from Protonated Triglycine

We present infrared multiple photon dissociation (IRMPD) spectra in the hydrogen stretching region of the simplest b fragment, b₂ from protonated triglycine, contrasted to that of protonated cyclo(Gly-Gly). Both spectra confirm the presence of intense, diagnostic vibrations linked to the site of proton attachment. Protonated cyclo(Gly-Gly) serves as a reference spectrum for the diketopiperazine structure, showing a diagnostic O-H⁺ stretch of the protonated carbonyl group at 3585 cm^–1. Conversely, b₂ from protonated triglycine exhibits a strong band at 3345 cm^–1, associated with the N-H stretching mode of the protonated oxazolone ring structure. Other weaker N-H stretches can also be discerned, such as the amino NH₂ and amide NH bands. These results demonstrate the usefulness of the hydrogen stretching region, and hence benchtop optical parametric oscillator/amplifier (OPO/A) set-ups, in making structural assignments of product ions in collision-induced dissociation (CID) of peptides.     

Electron ionization induced metastable decompositions of isomeric trimethylsilylmethanol, (CH3)3SiCH2OH, and methoxytrimethylsilane, (CH3)3SiOCH3

The metastable decompositions of trimethylsilylmethanol, (CH₃)₃SiCH₂OH (MW: 104, 1) and methoxytrimethylsilane, (CH₃)₃SiOCH₃ (MW: 104, 2) upon electron ionization have been investigated by use of mass-analyzed ion kinetic energy (MIKE) spectroscopy and D labeling. The metastable ions of 1 ^·+ decompose to give the fragment ions m/z 89 (CH ₃ ^· loss) and 73 (^·CH₂OH loss), whereas those of 2 ^·+ only yield the fragment ion m/z 89 (CH ₃ ^· loss). The latter fragment ion is generated by loss of a methyl radical from the trimethylsilyl group via a simple cleavage reaction as shown by D labeling. However, the fragment ions m/z 89 and 73 from 1 ^·+ are generated following an almost statistical exchange of the original methyl and methylene hydrogen atoms in the molecular ion as shown also by D labeling. This exchange indicates a complex rearrangement of the molecular ion of 1 ^·+ prior to metastable decomposition for which as key step a 1,2-trimethylsilyl group migration from carbon to oxygen is suggested. A different behavior is also found between the source-generated m/z 89 ions from 1 ^·+ which decompose in the metastable time region to give ions m/z 61 by loss of ethylene and those from 2 ^·+ which decompose in the metastable region to yield ions m/z 59 by elimination of formaldehyde.     

Investigation of UV matrix-assisted laser desorption fourier transform mass spectrometry for peptides

Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA Ultraviolet matrix-assisted laser desorption can be used to enhance formation of [M + H]⁺, [M + Na]⁺, and [M + K)⁺ ions from small peptides for Fourier transform mass spectrometry (FTMS). In accord with laser desorption (LD) time-of-flight experiments, matrices such as nicotinic acid and 2-pyrazinecarboxylic acid exhibit strong enhancement effects (i.e., formation of abundant protonated and cationized molecules for the analyte with virtually no fragment ions) for 266 nm LD/FTMS, whereas pyrazinedicarboxylic acid provides no matrix enhancement at this wavelength. Both sinapinic acid and coumarin-120 provide strong matrix enhancement effects for the 355-nm LD of peptides. For the small peptides examined in this study, no significant differences in the abundance of fragment ions were observed between the 266- and 355-nm wavelengths. Matrix-assisted LD/FTMS is useful for the generation and characterization of ions corresponding to protonated and cationized molecules from virtually all biological compounds with molecular weights up to 2000. The lack of observation of biological ions with m/ z > 2500 may be related to inefficient trapping of these laser-desorbed ions or instrumental detection limitations of FTMS and is under further investigation.     

Role of the UV absorber as a matrix in matrix‐assisted laser desorption/ionization mass spectrometric analysis of a mixture of a UV absorber and a stabilizer

A mixture of a UV absorber (Tinuvin 234 or Tinuvin 329) and a UV stabilizer (Tinuvin 770) was analyzed using matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) without any matrix. Fragmentation patterns of the UV absorbers and stabilizer were also investigated. The mass spectra showed the [M+H]⁺ ions and some fragment ions. Tinuvin 234, Tinuvin 329, and Tinuvin 770 generated three (m/z 119, 370, 432), one (m/z 252), and two (m/z 124 and 140) fragment ions, repectively. These fragment ions can be used to identify the chemical structures of the UV absorbers and stabilizer. Since the UV absorber performed a role as the matrix, the ion abundance of the UV stabilizer was enhanced by mixing with the UV absorber. When organic materials extracted from polypropylene (PP) containing the UV absorber and stabilizer were directly analyzed using MALDI‐MS without any matrix, the protonated molecule of the UV stabilizer was detected in abundance but the product ions of the UV absorber were not observed. When 2,5‐dihydroxybenzoic acid was used as a matrix, the protonated molecule of the UV absorber was observed. Copyright © 2010 John Wiley & Sons, Ltd.     

Use of liquid chromatography/tandem mass spectrometry and online databases for identification of phosphocholines and lysophosphatidylcholines in human red blood cells

In this work a systematic strategy integrating liquid chromatography/tandem mass spectrometry (LC/MS/MS) and online databases was developed to identify phosphocholines (PC) and lysophosphatidylcholines (LPC) in human red blood cells (RBCs). First of all, the neutral loss scan of 59 and the precursor ion scan of m/z 184 were performed to find out the possible lipids with phosphocholine head‐group structure in RBCs. The acquired [M+H]⁺ and [M+Na]⁺ adduct ions were then identified online using the Human Metabolome Database (HMDB) and the LIPID MAPS, which were then further confirmed by their MS/MS fragmentation. Based on the comparison of chemical structures of the detected PC and LPC with their corresponding MS/MS fragmentation pathways, several new diagnostic fragments or fragmentation pathway were found. These include, (1) the neutral losses of 183 could be used as a diagnostic fragmentation to discriminate PC and LPC; (2) product ions at m/z 104 could be used to distinguish LPC and their sn‐2 isomers; (3) fragment ions at m/z 184 are characteristic fragmentation that could be used for discrimination of sodiated ions [M+Na]⁺ and protonated ions [M+H]⁺, especially for co‐eluted PC or LPC with a molecular weight difference of 22. The structures of the above‐mentioned fragment ions were confirmed by quadrupole time‐of‐flight (Q‐TOF) MS. Furthermore, a PC and LPC focused LC/MS semi‐quantification approach was also developed and validated. This approach could be useful for future lipidomic study. Copyright © 2009 John Wiley & Sons, Ltd.     

Fragmentation of protonated O,O-dimethyl O-aryl phosphorothionates in tandem mass spectral analysis

A study was carried out on the fragmentation of 12 protonated O,O-dimethyl O-aryl phosphorothionates by tandem quadrupole mass spectrometry. Some of the studied compounds are used in agriculture as pesticides. Energy-resolved and pressure-resolved experiments were performed on the [M + H]⁺ ions to investigate the dissociation behavior of the ions with various amounts of internal energy. On collisionally activated dissociation, the [M + H]⁺ ions decompose to yield the [M + H ? CH₃OH]⁺, (CH₃O)₂PS⁺ (m/z 125), and (CH₃O)₂PO⁺ (m/z 109) ions as major fragments. The ions [M + H ? CH₃OH]⁺ and (CH₃O)₂PS⁺ probably arise from the [M + H]⁺ ions of the O,O-dimethyl O-aryl phosphorothionates with the proton on the sulfur or on the oxygen of the phenoxy group. The origin of the hydroxy proton of the methanol fragment was in many cases, surprisingly, the phenyl group and not the reagent gas. This was confirmed by using deuterated isobutane, C₄D₁₀, as reagent gas in Cl. The fragment ions (CH₃O)₂PO⁺ and [ZPhS]⁺ are the results of thiono-thiolo rearrangement reaction. The precursor ion for the ion (CH₃O)₂PO⁺ arises from most compounds upon chemical ionization, whereas the precursor ion for the ion [ZPhS]⁺ arises only from a few compounds upon chemical ionization. The observed fragments imply that several sites carry the extra proton and that these sites get the proton usually upon ionization. The stability order and some characteristics of three protomers of O,O-dimethyl O-phenyl phosphorothionate were investigated by ab initio calculations at the RHF/3-21G* level of theory.     

Protonated Dipeptide Losses from b 5 and b 4 Ions of Side Chain Hydroxyl Group Containing Pentapeptides

In this study, C-terminal protonated dipeptide eliminations were reported for both b ₅ and b ₄ ions of side chain hydroxyl group (–OH) containing pentapeptides. The study utilized the model C-terminal amidated pentapeptides having sequences of XGGFL and AXVYI, where X represents serine (S), threonine (T), glutamic acid (E), aspartic acid (D), or tyrosine (Y) residue. Upon low-energy collision-induced dissociation (CID) of XGGFL (where X?=?S, T, E, D, and Y) model peptide series, the ions at m/z 279 and 223 were observed as common fragments in all b ₅ and b ₄ ion (except b ₄ ion of YGGFL) mass spectra, respectively. By contrast, peptides, namely S_MeGGFL-NH₂ and E_OMeGGFL-NH₂, did not show either the ion at m/z 279 or the ion at m/z 223. It is shown that the side chain hydroxyl group is required for the possible mechanism to take place that furnishes the protonated dipeptide loss from b ₅ and b ₄ ions. In addition, the ions at m/z 295 and 281 were detected as common fragments in all b ₅ and b ₄ ion (except b ₄ ion of AYVYI) mass spectra, respectively, for AXVYI model peptide series. The MS⁴ experiments exhibited that the fragment ions at m/z 279, 223, 295, and 281 entirely reflect the same fragmentation behavior of [M?+?H]⁺ ion generated from commercial dipeptides FL-OH, GF-OH, YI-OH, and VY-OH. These novel eliminations reported here for b ₅ and b ₄ ions can be useful in assigning the correct and reliable peptide sequences for high-throughput proteomic studies.

Separation and identification of structural isomers by quadrupole collision-induced dissociation-hydrogen/deuterium exchange-infrared multiphoton dissociation (QCID-HDX-IRMPD)

A new approach that uses a hybrid Q-FTICR instrument and combines quadrupole collision-induced dissociation, hydrogen-deuterium exchange, and infrared multiphoton dissociation (QCID-HDX-IRMPD) has been shown to effectively separate and differentiate isomeric fragment ion structures present at the same m/z. This method was used to study protonated YAGFL-OH (free acid), YAGFL-NH₂ (amide), cyclic YAGFL, and YAGFL-OCH₃ (methyl ester). QCID-HDX of m/z 552.28 (C₂₉H₃₈N₅O₆) from YAGFL-OH reveals at least two distributions of ions corresponding to the b₅ ion and a non-C-terminal water loss ion structure. Subsequent IRMPD fragmentation of each population shows distinct fragmentation patterns, reflecting the different structures from which they arise. This contrasts with data for YAGFL-NH₂ and YAGFL-OCH₃, which do not show two distinct H/D exchange populations for the C₂₉H₃₈N₅O₆ structure formed by NH₃ and HOCH₃ loss, respectively. Relative extents of exchange for C₂₉H₃₈N₅O₆ ions from six sequence isomers (YAGFL, AGFLY, GFLYA, FLYAG, LYAGF, and LFGAY) show a sequence dependence of relative isomer abundance. Supporting action IRMPD spectroscopy data are also presented herein and also show that multiple structures are present for the C₂₉H₃₈N₅O₆ species from YAGFL-OH.     

Analysis of long-chain fatty acyl coenzyme a thioesters by negative ion fast-atom bombardment mass spectrometry and tandem mass spectrometry

Long-chain acyl Coenzyme A (CoA) is essentially composed of three major chemical groups, fatty acyl-, phosphopantetheino-, and 3′, 5′,-adenosine diphospho-moieties. The negative ion fast-atom bombardment mass spectrometry spectra of long-chain acyl CoA thioesters were characterized by the formation of abundant [M ? H]^? and two distinct classes of fragment ions, one class which retained the acyl group and another class which is related to CoA that contains the phosphopantethene and adenine. The ions which retained the acyl group in the spectrum of palmitoyl CoA appeared at m/z 675, 657, 595, and 577 and were found to decompose by loss of alkylketene observed at m/z 357 and 339. Those ions which retained the adenine group were observed at m/z 426 and 408. In contrast to these ions observed following fast-atom bombardment ionization, tandem mass spectrometry of the [M ? H]^?, from palmitoyl CoA (m/z 1004), yielded the adenine-containing ions as major products and the acyl-containing ions were of low abundance or not detected. These results suggested that the formation of many characteristic ions observed in direct FAB analysis occurred during the desorption process. The unique relationship between ions which involved the transition from acyl-containing ions to only CoA-containing ions by the loss of alkylketene allowed the development of tandem mass spectrometry protocols for the analysis of acyl CoA mixtures. Precursor scans of either m/z 357 or 339 yielded the identification of each species in a complex mixture. Identification of specific species was obtained with a neutral loss scan of the mass for a specific alkylketene.     

Density Functional Theory and Mass Spectrometry of Phthalate Fragmentations Mechanisms: Modeling Hyperconjugated Carbocation and Radical Cation Complexes with Neutral Molecules

This is the first ab initio study of the energetics of the fragmentation mechanisms of phthalate, by mass spectrometry, leading to protonated phthalic anhydride (m/z 149). Phthalates fragment by two major pathways; namely, the McLafferty + 1 rearrangement and the loss of alkoxy. Both pathways involve a carbonyl oxygen attack to the ortho-carbonyl carbon leading to structures with tetrahedral carbon intermediates that eventually give m/z 149. These pathways were studied by collision induced dissociation (CID) using triple quadrupole mass spectrometry. The proposed McLafferty + 1 pathway proceeds through a distonic M^•+, leading to the loss of an allylic-stabilized alkene radical. The McLafferty rearrangement step proceeds through a six-membered ring transition state with a small activation energy ranging 0.4–6.2 kcal/mol; the transfer of a second H from the distonic ion of the rearrangement step proceeds through a radical cation molecule complex. Based on quantum chemical modeling of the cation molecule complexes, two kinds of cation molecule complexes were identified as radical cation molecule complex and hyperconjugated cation molecule complex. This distinction is based on the cation and simplifies future modeling of similar complexes. Optimization of important fragments in these pathways showed cyclized and hydrogen-bonded structures to be favored. An exception was the optimized structure of the protonated phthalic anhydride (m/z 149) that showed a structure with an open anhydride ring.     

How Does Acetylcholine Lose Trimethylamine? A Density Functional Theory Study of Four Competing Mechanisms

Low-energy collision-induced dissociation (CID) of acetylcholine (ACh) yields only two fragment ions: the dominant C(4)H(7)O(2)(+) ion at m/z 87, arising from trimethylamine loss; and protonated trimethylamine at m/z 60. Since the literature is replete with conflicting mechanisms for the loss of trimethylamine from ACh, in this article density functional theory (DFT) calculations are used to assess four competing mechanisms: (1) Path A involves a neighboring group attack to form a five-membered ring product, 2-methyl-1,3-dioxolan-2-ylium cation; (2) Path B is a neighboring group attack to form a three-membered ring product, 1-methyl-oxiranium ion; (3) Path C involves an intramolecular elimination reaction to form CO protonated vinylacetate; and (4) Path D is a 1,2-hydride migration reaction forming CH(2)-protonated vinylacetate. At the MP2/6-311++G(2d,p)//B3-LYP/6-31+G(d,p) level of theory path A is the kinetically favored pathway, with a transition-state energy barrier of 37.7 kcal mol(-1) relative to the most stable conformer of ACh. The lowest energy pathway for the formation of protonated trimethylamine was also calculated to proceed via path A, involving proton transfer within the ion-molecule complex intermediate, with the exocylic methyl group being the proton donor. To confirm the site of proton transfer, low-energy CID of acetyl-d(3)-choline (d(3)-ACh) was carried out, which revealed loss of trimethylamine and the formation of Me(3)ND(+).     

Quantification of Competing H3PO4 Versus HPO3 + H2O Neutral Losses from Regioselective 18O-Labeled Phosphopeptides

Abundant neutral losses of 98 Da are often observed upon ion trap CID-MS/MS of protonated phosphopeptide ions. Two competing fragmentation pathways are involved in this process, namely, the direct loss of H₃PO₄ from the phosphorylated residue and the combined losses of HPO₃ and H₂O from the phosphorylation site and from an additional site within the peptide, respectively. These competing pathways produce product ions with different structures but the same m/z values, potentially limiting the utility of CID-MS³ for phosphorylation site localization. To quantify the relative contributions of these pathways and to determine the conditions under which each pathway predominates, we have examined the ion trap CID-MS/MS fragmentation of a series of regioselective ¹⁸O-phosphate ester labeled phosphopeptides prepared using novel solution-phase amino acid synthesis and solid-phase peptide synthesis methodologies. By comparing the intensity of the –100 Da (–H₃PO₃ ¹⁸O) versus –98 Da (–[HPO₃ + H₂O]) neutral loss product ions formed upon MS/MS, quantification of the two pathways was achieved. Factors that affect the extent of formation of the competing neutral losses were investigated, with the combined loss pathway predominantly occurring under conditions of limited proton mobility, and with increased combined losses observed for phosphothreonine compared with phosphoserine-containing peptides. The combined loss pathway was found to be less dominant under ion activation conditions associated with HCD-MS/MS. Finally, the contribution of carboxylic acid functional groups and backbone amide bonds to the water loss in the combined loss fragmentation pathway was determined via methyl esterification and by examination of a phosphopeptide lacking side-chain hydroxyl groups.

Elucidation de structure et comportement de produits naturels sous ionisation chimique—I: Mise en evidence de la cinerubine a par etude des decompositions unimoleculaires des ions produits en phase gazeuse par iBu+

Positive ion chemical ionization mass spectrometry by ⁱBu⁺ is used to determine the structure of anthracycline 4915 A. The metastable decompositions of protonated molecular ion (m/z 828) and fragment ions (aglycone and sugar moieties) show that the antibiotic in cinerubine A. Unimolecular decompositions occuring in the first field-free region are detected by “Linked Scan méthods” B/E and B²/E.