Analysis of protonated and alkali metal cationized aminoglycoside antibiotics by collision-activated dissociation and infrared multi-photon dissociation in the quadrupole ion trap

Nine aminoglycoside antibiotics were analyzed in two quadrupole ion trap mass spectrometers using electrospray ionization. Structural information was obtained via collision-activated dissociation (CAD) and infrared multi-photon dissociation (IRMPD) of the protonated species. Several of the compounds, having multiple basic sites, preferred the doubly protonated form while some existed in the singly charged state or were distributed between single and doubly protonated species, allowing comparison of the fragmentation patterns of the two charge states. In general, IRMPD is as efficient as CAD, produces more low-mass fragment ions, and is more universally applied owing to its low dependence on trapping, pressure and tuning conditions. Alkali metal complexation using Li(+) and Na(+) was probed as a means of producing different fragmentation patterns, but in most cases the resulting fragmentation patterns were simplified versions of those obtained for the protonated analogs.     

Dehydration versus deamination of N-terminal glutamine in collision-induced dissociation of protonated peptides

Some of the most prominent "neutral losses" in peptide ion fragmentation are the loss of ammonia and water from N-terminal glutamine. These processes are studied by electrospray ionization mass spectrometry in singly- and doubly-protonated peptide ions undergoing collision-induced dissociation in a triple quadrupole and in an ion trap instrument. For this study, four sets of peptides were synthesized: (1) QLLLPLLLK and similar peptides with K replaced by R, H, or L, and Q replaced by a number of amino acids, (2) QLnK (n = 0, 1, 3, 5, 7, 9, 11), (3) QLnR (n = 0, 1, 3, 5, 7, 9), and (4) QLn (n = 1, 2, 3, 4, 8). The results for QLLLPLLLK and QLLLPLLLR show that the singly protonated ions undergo loss of ammonia and to a smaller extent loss of water, whereas the doubly protonated ions undergo predominant loss of water. The fast fragmentation next to P (forming the y5 ion) occurs to a larger extent than the neutral losses from the singly protonated ions but much less than the water loss from the doubly protonated ions. The results from these and other peptides show that, in general, when N-terminal glutamine peptides have no "mobile protons", that is, the number of charges on the peptide is no greater than the number of basic amino acids (K, R, H), deamination is the predominant neutral loss fragmentation, but when mobile protons are present the predominant process is the loss of water. Both of these processes are faster than backbone fragmentation at the proline. These results are rationalized on the basis of resonance stabilization of the two types of five-membered ring products that would be formed in the neutral loss processes; the singly protonated ion yields the more stable neutral pyrrolidinone ring whereas the doubly protonated ion yields the protonated aminopyrroline ring (see Schemes). The generality of these trends is confirmed by analyzing an MS/MS spectra library of peptides derived from tryptic digests of yeast. In the absence of mobile protons, glutamine deamination is the most rapid neutral loss process. For peptides with mobile protons, dehydration from glutamine is far more rapid than from any other amino acid. Most strikingly, end terminal glutamine is by far the most labile source of neutral loss in excess-proton peptides, but not highly exceptional when mobile protons are not available. In addition, rates of deamination are faster in lysine versus arginine C-terminus peptides and 20 times faster in positively charged than negatively charged peptides, demonstrating that these formal neutral loss reactions are not "neutral reactions" but depend on charge state and stability.     

A novel tandem quadrupole mass spectrometer allowing gaseous collisional activation and surface induced dissociation.

A novel tandem quadrupole mass spectrometer is described that enables gaseous collision-induced dissociation (CID) and surface-induced dissociation (SID) experiments. The instrument consists of a commercially available triple quadrupole mass spectrometer connected to an SID region and an additional, orthogonal quadrupole mass analyser. The performance of the instrument was evaluated using leucine-enkephalin, allowing a comparison between CID and SID, and with previous reports of other SID instruments. The reproducibility of SID data was assessed by replicate determinations of the collision energy required for 50% dissociation of leucine-enkephalin; excellent precision was observed (standard deviation of 0.6 eV) though, unexpectedly, the reproducibility of the equivalent figure for CID was superior. Several peptides were analysed using SID in conjunction with liquid secondary-ion mass spectrometry or electrospray; a comparison of the fragmentation of singly protonated peptide ions and the further dissociation of y-type fragments was consistent with the equivalence of the latter fragments to protonated peptides. Few product ions attributable to high-energy cleavages of amino acid side-chains were observed. The SID properties were investigated of a series of peptides differing only in the derivatization of a cysteine residue; similar decomposition efficiencies were observed for all except the cysteic acid analogue, which demonstrated significantly more facile fragmentation.     

Substituent effects on the gas-phase fragmentation reactions of sulfonium ion containing peptides

The multistage mass spectrometric (MS/MS and MS3) gas-phase fragmentation reactions of methionine side-chain sulfonium ion containing peptides formed by reaction with a series of para-substituted phenacyl bromide (XBr where X=CH2COC6H4R, and R=--COOH, --COOCH3, --H, --CH3 and --CH2CH3) alkylating reagents have been examined in a linear quadrupole ion trap mass spectrometer. MS/MS of the singly (M+) and multiply ([M++nH](n+1)+) charged precursor ions results in exclusive dissociation at the fixed charge containing side chain, independently of the amino acid composition and precursor ion charge state (i.e., proton mobility). However, loss of the methylphenacyl sulfide side-chain fragment as a neutral versus charged (protonated) species was observed to be highly dependent on the proton mobility of the precursor ion, and the identity of the phenacyl group para-substituent. Molecular orbital calculations were performed at the B3LYP/6-31+G** level of theory to calculate the theoretical proton affinities of the neutral side-chain fragments. The log of the ratio of neutral versus protonated side-chain fragment losses from the derivatized side chain were found to exhibit a linear dependence on the proton affinity of the side-chain fragmentation product, as well as the proton affinities of the peptide product ions. Finally, MS3 dissociation of the nominally identical neutral and protonated loss product ions formed by MS/MS of the [M++H]2+ and [M++2H]3+ precursor ions, respectively, from the peptide GAILM(X)GAILK revealed significant differences in the abundances of the resultant product ions. These results suggest that the protonated peptide product ions formed by gas-phase fragmentation of sulfonium ion containing precursors in an ion trap mass spectrometer do not necessarily undergo intramolecular proton 'scrambling' prior to their further dissociation, in contrast to that previously demonstrated for peptide ions introduced by external ionization sources.     

Specific UV photodissociation of tyrosyl-containing peptides in multistage mass spectrometry

UV photodissociation (UVPD) at 262 nm has been carried out on protonated tyrosyl-containing peptides formed by trypsin digestion of apo-transferrin. Under UVPD, the main event is the fragmentation of the C(alpha)-C(beta) bond of the tyrosyl residues leading to a radical ion 107 Da below the precursor ion. The dissociation rate of this specific cleavage appears to be strongly dependent on the peptide sequence and is more prominent on the singly protonated species than on the doubly protonated state. The fragmentation spectra resulting from collisional activation of the protonated even-electron native peptides and of the odd-electron radical species prepared by UVPD are dominated by y-type backbone cleavages. A comparison of their respective y-ion pattern shows complementarities since the combination of both increases the sequence coverage of the peptide sequence. The specific detection of the neutral loss of 107 Da from peptides witnesses the content of at least one tyrosyl residue and, though preliminary, is proposed as a potential new filtering strategy during protein database searching.     

Electrospray Ionization Tandem Mass Spectrometry of Ammonium Cationized Polyethers

Quaternary ammonium salts (Quats) and amines are known to facilitate the MS analysis of high molar mass polyethers by forming low charge state adduct ions. The formation, stability, and behavior upon collision-induced dissociation (CID) of adduct ions of polyethers with a variety of Quats and amines were studied by electrospray ionization quadrupole time-of-flight, quadrupole ion trap, and linear ion trap tandem mass spectrometry (MS/MS). The linear ion trap instrument was part of an Orbitrap hybrid mass spectrometer that allowed accurate mass MS/MS measurements. The Quats and amines studied were of different degree of substitution, structure, and size. The stability of the adduct ions was related to the structure of the cation, especially the amine’s degree of substitution. CID of singly/doubly charged primary and tertiary ammonium cationized polymers resulted in the neutral loss of the amine followed by fragmentation of the protonated product ions. The latter reveals information about the monomer unit, polymer sequence, and endgroup structure. In addition, the detection of product ions retaining the ammonium ion was observed. The predominant process in the CID of singly charged quaternary ammonium cationized polymers was cation detachment, whereas their doubly charged adduct ions provided the same information as the primary and tertiary ammonium cationized adduct ions. This study shows the potential of specific amines as tools for the structural elucidation of high molar mass polyethers.     

Investigation of fragmentation behaviors of steroidal drugs with Li+, Na+, K+ adducts by tandem mass spectrometry aided with computational analysis

In this study, we have investigated the fragmentation of the widely used steroidal pharmaceutical drugs (n = 14), complexed by a singly charged proton or alkali metal ion (Li⁺, Na⁺, K⁺) using Ion trap and quadrupole time-of-flight mass spectrometers. Spectra were collected by LC-MS/MS analysis using system automated collision energy i.e., of 25–60 eV. Theoretical calculations were also calculated using DFT software. The metal complexes showed different fragmentation pathways not commonly observed for protonated compounds. There was a distinct difference observed in the relative intensities of some common fragments for free vs. metallated drugs. Some major fragments from protonated and lithium adducts showed close resemblance, while sodium and potassium adducts showed different fragments. Theoretical calculations showed a distinct difference in the position of attachment of proton and metals. This adducts ion fragmentation information will be helpful for the identification of these compounds in complex samples.     

RNA fragmentation in MALDI mass spectrometry studied by H/D-exchange: Mechanisms of general applicability to nucleic acids

To reveal the gas-phase chemistry of RNA and DNA fragmentation during MALDI mass spectrometry in positive ion mode, we performed hydrogen/deuterium exchange on a series of RNA and DNA tetranucleotides and studied their fragmentation patterns on a high-resolution MALDI TOF-TOF instrument. We were specifically interested in elucidating the remarkably different fragmentation behavior of RNA and DNA, i.e., the characteristic and abundant production of c- and y-ions from RNA versus a dominating generation of (a-B)- and w-ions from DNA analytes. The analysis yielded important information on all significant backbone cleavages as well as nucleobase losses. Based on this, we suggest common fragmentation mechanisms for RNA and DNA as well as an important RNA-specific reaction requiring a 2'-hydroxyl group, leading to c- and y-ions. The data is viewed and discussed in the context of previously published data to obtain a coherent picture of the fragmentation of singly protonated nucleic acids.     

Metastable atom‐activated dissociation mass spectrometry: leucine/isoleucine differentiation and ring cleavage of proline residues

Extensive backbone fragmentation resulting in a‐, b‐, c‐, x‐, y‐ and z‐type ions is observed of singly and doubly charged peptide ions through their interaction with a high kinetic energy beam of argon or helium metastable atoms in a modified quadrupole ion trap mass spectrometer. The ability to determine phosphorylation‐sites confirms the observation with previous reports and we report the new ability to distinguish between leucine and isoleucine residues and the ability to cleave two covalent bonds of the proline ring resulting in a‐, b‐, x‐, y‐, z‐ and w‐type ions. The fragmentation spectra indicate that fragmentation occurs through nonergodic radical ion chemistry akin to electron capture dissociation (ECD), electron transfer dissociation (ETD) and electron ionization dissociation mechanisms. However, metastable atom‐activated dissociation mass spectrometry demonstrates three apparent benefits to ECD and ETD: (1) the ability to fragment singly charged precursor ions, (2) the ability to fragment negatively charged ions and (3) the ability to cleave the proline ring that requires the cleavage of two covalent bonds. Helium metastable atoms generated more fragment ions than argon metastable atoms for both substance P and bradykinin regardless of the precursor ion charge state. Reaction times less than 250 ms and efficiencies approaching 5% are compatible with on‐line fragmentation, as would be desirable for bottom‐up proteomics applications. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

Structural analysis of permethylated oligosaccharides using electrospray ionization quadrupole time-of-flight tandem mass spectrometry and deutero-reduction

Deutero-reduced permethylated oligosaccharides were analyzed by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) using a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer, fitted with a nanoflow ESI source. Under these ionization conditions such derivatives preferentially form sodiated molecular species in addition to protonated molecular species. Under collision-induced dissociation, protonated and sodiated molecular species yield simple and predictable fragment mass spectra. A systematic study was conducted on a series of deutero-reduced permethylated glycans to allow rationalization of the fragmentation processes. MS/MS spectra were characterized by fragments resulting from the cleavage of glycosidic bonds. These fragments originating from both the reducing and the non-reducing ends of the glycan yield information on sequence and branching. Furthermore, the substituent 3-linked to a HexNAc unit was readily eliminated. Special attention was devoted to a systematic study of fucosylated glycans. The fucosylated deutero-reduced permethylated glycans were submitted to an acidic hydrolysis, releasing specifically the fucosyl residues. The nascent free hydroxyl groups were subsequently CD3-labelled in order to determine the positions initially bearing the fucosyl residues along the oligosaccharide backbone. This methodology was finally applied to characterize a glycan pool enzymatically released from glycoproteins. The present data show that structural elucidation can be achieved at the 50 fmol level.     

Structural determination of the novel fragmentation routes of zwitteronic morphine opiate antagonists naloxonazine and naloxone hydrochlorides using electrospray ionization tandem mass spectrometry

Electrospray ionization quadrupole time-of-flight (ESI-QqToF) mass spectra of the zwitteronic salts naloxonazine dihydrochloride 1 and naloxone hydrochloride 2, a common series of morphine opiate receptor antagonists, were recorded using different declustering potentials. The singly charged ion [M+H-2HCl](+) at m/z 651.3170 and the doubly charged ion [M+2H-2HCl](2+) at m/z 326.1700 were noted for naloxonazine dihydrochloride 1; and the singly charged ion [M+H-HCl](+) at m/z 328.1541 was observed for naloxone hydrochloride 2. Low-energy collision-induced dissociation tandem mass spectrometry (CID-MS/MS) experiments established the fragmentation routes of these compounds. In addition to the characteristic diagnostic product ions obtained, we noticed the formation of a series of radical product ions for the zwitteronic compounds 1 and 2, and also the formation of a distonic ion product formed from the singly charged ion [M+H-HCl](+) of naloxone hydrochloride 2. Confirmation of the various established fragmentation routes was effected by conducting a series of ESI-CID-QqTof-MS/MS product ion scans, which were initiated by CID in the atmospheric pressure/vacuum interface using a higher declustering potential. Deuterium labeling was also performed on the zwitteronic salts 1 and 2, in which the hydrogen atoms of the OH and NH groups were exchanged with deuterium atoms. Low-energy CID-QqTof-MS/MS product ion scans of the singly charged and doubly charged deuteriated molecules confirmed the initial fragmentation patterns proposed for the protonated molecules. Precursor ion scan analyses were also performed with a conventional quadrupole-hexapole-quadrupole tandem mass spectrometer and allowed the confirmation of the genesis of some diagnostic ions.     

Multiple losses of neutral C14H14 in the tandem mass spectrometry of several perbenzyl ether intermediates in the synthesis of green tea constituents

Electrospray and matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS) experiments were used to investigate an unusual fragmentation in collision-induced dissociation (CID) of sodiated and potassiated perbenzyl ether intermediates obtained in the total synthesis of gallate ester constituents of green tea. Prominent fragments correspond to multiple sequential losses of neutral C14H14 that were not observed in the protonated and ammoniated species, that instead present fragment ion series in which members are separated by C7H6. High-resolution MALDI quadrupole time-of-flight (Q-TOF) and electrospray-Fourier transform mass spectrometry (FTMS) were used to confirm elemental compositions of these and related ions.     

Fragmentation studies on monensin A by sequential electrospray mass spectrometry

Monensin A was studied by electrospray ionisation sequential mass spectrometry (ESI-MSn) and all fragments were confirmed by accurate-mass measurements. Analyses were performed on both a quadrupole time-of-flight (Q-tof) and a Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer. MSn analysis shows that depending on sample preparation the ion at m/z 671 consists of two different ions with the same accurate-mass. It is either the monensin protonated parent ion or a different ion structure derived from the loss of water from the water adduct of monensin. Both ions show different fragmentation patterns. Major fragment ions from the protonated parent ion were produced by Grob-Wharton type fragmentations in addition to various simple neutral losses. The fragmentation pathways of the two different m/z 671 ions are proposed.     

How does a CC double bond cleave in the gas phase? Fragmentation of protonated ketotifen in mass spectrometry

In the literature, it is reported that the protonated ketotifen mainly undergoes C?C double bond cleavage in electrospray ionization tandem mass spectrometry (ESI‐MS/MS); however, there is no explanation on the mechanism of this fragmentation reaction. Therefore, we carried out a combined experimental and theoretical study on this interesting fragmentation reaction. The fragmentation of protonated ketotifen (m/z 310) always generated a dominant fragment ion at m/z 96 in different electrospray ionization mass spectrometers (ion trap, triple quadrupole and linear trap quadrupole (LTQ)‐orbitrap). The mechanism of the generation of this product ion (m/z 96) through the C?C double bond cleavage was proposed to be a sequential hydrogen migration process (including proton transfer, continuous two‐step 1,2‐hydride transfer and ion‐neutral complex‐mediated hydride transfer). This mechanism was supported by density functional theory (DFT) calculations and a deuterium labeling experiment. DFT calculations also showed that the formation of the product ion m/z 96 was most favorable in terms of energy. This study provides a reasonable explanation for the fragmentation of protonated ketotifen in ESI‐MS/MS, and the fragmentation mechanism is suitable to explain other C?C double bond cleavage reactions in mass spectrometry. Copyright © 2016 John Wiley & Sons, Ltd.     

Use of doubly protonated molecules in the analysis of cathedulins in crude extracts of khat (Catha edulis) by liquid chromatography/serial mass spectrometry

Analysis of crude methanolic extracts of fresh khat (Catha edulis) by liquid chromatography/mass spectrometry (LC/MS) revealed the presence of 62 cathedulin alkaloids (compared with 15 published structures). Many cathedulins generated doubly protonated molecules following electrospray ionisation and the ratio of doubly to singly protonated species could be manipulated by adjusting the electrospray capillary position and source conditions. By selecting the doubly protonated species for serial mass spectrometric analysis (MS/MS), it was possible to use an ion trap mass spectrometer to observe singly charged product ions at lower m/z values than ion trap MS/MS analysis of [M+H](+) would have allowed. These spectra were particularly valuable in elucidating the acylation patterns of cathedulins where MS/MS analysis of [M+H](+) resulted in loss of a large neutral species to yield a small singly charged fragment below the lower limit for ion trapping. Acylation patterns for most of the 62 cathedulins are proposed from mass spectrometric analysis, and the data obtained for a major unreported cathedulin of mass 1001 Da suggest that it belongs to a new group of cathedulins having a cathate dilactone bridge but not an evoninate bridge.     

Neutral loss of amino acid residues from protonated peptides in collision-induced dissociation generates N- or C-terminal sequence ladders

The widespread occurrence of the neutral loss of one to six amino acid residues as neutral fragments from doubly protonated tryptic peptides is documented for 23 peptides with individual sequences. Neutral loss of amino acids from the N-terminus of doubly charged tryptic peptides results in doubly charged y-ions, forming a ladder-like series with the ions [M + 2H](2+) = y(max) (2+), y(max - 1) (2+), y(max - 2) (2+), etc. An internal residue such as histidine, proline, lysine or arginine appears to favor this type of fragmentation, although it was sometimes also observed for peptides without this structure. For doubly protonated non-tryptic peptides with one of these residues at or near the N-terminus, we observed neutral loss from the C-terminus, resulting in a doubly charged b-type ion ladder. The analyses were performed by Q-TOF tandem mass spectrometry, facilitating the recognition of neutral loss ladders by their 2+ charge state and the conversion of the observed mass differences into reliable sequence information. It is shown that the neutral loss of amino acid residues requires low collision offset values, a simple mechanistic explanation based on established fragmentation rules is proposed and the utility of this neutral loss fragmentation pathway as an additional source for dependable peptide sequence information is documented.     

Facile sequencing of oligosaccharides by matrix-assisted laser desorption/ionisation on a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer.

N-Linked oligosaccharide mixtures released from a number of standard glycoproteins were derivatised with 3-acetylamino-6-acetylaminoacridine (AA-Ac) using reductive amination. Analysis of these mixtures using an experimental matrix-assisted laser desorption/ionisation (MALDI) hybrid quadrupole orthogonal acceleration time-of-flight (Q-TOF) mass spectrometer provided detailed information about the mass distribution of the glycan derivatives. Collision-induced dissociation of the singly protonated [M + H](+) ions also gave rise to a number of product ions produced by the sequential cleavage of the glycosidic linkages. As fragmentation of the positively charged species occurred predominantly in one direction, i.e., from the non-reducing end of the glycan to the AA-Ac moiety, a considerable amount of information could be obtained with ease about the sequence in which the sugar residues were attached to one another. This derivatisation procedure and mass spectrometric methodology were applied successfully to neutral and acidic glycans released from proteins separated by gel electrophoresis.     

Mechanisms of glycerol dehydration

Dehydration of neutral and protonated glycerol was investigated using quantum mechanical calculations (CBS-QB3). Calculations on neutral glycerol show that there is a high barrier for simple 1,2-dehydration, E(a)=70.9 kcal mol(-1), which is lowered to 65.2 kcal mol(-1) for pericyclic 1,3-dehydration. In contrast, the barriers for dehydration of protonated glycerol are much lower. Dehydration mechanisms involving hydride transfer, pinacol rearrangement, or substitution reactions have barriers between 20 and 25 kcal mol(-1). Loss of water from glycerol via substitution results in either oxirane or oxetane intermediates, which can inter-convert over a low barrier. Subsequent decomposition of these intermediates proceeds via either a second dehydration step or loss of formaldehyde. The computed mechanisms for decomposition of protonated glycerol are supported by the gas-phase fragmentation of protonated glycerol observed using a triple--quadrupole mass spectrometer.     

Fast atom bombardment tandem mass spectrometry employing ion-molecule reactions for the differentiation of phospholipid classes

Fast atom bombardment tandem mass spectrometry, employing ion-molecule reactions with ethyl vinyl ether in a triple-quadrupole mass spectrometer, is used to differentiate classes of phospholipids. The phospholipids are desorbed and ionized by fast atom bombardment, mass-selected by the first quadrupole, and reacted with ethyl vinyl ether in the second quadrupole; the resulting product ions are analyzed by the third quadrupole. The protonated molecules and reaction product ions observed permit the differentiation of various phospholipid classes. The pattern of addition reaction products formed is shown to depend solely on the functionality of the lipid polar head group and not on the fatty acyl constituents. Neutral gain scans that are specific for each phospholipid class are performed. Ion dissociation products are observed in the same scan as the ion reaction products to provide data on the fatty acyl composition and position on the glycerophosphate core along with the phospholipid class. Although this method is less sensitive than neutral loss scanning for most phospholipid classes, it can (1) identify phospholipids that do not readily lose their head group as a neutral fragment and (2) detect phospholipids in mixtures containing species that give interfering neutral losses.