In-source decay of hyperbranched polyesteramides in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Hyperbranched polyesteramides (DA2), prepared from hexahydrophthalic anhydride (D) and diisopropanolamine (A) have been characterized, by use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), field desorption (FD)-MS, and electrospray ionization (ESI)-MS. MALDI of polyesteramides produces protonated molecules. The spectra show a complex chemical composition distribution and end-group distribution which are mainly composed of two series of homologous oligomers DnA(n)+1 - mH2O and DnA(n) - mH2O, where m = 1-2. Signals from protonated molecules DnAn+1 and DnAn are almost absent in the MALDI spectrum, whereas these ions are responsible for the base peak of DnA(n)+1 - mH2O and DnA(n) - mH2O (m = 1-2) clusters in the ESI spectrum. The absence of -OH end-groups signals in the MALDI spectrum is due to a metastable decay of protonated DnA(n)+1 and DnAn ions in the ion source of the MALDI mass spectrometer prior to ion extraction. In-source decay results in the formation of protonated lower DnA(n)+1 - mH2O and DnA(n) - mH2O oligomers and their corresponding neutrals, leading to wrong conclusions concerning the relative end-group distribution as a function of the degree of polymerization and the chemical composition.     

Successful MALDI-MS analysis of synthetic polymers with labile end-groups: the case of nitroxide-mediated polymerization using the MAMA-SG1 alkoxyamine

A sample pretreatment was evaluated to enable the production of intact cationic species of synthetic polymers holding a labile end-group using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. More specifically, polymers obtained by nitroxide-mediated polymerization involving the MAMA-SG1 alkoxyamine were stirred for a few hours in trifluoroacetic acid (TFA) to induce the substitution of a tert-butyl group on the nitrogen of nitroxide end-group by a hydrogen atom. Nuclear magnetic resonance, electrospray ionization tandem mass spectrometry, and theoretical calculations were combined to scrutinize this sample pretreatment from both mechanistic and energetic points of view. The substitution reaction was found to increase the dissociation energy of the fragile C-ON bond to a sufficient extent to prevent this bond to be spontaneously cleaved during MALDI analysis. This TFA treatment is shown to be very efficient regardless of the nature of the polymer, as evidenced by reliable MALDI mass spectrometric data obtained for poly(ethylene oxide), polystyrene and poly(butylacrylate).     

Theoretical investigation of low detection sensitivity for underivatized carbohydrates in ESI and MALDI

Electrospray ionization (ESI) and matrix‐assisted laser desorption/ionization (MALDI) mainly generate protonated ions from peptides and proteins but sodiated (or potassiated) ions from carbohydrates. The ion intensities of sodiated (or potassiated) carbohydrates generated by ESI and MALDI are generally lower than those of protonated peptides and proteins. Ab initio calculations and transition state theory were used to investigate the reasons for the low detection sensitivity for underivatized carbohydrates. We used glucose and cellobiose as examples and showed that the low detection sensitivity is partly attributable to the following factors. First, glucose exhibits a low proton affinity. Most protons generated by ESI or MALDI attach to water clusters and matrix molecules. Second, protonated glucose and cellobiose can easily undergo dehydration reactions. Third, the sodiation affinities of glucose and cellobiose are small. Some sodiated glucose and cellobiose dissociate into the sodium cations and neutral carbohydrates during ESI or MALDI process. The increase of detection sensitivity of carbohydrates in mass spectrometry by various methods can be rationalized according to these factors. Copyright © 2016 John Wiley & Sons, Ltd.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of lipids: ionization and prompt fragmentation patterns

Ionization and prompt fragmentation patterns of triacylglycerols, phospholipids (PLs) and galactolipids were investigated using matrix-assisted laser desorption/ionization (MALDI). Positive ions of non-nitrogen-containing lipids appeared only in the sodiated form, while nitrogen-containing lipids were detected as both sodiated and protonated adducts. Lipids containing acidic hydroxyls were detected as multiple sodium adducts or deprotonated ions in the positive and negative modes, respectively, with the exception of phosphatidylcholines. The positive MALDI spectra of triacylglycerols contained prompt fragments equivalent to the loss of RCOO(-) from the neutral molecules. Prompt fragment ions [PL-polar head](+) were observed in the positive MALDI spectra of all phospholipids except phosphatidylcholines. The phosphatidylcholines produced only a minor positive fragment corresponding to the head group itself (m/z 184). Galactolipids did not undergo prompt fragmentation. Post-source decay (PSD) was used to examine the source of prompt fragments. PSD fragment patterns indicated that the lipid prompt fragment ions did not originate from the observed molecular ions (sodiated or protonated), and suggested that the prompt fragmentation followed the formation of highly unstable, probably protonated, precursor ions. Pathways leading to the formation of prompt fragment ions are proposed.     

Electrospray and matrix-assisted laser desorption/ionization mass spectral characterization of linear single nylon-6 oligomers

Synthetic nylon-6 single molecular mass oligomers were studied by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry. These oligomers, considered as model compounds for the study of nylon-6 polymers, gave good mass spectrometric results using both MALDI and ESI. In spite of the gentle nature of both techniques, the MALDI and ESI spectra showed evidence of end-group cleavage from the oligomer chains. MALDI-MS was found to give similar fragmentation patterns for all of the oligomer samples. An increase in doubly charged ion signals with increasing oligomer mass was observed in the ESI mass spectra, as was end-group fragmentation. Signals from oligomer clusters were observed in ESI-MS for the dimer, tetramer and hexamer, most likely due to non-covalent bonding among the low-mass oligomer molecules.     

Matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectra of poly(butylene adipate)

Matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS/MS) was employed to analyze four poly(butylene adipate) (PBAd) oligomers and to investigate their fragmentation pathways as a continuation of our work on the MALDI-TOF/TOF-MS/MS study of synthetic polymers. MALDI-TOF/TOF-MS/MS analysis was performed on oligomers terminated by carboxyl and hydroxyl groups, methyl adipate and hydroxyl groups, dihydroxyl groups, and dicarboxyl groups. The sodium adducts of these oligomers were selected as precursor ions. Different end groups do not influence the fragmentation of sodiated polyester oligomers and similar series of product ions were observed in all the MALDI-TOF/TOF-MS/MS spectra. According to the structures of the most abundant product ions identified in the present work, three fragmentation pathways have been proposed to occur most frequently in PBAd: beta-hydrogen-transfer rearrangement, leading to the selective cleavage of the --O--CH(2)-- bonds; --CH(2)--CH(2)-- (beta--beta) bond cleavage in the adipate moiety; and ester bond scission.     

Double cation adduction in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of electron deficient anthraquinone derivatives

Six anthraquinone derivatives were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS). Clear (pseudo) molecular ions were observed for all the compounds. Interestingly, for some derivatives, strong ions with double cation adduction were also recorded in the positive mode. It is remarkable that all these ions are singly charged. In this work, possible mechanisms for the double cation adduction were investigated and discussed. It appears that the double cation adduction was due to the electron deficient nature of the derivatives, and formed by taking up two singly charged cations and one electron. Substituents on the anthraquinone ring were found to have a significant effect on the double cation adduction. In contrast, no considerable influence of the acidity of MALDI matrix/solution was observed, even on the double proton adduction. Furthermore, it was demonstrated that double cation adduction might occur in the MALDI gas-phase plume. In addition to the anthraquinones, three more electron deficient compounds of different types, i.e. a perylene bisimide derivative (PB), 3,7-decanoylamino-4,8-dihydrobenzo[1,2-b:4,5-b']dithiophene-4,8-dione (TQ) and 6,6-phenyl C61-butyric acid methyl ester (PCBM), were also analyzed with MALDI TOF MS. The results indicate that the 'abnormal' double cation adduction might be a 'normal' phenomenon in the MALDI TOF MS analysis of many electron deficient compounds.     

Successful MALDI‐MS Analysis of Synthetic Polymers with Labile End‐Groups: The Case of Nitroxide‐Mediated Polymerization Using the MAMA‐SG1 Alkoxyamine

A sample pretreatment was evaluated to enable the production of intact cationic species of synthetic polymers holding a labile end‐group using matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry. More specifically, polymers obtained by nitroxide‐mediated polymerization involving the MAMA‐SG1 alkoxyamine were stirred for a few hours in trifluoroacetic acid (TFA) to induce the substitution of a tert‐butyl group on the nitrogen of nitroxide end‐group by a hydrogen atom. Nuclear magnetic resonance, electrospray ionization tandem mass spectrometry, and theoretical calculations were combined to scrutinize this sample pretreatment from both mechanistic and energetic points of view. The substitution reaction was found to increase the dissociation energy of the fragile C? ON bond to a sufficient extent to prevent this bond to be spontaneously cleaved during MALDI analysis. This TFA treatment is shown to be very efficient regardless of the nature of the polymer, as evidenced by reliable MALDI mass spectrometric data obtained for poly(ethylene oxide), polystyrene and poly(butylacrylate).     

Reducing fragmentation observed in the matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of triacylglycerols in vegetable oils

Edible oils consist primarily of triacylglycerols (TAGs). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra of the oils are typically dominated by sodium adducts of these TAGs but also show prominent fragment ions (that do not contain sodium), which can interfere with analytical measurements of other components in oils. The fragments seemingly correspond to the loss of a fatty acid moiety from the sodiated TAGs as a sodium salt: RCOONa. However, a previous study suggested that the fragments actually arise from nearly complete fragmentation of unseen protonated TAGs. These authors suggested that the fragmentation occurs so rapidly and completely that protonated TAGs are not normally observed in the spectra of these oils. In this paper, we present evidence to support their theory and also demonstrate an approach to eliminate these interfering ions from the MALDI-TOF mass spectra via addition of a base to the matrix/sample mixture. The added base does not impede formation of the sodiated TAGs, but does significantly reduce the amount of fragments observed. We propose that this occurs by depleting the H+ ions from the matrix, thus preventing the formation of significant numbers of protonated TAGs in the first place. For measurements by MALDI-TOF, the relative abundances of the fragment ions are related to the strength of the base, and can be almost completely eliminated. However, in longer time-scale experiments such as in post-source decay and Fourier transform mass spectrometry, sodiated and non-sodiated diacylglycerol (DAG)-like fragments are present in spectra, regardless of whether or not a base is added to the sample.     

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.     

Tandem mass spectrometry of poly(ethylene imine)s by electrospray ionization (ESI) and matrix‐assisted laser desorption/ionization (MALDI)

In this contribution, linear poly(ethylene imine) (PEI) polymers, which are of importance in gene delivery, are investigated in detail by using electrospray ionization‐quadrupole‐time of flight (ESI‐Q‐TOF) and matrix‐assisted laser desorption/ionization‐time of flight (MALDI‐TOF) mass spectrometry (MS). The analyzed PEIs with different end groups were synthesized using the polymerization of substituted 2‐oxazoline via a living cationic ring‐opening polymerization (CROP) and a subsequent hydrolysis under acidic conditions. The main goal of this study was to identify linear PEI polymers in a detailed way to gain information about their fragmentation pathways. For this purpose, a detailed characterization of three different linear PEIs was performed by using ESI‐Q‐TOF and MALDI‐TOF MS in combination with collision‐induced dissociation (CID) experiments. In ESI‐MS as well as MALDI‐MS analysis, the obtained spectra of PEIs resulted in fitting mass distributions for the investigated PEIs. In the tandem MS analysis, a 1,2‐hydride shift with a charge‐remote rearrangement via a four‐membered cyclic transition state, as well as charge‐induced fragmentation reactions, was proposed as the main fragmentation mechanisms according to the obtained fragmentation products from the protonated parent peaks. In addition, heterolytic and homolytic cleavages were proposed as alternative fragmentation pathways. Moreover, a 1,4‐hydrogen elimination was proposed to explain different fragmentation products obtained from the sodiated parent peaks. Copyright © 2012 John Wiley & Sons, Ltd.     

End-group characterization of poly(styrene sulfonate sodium salt) by activated electron photo-detachment dissociation

Tandem mass spectrometry of poly(styrene sulfonate sodium salt) (PSS) was performed after activated electron photo-detachment dissociation (activated EPD). In this technique, doubly charged PSS oligomers were first produced in negative mode electrospray ionization, then oxidized into radical anions upon electron photo-detachment using a 220 nm laser wavelength, and further activated by collision. In contrast to the collision-induced dissociation (CID) of negatively charged PSS oligomers, which does not provide informative data with regard to the end-groups, activated-EPD is shown here to promote radical-induced dissociation reactions thanks to the oxidation of a sulfonate group upon laser irradiation. Major product ions generated after backbone bond cleavages contained one or the other chain terminations and could be accounted for by two main mechanisms. Moreover, each of the proposed dissociation reactions was shown to generate two distinct fragments, depending on the location of the oxidized monomer near one or the other chain terminal moieties. As a result, a combination of these two fragments allowed a straightforward mass characterization of each end-group.     

Matrix-assisted and polymer-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of low molecular weight polystyrenes and polyethylene glycols

Recently, matrices based on oligomers of dioxin and thiophene (polymer-assisted laser desorption/ionization (PALDI)) have been described for mass spectrometric (MS) analysis of low molecular weight compounds (Woldegiorgis A, von Kieseritzky F, Dahlstedt E, Hellberg J, Brinck T, Roeraade J. Rapid Commun. Mass Spectrom. 2004; 18: 841-852). In this paper, we report the use of PALDI matrices for low molecular weight polymers. An evaluation with polystyrene and polyethylene glycol showed that no charge transfer ionization occurs. Ionization is mediated through metal ion adduction. Comparison of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) data for two very low molecular weight polymers with data obtained from size-exclusion chromatography (SEC) revealed a systematic difference regarding mean molecular weight and dispersity. Further, the mass spectra obtained with PALDI matrices had a higher signal-to-noise ratio than the spectra obtained with conventional matrices. For polymers with higher molecular weights (>1500 Da), the conventional matrices gave better performance. For evaluation of the MALDI spectra, three non-linear mathematical models were evaluated to model the cumulative distributions of the different oligomers and their maximal values of Mw, Mn and PDI. Models based on sigmoidal or Boltzmann equations proved to be most suitable. Objective modeling tools are necessary to compare different sample and instrumental conditions during method optimization of MALDI analysis of polymers, since the bias between MALDI and SEC data can be misleading.     

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.     

Gas-Phase cationization and protonation of neutrals generated by matrix-assisted laser desorption

The ionization mechanisms involved in matrix-assisted ultraviolet laser desorption/ionization (MALDI) were studied with a time-of-flight mass spectrometer. When protonated or cationized quasimolecular ions generated by MALDI are not extracted promptly, their abundance is a function of the delay time between laser irradiation and ion extraction, maximizing at an optimum delay time (DTM) of a few hundred nanoseconds. The ion abundance at DTM exceeds that of prompt extraction by a factor of 2 or more. Increasing the cation density near the sample surface reduces the DTM, whereas increasing the desorption laser irradiance has the opposite effect. The enhancement suggests extensive gas-phase ion-molecule reactions after irradiation by the desorption laser has ceased.     

Qualitative and quantitative end-group analysis of a small molecular weight polyester by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was used for qualitative and quantitative end-group analysis of a small molecular weight polyester, poly(2-butyl-2-ethyl-1,3-propylene phthalate). The presence of carboxyl-terminated linear and cyclic polyester oligomers was confirmed with the help of simple sample preparation methods. The presence of carboxyl end-groups in the polyester chains was verified through their formation of carboxylate salts with alkali metal cations. Cyclic oligomers were identified through deuterium exchange of the exchangeable protons of the polyester. Various inorganic salts were tested for salt formation of the carboxyl end-groups, but only the alkali metal salts proved effective. The influence of the alkali metal salts on the results of the quantitative end-group analysis was also studied. The relative amounts of differently terminated and cyclic oligomers were calculated when the alkali metal salts were used with different matrices. The results showed that both the salts and the matrices used in sample preparation can have a marked effect on the quantitative results of the end-group analysis. The measurements were carried out using 2,5-dihydroxybenzoic acid (DHB), 1,8, 9-trihydroxyanthracene (dithranol), and 2-(4-hydroxyphenylazo)benzoic acid (HABA) as matrix compounds. Dithranol and HABA repeatably exhibited similar results, and these results differed from those obtained with DHB probably because of the different ionization mechanisms in the MALDI process. Copyright-Copyright 2000 John Wiley & Sons, Ltd.     

Hydrocarbon polymer analysis by external MALDI fourier transform and reflectron time of flight mass spectrometry

The traditional solvent-based matrix-assisted laser desorption ionization (MALDI) preparation method has been used to analyze nonpolar polymers of various molecular weights. High resolution silver cationized oligomers with masses of up to 12 KDa were measured using 9.4 tesla Fourier transform mass spectrometry (FTMS) with an external ionization source. It was observed that when time-of-flight mass spectrometry was used, the spectra of polyethylene polymers showed abundant low mass fragment ions. However, these fragments were absent from the FTMS spectra.     

The detection of red pigment-concentrating hormone (RPCH) in crustacean eyestalk tissues using matrix-assisted laser desorption/ionization-Fourier transform mass spectrometry: [M + Na]+ ion formation in dried droplet tissue preparations

Red pigment-concentrating hormone (RPCH), an octapeptide found in crustaceans and insects with the sequence pGlu-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NH2, is an N- and C-terminally blocked uncharged peptide. These structural features are shared with many members of the larger adipokinetic hormone (AKH)/RPCH peptide family in insects. We have applied vacuum UV matrix-assisted laser desorption/ionization (MALDI)-Fourier transform ion cyclotron mass spectrometry (FTMS) to the direct analysis of crustacean sinus gland tissues, using 2,5-dihydroxybenzoic acid (DHB) as the MALDI matrix, and have found that RPCH is detected in the cationized, [M + Na]+, form under conditions where other peptides in the direct tissue spectra are protonated without accompanying [M + Na]+ or [M + K]+ satellite peaks. The [M + H]+ ion for RPCH is not detected in tissue samples or for an RPCH standard, even when care is taken to eliminate metal ions. This behavior is not unprecedented; however, both direct tissue spectra and SORI-CID spectra provide no clues to suggest that the ionizing agent is a metal cation. In this communication, we characterize the MALDI-FTMS ionization and SORI-CID mass spectra of the [M + Na]+ and [M + K]+ ions from RPCH, and report on the detection of this neuropeptide in sinus gland tissues from the lobster Homarus americanus and the kelp crab Pugettia producta. We describe two strategies, an on-probe extraction procedure and a salt-doping approach, that can be applied to previously analyzed MALDI tissue samples to enhance and unmask sodiated peptides that may otherwise be mistaken for novel neuropeptides.     

Decomposition of protein nitrosothiolsin matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry

The S-nitrosylation of proteins is involved in the trafficking of nitric oxide (NO) in intra- and extracellular milieus. To establish a mass spectrometric method for identifying this post-translational modification of proteins, a synthetic peptide and transthyretin were S-nitrosylated in vitro and analyzed by electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The intact molecular ion species of nitrosylated compounds was identified in the ESI mass spectrum without elimination of the NO group. However, the labile nature of the S-NO bond was evident when the in-source fragmentation efficiently generated [M + H - 30](+) ions. The decomposition was prominent for multiply charged transthyretin ions with high charge states under ordinary ESI conditions, indicating that the application of minimum nozzle potentials was essential for delineating the stoichiometry of nitrosylation in proteins. With MALDI, the S-NO bond cleavage occurred during the ionization process, and the subsequent reduction generated [M + H - 29](+) ions.