Tandem mass spectrometry of synthetic polymers

The detailed characterization of macromolecules plays an important role for synthetic chemists to define and specify the structure and properties of the successfully synthesized polymers. The search for new characterization techniques for polymers is essential for the continuation of the development of improved synthesis methods. The application of tandem mass spectrometry for the detailed characterization of synthetic polymers using the soft ionization techniques matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) and electrospray ionization mass spectrometry (ESI‐MS), which became the basic tools in proteomics, has greatly been increased in recent years and is summarized in this perspective. Examples of a variety of homopolymers, such as poly(methyl methacrylate), poly(ethylene glycol), as well as copolymers, e.g. copolyesters, are given. The advanced mass spectrometric techniques described in this review will presumably become one of the basic tools in polymer chemistry in the near future. Copyright © 2009 John Wiley & Sons, Ltd.     

Structural characterization of polyethers using matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry

Fragmentation of polyethers, such as poly(ethylene glycol) (PEG), poly(propylene glycol) and poly(tetramethylene glycol) was analyzed by matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) using a quadrupole ion trap time-of-flight mass spectrometer (QIT-ToF). The Li adduct ion provided more abundant fragments than the Na and K adduct ions in the MS/MS spectra. A previous study had demonstrated four series fragments of hydroxyl-, vinyl- and formyl-terminated ions, as well as distonic cations, in high-energy fast atom bombardment MS/MS and MALDI collision-induced dissociation measurements of poly(ethylene glycol). In the present study, the low-energy MS/MS measurements using MALDI-QIT-ToF, showed hydroxyl-, vinyl- and formyl- terminated fragments with or without other fragment groups, but not distonic cations. The fragmentation depended on the types of polyethers examined. MS/MS measurements using MALDI-QIT-ToF are expected to allow structural characterization of unknown components of polyethers.     

Comparison of negative and positive ion electrospray tandem mass spectrometry for the liquid chromatography tandem mass spectrometry analysis of oxidized deoxynucleosides

Oxidized deoxynucleosides are widely used as biomarkers for DNA oxidation and oxidative stress assessment. Although gas chromatography mass spectrometry is widely used for the measurement of multiple DNA lesions, this approach requires complex sample preparation contributing to possible artifactual oxidation. To address these issues, a high performance liquid chromatography (HPLC)-tandem mass spectrometric (LC-MS/MS) method was developed to measure 8-hydroxy-2'-deoxyguanosine (8-OH-dG), 8-hydroxy-2'-deoxyadenosine (8-OH-dA), 2-hydroxy-2'-deoxyadenosine (2-OH-dA), thymidine glycol (TG), and 5-hydroxy-methyl-2'-deoxyuridine (HMDU) in DNA samples with fast sample preparation. In order to selectively monitor the product ions of these precursors with optimum sensitivity for use during quantitative LC-MS/MS analysis, unique and abundant fragment ions had to be identified during MS/MS with collision-induced dissociation (CID). Positive and negative ion electrospray tandem mass spectra with CID were compared for the analysis of these five oxidized deoxynucleosides. The most abundant fragment ions were usually formed by cleavage of the glycosidic bond in both positive and negative ion modes. However, in the negative ion electrospray tandem mass spectra of 8-OH-dG, 2-OH-dA, and 8-OH-dA, cleavage of two bonds within the sugar ring produced abundant S1 type ions with loss of a neutral molecule weighing 90 u, [M - H - 90]-. The signal-to-noise ratio was similar for negative and positive ion electrospray MS/MS except in the case of thymidine glycol where the signal-to-noise was 100 times greater in negative ionization mode. Therefore, negative ion electrospray tandem mass spectrometry with CID would be preferred to positive ion mode for the analysis of sets of oxidized deoxynucleosides that include thymidine glycol. Investigation of the fragmentation pathways indicated some new general rules for the fragmentation of negatively charged oxidized nucleosides. When purine nucleosides contain a hydroxyl group in the C8 position, an S1 type product ion will dominate the product ions due to a six-membered ring hydrogen transfer process. Finally, a new type of fragment ion formed by elimination of a neutral molecule weighing 48 (CO2H4) from the sugar moiety was observed for all three oxidized purine nucleosides.     

Investigating the effect of mixing ratio on molar mass distributions of synthetic polymers determined by MALDI-TOF mass spectrometry using design of experiments

It is well known that the mixing ratio affects the molar mass distribution of synthetic polymers determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Surely, the molar mixing ratio determines whether a mass spectrum will be obtained or not. However, depending on the mass range, several effects such as multimer formation occur, which might be a source of errors in molar mass distribution calculations. In this study, the effect of mixing ratio was investigated for several synthetic polymers, including polystyrene (PS), poly(dimethylsiloxane) (PDMS), poly(ethylene glycol) (PEG), and poly (methyl methacrylate) (PMMA) using statistical designs of experiments. The 2³ full factorial design was found to be suitable in the study of more than 1000 samples. The obtained MALDI mass spectra as well as the ANOVA statistics show that the mixing ratio affects the molar mass distribution. The optimal mixing ratio for a defined synthetic polymer depends on the studied combination (matrix, cationization reagent, solvent).     

Liquid chromatography–tandem mass spectrometry of porphyrins and porphyrinogens in biological materials: separation and identification of interfering poly(ethylene) glycol by travelling wave ion mobility spectrometry/tandem mass spectrometry

Biological and clinical samples for porphyrin and porphyrinogen analyses by liquid chromatography–tandem mass spectrometry (LC‐MS/MS) are often contaminated with poly(ethylene)glycol (PEG), which complicates the interpretation of mass spectra and characterisation of new porphyrin metabolites. Two contaminating PEG molecules (m/z 833 and m/z 835) were completely separated from uroporphyrin I (m/z 831) by travelling wave ion mobility spectrometry and characterised by tandem mass spectrometry. One of the PEG species (m/z 835) also co‐eluted with uroporphyrinogen I (m/z 837) and was unresolvable by travelling wave ion mobility spectrometry/MS, therefore contaminating the MS/MS mass spectra owing to isotope distribution. These PEG species, with the [M + H]⁺ ions at m/z at 833 and/or m/z 835, co‐eluted with uroporphyrin I and uroporphyrinogen I by LC‐MS/MS and could be wrongly identified as uroporphomethenes. Copyright © 2013 John Wiley & Sons, Ltd.     

Tandem mass spectrometry of lithium-attachment ions from polyglycols

A detailed study has been carried out of the fast atom bombardment tandem mass spectrometry (MS/MS) behavior of lithium-attachment ions from three glycol polymers: linear poly(ethylene glycol), linear poly(propylene glycol), and an ethoxylated fatty alcohol. Collisional activation was carried out in the “collision octapole” of a BEoQ hybrid mass spectrometer at a translational energy of 50 eV, with collision gas air. It was found that [M + Li]⁺ ions provide a number of advantages as precursors for practical MS/MS analysis as compared to the use of [M + H]⁺ or [M + Na]⁺ ions. First, [M + Li]⁺ ions are much more intense than the corresponding [M + H]⁺ ions. Second, [M + Li]⁺ ions dissociate to lithiated organic fragments with reasonable efficiency, which is not the case with [M + Na]⁺ precursors. Third, product ions are generally formed over the entire mass range for low molecular weight polyglycols. The most intense product ions are lithiated, linear polyglycol oligomers. These ions are formed via internal hydrogen transfer reactions which are facilitated by lithium (charge-induced). Two series of less intense product ions are formed via charge-remote fragmentations involving l,4-hydrogen elimination. A fourth product ion series consists of lithiated radical cations; these form via homolytic bond cleavages near chain ends. Overall, MS/MS analysis of [M + Li]⁺ polyglycol ions proved to be quite useful for chemical structure elucidation.     

Tandem mass spectrometry of aminated fullerene derivatives

The products of the reaction between fullerenes (C₆₀/C₇₀) and dimethylamine were investigated by fast atom bombardment (FAB) mass spectrometry and tandem mass spectrometry (MS/MS). The FAB mass spectrum shows peaks corresponding to the addition of up to eight dimethylamine species, exclusively to C₇₀. MS/MS reveals an unusual fragmentation pattern. The mass spectrum of the reaction products, together with a number of tandem mass spectra, are shown.     

The influence of electrospray deposition in matrix-assisted laser desorption/ionization mass spectrometry sample preparation for synthetic polymers

Although electrospray sample deposition in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) sample preparation increases the repeatability of both the MALDI signal intensity and the measured molecular mass distribution (MMD), the electrospray sample deposition method may influence the apparent MMD of a synthetic polymer. The MMDs of three polymers of differing thermal stability, polystyrene (PS), poly(ethylene glycol) (PEG), and poly(propylene glycol) (PPG), were studied by MALDI time-of-flight (TOF) MS as the electrospray deposition voltage was varied. The MMDs obtained using the electrospray deposition method were compared with those obtained for hand-spotted samples. No change was observed in the measured polymer MMD when the electrospray deposition voltage was varied in the analysis of PS, but those of PEG and PPG changed at higher electrospray voltages due to increased ion fragmentation. It was also shown that the fragmentation in the hand-spotted samples is dependent on the matrix used in sample preparation.     

Atmospheric pressure matrix-assisted laser desorption/ionisation ion trap mass spectrometry of synthetic polymers: a comparison with vacuum matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry

Atmospheric pressure matrix-assisted laser desorption/ionisation quadrupole ion trap (AP-MALDI/QIT) mass spectrometry has been investigated for the analysis of polyethylene glycol (PEG 1500) and a hyperbranched polymer (polyglycidol) in the presence of alkali-metal salts. Mass spectra of PEG 1500 obtained at atmospheric pressure showed dimetallated matrix/analyte adducts, in addition to the expected alkali-metal/PEG ions, for all matrix/alkali-metal salt combinations. The relative intensities of the desorbed ions were dependent on the matrix, the alkali-metal salt added to aid cationisation and the ion trap interface conditions [capillary temperature, in-source collisionally-induced dissociation (CID)]. These data indicate that the adducts are rapidly stabilised by collisional cooling enabling them to be transferred into the ion trap. Experiments using identical sample preparation conditions were carried out on a vacuum MALDI time-of-flight (ToF) mass spectrometer. In all cases, vacuum MALDI-ToF spectra showed only alkali-metal/PEG ions and no matrix/analyte adducts. The tandem mass spectrometry (MS/MS) capability of the ion trap has been demonstrated for a lithiated polyglycol yielding a rich fragment-ion spectrum. Analysis of the hyperbranched polymer polyglycidol by AP-MALDI/QIT reveals the characteristic ion series for these polymers as also observed under vacuum MALDI-ToF conditions.     

Automated multiple-layer spotting for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of synthetic polymers utilizing ink-jet printing technology

Recently, a new multiple-layer matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) sample spotting technique for poly(ethylene glycol), offering improved analysis possibilities, was described. In this contribution the application of ink-jet printing to automated, multiple-layer MALDI-TOFMS sample preparation of synthetic polymers is presented, allowing accurate deposition of matrix, additive and analyte solutions. The new sample preparation technique was evaluated for poly(ethylene glycol) as well as poly(methyl methacrylate) standards, and optimized settings for both synthetic polymers have been obtained.     

Lithium and transition metal ions enable low energy collision-induced dissociation of polyglycols in electrospray ionization mass spectrometry

Electrospray ionization tandem mass spectrometry has the potential to be widely used as a tool for polymer structural characterization. However, the backbones or molecular chains of many industrial polymers including functional polyglycols are often difficult to dissociate in tandem mass spectrometers using low energy collision-induced dissociation (CID). We present a method that uses Li+ and transition metal ions such as Ag+ as the cationization reagents for electrospray ionization in an ion trap mass spectrometer. It is shown that lithium and transition metal polyglycol adduct ions can be readily fragmented with low energy CID. Comparative results from different cationization reagents in their abilities of producing both MS spectra and CID spectra are shown. This method opens the possibility of using conventional and readily available low energy CID tandem MS to study polyglycol structures.     

Electrospray ionization mass spectrometry of AZT H-phosphonates conjugated with steroids

AZT H-phosphonates conjugated with steroids were synthesized and determined by positive and negative ion electrospray ionization mass spectrometry (ESI-MS) in conjunction with tandem mass spectrometry (MS/MS). The fragmentation pathways were investigated in detail. There are very different characteristic fragment ions in the positive and negative ion MS/MS spectra. The azide group of compounds 6a and 6b underwent either elimination of HN(3) or rearrangement to an amine in both positive and negative ion mass spectrometry.     

Analysis of the volatile compounds in Senecio scandens Buch-Ham by gas chromatography-tandem mass spectrometry based on diversified scan technologies

Static headspace gas chromatography-tandem mass spectrometry was used to identify volatile compounds from Senecio scandens Buch-Ham. The elemental composition of compounds was confirmed by exploiting the tandem mass spectra of isotopic peaks from the precursor ion. Some isomers were well distinguished by the diversified scan technologies of tandem mass spectrometry (MS/MS). The MS/MS included a product ion scan, a precursor ion scan and a neutral loss scan. The results showed that 46 volatile compounds were completely identified, and the great of majority compounds were α-pinene (11.93%), n-caproaldehyde (9.02%) and dehydrosabinene (6.22%). This qualitative method is convenient and accurate and can be considered as a complementary identification method for the qualitative analysis of volatile compounds in complex samples.     

Characteristic fragmentation behavior of phosphoamino acid conjugates with 3'-azido-3'-deoxythymidine by electrospray ionization tandem mass spectrometry

The conjugates of phosphoamino acids with 3'-azido-3'-deoxythymidine were synthesized and their structures were determined by various spectral methods. In positive and negative ion electrospray mass spectrometry (ESI-MS), the fragmentation pathways were investigated in conjunction with tandem mass spectrometry (MS/MS). The results showed that there were very different characteristic fragment ions in the positive ion MS/MS spectra and the negative ion MS/MS spectra.     

On-line capillary separations/tandem mass spectrometry for protein digest analysis by using an ion trap storage/reflectron time-of flight mass detector

Capillary separations interfaced to tandem mass spectrometry provide a very powerful tool for the characterization of biological macromolecules such as proteins and peptides. The development of real time data-dependent data acquisition has further enhanced the capability of this method. However, the application of this technique to fast capillary separations has been limited by the relatively slow spectral acquisition speed available on scanning mass spectrometers. In this work, an ion trap storage/reflectron time-of-flight mass spectrometer (IT/reTOF-MS) has been used as an on-line tandem mass detector for capillary high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) separations of peptide mixtures including a protein digest. By taking advantage of the nonscanning property of the time-of-flight mass spectrometer, a fast spectral acquisition rate has been achieved. This fast spectral acquisition rate, combined with a new protocol that speeds up tickle voltage optimization, has provided MS/MS spectra for multiple components in a hemoglobin digest during one liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) run. Further, the IT/reTOF-MS has the speed to provide MS/MS spectra for multiple components in a CE separation of a synthetic peptide mixture within one CE/MS/MS run.     

Direct laser desorption/ionization time-of-flight mass spectrometry of conjugated polymers

Two conjugated polymers (CPs), poly(9,9-dioctylfluorene) (PF) and poly(3-octylthiophene) (PT) were analyzed by direct laser desorption/ionization time-of-flight mass spectrometry (LDI-ToF MS). Because of their strong absorption near the wavelength of the laser (337 nm), easy and transient energy transfer properties and sufficient thermal stability, CPs can be desorbed and ionized directly without a matrix. For comparison, these two polymers were also analyzed using matrix-assisted laser desorption/ionization (MALDI)-ToF MS in the positive reflectron mode. The results revealed that they are very similar in terms of quality and resolution. All results demonstrate that LDI-ToF MS is an alternative method for the mass characterization of some conjugated systems, thereby simplifying the process of sample preparation and result analysis.     

Solvent selection for matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometric analysis of synthetic polymers employing solubility parameters

The principle relating to the selection of a proper matrix, cationization reagent, and solvent for matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) of synthetic polymers is still a topic of research. In this work we focused on the selection of a suitable MALDI solvent. Polystyrene PS7600 and poly(ethylene glycol) PEG4820 were analyzed by MALDI‐TOF MS using various solvents which were selected based on the Hansen solubility parameter system. For polystyrene (PS), dithranol was used as the matrix and silver trifluoroacetate as the cationization reagent whereas, for poly(ethylene glycol) (PEG), the combination of 2,5‐dihydroxybenzoic acid and sodium trifluoroacetate was used for all experiments. When employing solvents which dissolve PS and PEG, reliable MALDI mass spectra were obtained while samples in non‐solvents (solvents which are not able to dissolve the polymer) failed to provide spectra. It seems that the solubility of the matrix and the cationization reagent are less important than the polymer solubility. Copyright © 2010 John Wiley & Sons, Ltd.     

Structural analysis of diols by electrospray mass spectrometry on boric acid complexes

A method is presented to characterize diols using negative ion electrospray (ES) mass spectrometry in combination with collision-induced dissociation tandem mass spectrometry (MS/MS). The analyte diol is added to a solution containing an ethylene glycol/boric acid [2:1] complex and then subjected to infusion ES. The following boric acid complexes are formed: (i) a complex with two ethylene glycol molecules, (ii) a mixed ethylene glycol/analyte complex, and (iii) a complex with two analyte molecules. The first complex serves as a reference for the assessment of the extent of complex formation with the analyte. The ES mass spectra of acyclic vicinal diols all feature intense mixed complex signals, indicative of efficient complex formation. Chemical fine tuning is achieved by MS/MS experiments. Thus, although the (2R,3R)-(-)-2,3-butanediol and meso-2,3-butanediol stereo-isomers show the same complexation efficiency, MS/MS experiments reveal pronounced structure characteristic differences. By contrast, 1,3- and 1,4-diols are less prone to complex formation as they give only weak signals relative to the reference. For cyclic vicinal diols only the cis isomer produces an intense mixed complex, whose MS/MS spectrum is characteristically different from that of the trans form. The above procedure does not permit an unambiguous differentiation of acyclic polyhydroxy compounds like mannitol and sorbitol. However, structurally related methyl glycosides show characteristic MS/MS spectra. Our findings indicate that the above simple procedure may be useful to probe the presence and structure of diols and other polyols in aqueous solutions. Copyright 1999 John Wiley & Sons, Ltd.     

Tandem mass spectra of tryptic peptides at signal-to-background ratios approaching unity using electrospray ionization high-field asymmetric waveform ion mobility spectrometry/hybrid quadrupole time-of-flight mass spectrometry

High-field asymmetric waveform ion mobility spectrometry (FAIMS) has been coupled to a quadrupole time-of-flight mass spectrometer for the tandem mass spectrometric analysis of tryptic peptides of pig hemoglobin. Using FAIMS, low levels (fmol/microL) of multiply charged tryptic peptides were separated from relatively intense chemical background such that their tandem mass spectra (MS/MS) lacked many background-related fragment ions observed using a conventional ESI-QqTOFMS instrument. Substantial improvements in both first-order and tandem mass spectra were realized while maintaining approximately the same absolute intensities.     

Gas-phase chemistry of the negative ions of fully-protected peptides by high-resolution electrospray ionization tandem mass spectrometry

Fully-protected C-terminal free peptides can be conveniently analyzed by high-resolution electrospray tandem mass spectrometry (ESI-MS/MS) in a quadrupole quadrupole time-of-flight tandem hybrid mass spectrometer, operated in the negative (-) ionizaionization mode. The unusual choice of negative ions in mass spectrometry applications to peptide analysis was needed to obtain exhaustive sequence and structural data. The low-energy collision-induced dissociation (CID) experiments provided, in fact, tandem mass spectra displaying highly diagnostic fragments with a good signal-to-noise ratio. The method is applied to segments of porcine calcitonin (Cal), Cal (1016, 1), Cal (1724, 2) and Cal (2528, 3) whose [M H]- deprotonated molecular ions provided low-energy CID mass spectra which allow the evaluation either of the primary structure of the peptide and of the location of the side-chain protective groups. ESI (+) MS can be conveniently used, in the high resolution mode, to achieve precise information on the elemental composition of the examined peptides.