Mass Spectrometry and Tandem Mass Spectrometry: Fundamental Studies and Analytical Applications to Macromolecules
Mass spectrometry (MS) offers the potential to pursue research in gas phase ion chemistry, instrumentation development, and chemical analysis. Our group combines work in these areas to develop multidimensional MS methodologies for the characterization of synthetic polymers, biopolymers, and polymer-biomolecule interfaces and conjugates. Research is performed with online and offline LC/MS, tandem mass spectrometry (MS/MS), and ion mobility mass spectrometry (IM/MS) instrumentation equipped with vacuum and ambient ionization methods as well as a variety of MS/MS activation methods.
1. Synthetic Polymers
Synthetic polymers are found in a large array of products that have resulted in improved living conditions and better health. The physical and mechanical properties that make a synthetic polymer suitable for a particular application depend on the size, chemical composition, end groups, and architecture of the polymer. Our group uses established and new MS methods to determine the latter, chemical features, whose knowledge is essential for correlating polymer structure to specific properties and functions.
1a. Single-Stage MS
Single-stage (one-dimensional) mass spectrometry provides the accurate masses of the constituents of a polymer or copolymer, from which the corresponding compositions can be deduced with unsurpassed sensitivity, specificity, and speed. As a dispersive method, MS is ideally suited for the identification of the minor synthetic products and degradation products of polymer synthesis, which are difficult to detect by integrative methods, such as IR or NMR spectroscopy. Our group uses MALDI, ESI, and APCI mass spectrometry for the complete analysis of new synthetic polymers and materials; the minor and/or degradation products detected by MS are crucial for establishing polymerization mechanisms, confirming the origin of unknown samples, and comparing products prepared in different batches or by different methods.
Blends of differently sized polymers can be separated into their components or into narrower molecular weight mixtures by gel permeation chromatography (GPC). This liquid chromatography (LC) method separates based on hydrodynamic volume. On the other hand, interactive LC disperses on the basis of chemical composition, functionality, and mass. Our group investigates interactive LC for the analysis of copolymers and derivatized polymers that contain components of different polarities. For example, we recently found that reverse-phase LC of poly(ethylene glycol) / poly(propylene glycol) (PEG / PPG) copolymers can be optimized to yield fractions with constant (or very narrow) PPG content, as illustrated in Figure 1.
Fig. 1. (a) LC chromatogram of a random PEG/PPG copolymer (Mn = 970); (b) ESI mass spectrum of the fraction eluting at 20.7-21.1 min.
1c. Multistage MS
In tandem (MS/MS) or multistage (MSn) mass spectrometry, a specific precursor ion is mass-selected and induced to decompose into structurally indicative fragments, which are identified in a subsequent mass-analysis step. A series of studies in our group focuses on the acquisition, interpretation, and classification of the MS/MS and MSn mass spectra of synthetic polymers. Knowledge of the fragmentation mechanisms of polymer ions is the first step in the development of MS/MS and MSn strategies for the characterization of polymer sequences and architectures. We recently elucidated the fragmentation pathways of polystyrenes and polyacrylates and developed a protocol for the determination of branched architectures (Figure 2).
Fig. 2. MALDI-MS/MS spectrum of the [4-mer + Li]+ from a hyperbranched polyacrylate (synthesis by Pugh et al.). The fragments marked by green stars diagnose the branched architecture shown, and the ones marked by violet stars diagnose a differently branched isomer. R = (CH2)11-O-C6H4-p-C6H4-p-CN.
Precursor ions are induced to decompose by collisionally activated dissociation, CAD, or electron transfer dissociation, ETD. The former can be applied to singly as well as multiply charged precursor ions, whereas the latter is only applicable to ions with more than one charge. ETD is widely used in peptide sequencing. Current research in our group tests the advantages of ETD vs. CAD for synthetic polymers containing unsaturated functionalities.
1d. Degradation / MS
Considerable research efforts are devoted to the development of selective degradation / mass spectrometry techniques for the characterization of polymers that are not analyzable directly by MS or other spectroscopic means. Mild thermal degradation is employed to cleave large or complex polymer systems to smaller oligomers, which can subsequently be identified by MS and MSn, to thereby gain structural data about the original polymer. With (co)polymers containing biodegradable constituents, enzymatic digestion provides a means to cleave selectively the biodegradable components. The mass spectra obtained from the degradation products provide valuable structural insight about the composition of large polymer networks or complex formulations, which are very challenging to characterize otherwise.
1e. Ion Mobility Mass Spectrometry (IM/MS)
Fig. 3. (a) Ion mobility vs. m/z of the ions formed by ESI of a pegylated sorbitan trioleate; (b,c) mass spectra of the separated charge states.
In IM/MS, ions travel through a drift cell pressurized with a carrier gas. The drift time through this cell depends on the mass, charge, and shape of the ions. Hence, it allows one to differentiate isomeric or isobaric compositions with different geometries. IM/MS is a powerful method for the analysis of protein conformations. Our group explores its power to simplify the complexity of polymer mass spectra, to identify minor impurities and byproducts which are often buried under the signals produced from the major polymer components, and to distinguish (co)polymer isomers and isobars. Figure 3 shows the distributions of drift times vs. mass-to-charge ratios of the ions formed upon ESI of a pegylated sorbitan trioleate. The spectra within the slice at m/z ~1,000 indicate the presence of singly and doubly charged oligomers with significantly different drift times (and, hence, geometries). Such separation allows for definitive identification of the surfactant's components, even the minor ones, which is impossible from the simple mass spectrum alone.
1f. Polymer-Biomolecule Interactions and Interfaces
Fig. 4. ESI mass spectrum of a poly(ethylene imine) - adenosine monophosphate mixture, showing the formation of noncovalent complexes between the polymer and the nucleotide. PEI is used for nucleic acid transport in vivo.
Synthetic polymers are widely used for biomedical purposes, for example, in the manufacture of stents and implants, drug delivery systems, biosensors, and corrective devices. Biomolecules can adhere on the surface of such polymers by adsorption and adsorbed biomolecules can affect the biocompatibility and in vivo performance of the polymer hosts. Our group exposes polymers engineered for biomedical use to biological solutions and characterizes the adsorbed biopolymers either by extracting them from the surfaces and analyzing them by MSn and LC/MSn methods, or by direct surface MALDI imaging techniques. In control studies, the adsorption of single biomolecules, biopolymers, or analogs is investigated. These studies aim at assessing the biocompatibility of the new materials and also reveal fundamental information about the guest-host interactions that can develop in these assemblies (Figure 4).
Our group is an active participant in the Integrated Biosciences Program, an interdisciplinary combination of integrative biology, biochemistry, bioinformatics, and bioengineering. In synergistic research with groups in the Departments of Biology and Polymer Science, we employ mass spectrometry methods for the characterization of proteins, lipids, and their glycoconjugates. Ongoing investigations aim at the identification of the proteins present in gecko foot pads using comparative proteomics approaches. Concurrent work explores new mass spectrometry methods for the analysis of peptide sequences and glycolipid structures and examines fundamental aspects of the tandem mass spectrometry characteristics of such biopolymers.
- Gerișlioǧlu, S.; Wesdemiotis, C. Chain-end and backbone analysis of poly(N-isopropylacrylamide)s using sequential electron transfer dissociation and collisionally activated dissociation. J. Mass Spectrom. 2017, 413, 61-68 (DOI: 10.1016/j.ijms.2016.08.001).
- Alalwiat, A.; Tang, W.; Gerișlioǧlu, S.; Becker, M.L.; Wesdemiotis, C. Mass spectrometry and ion mobility characterization of bioactive peptide - synthetic polymer conjugates. Chem. 2017, 89, 1170-1177 (DOI: 10.1021/acs.analchem.6b03553).
- Liu, Y.; Lee, J.; Mansfield, K.M.; Ko, J.H.; Sallam, S.; Wesdemiotis, C.; Maynard, H.D. Trehalose glycopolymer enhances both solution stability and pharmacokinetics of a therapeutic protein. Bioconjugate Chem. 2017, 28, 836-845 (DOI: 10.1021/acs.bioconjchem.6b00659).
- Wesdemiotis, C. Multidimensional mass spectrometry of synthetic polymers and advanced materials. Chem. Int. Ed. 2017, 56, 1452-1464 (DOI: 10.1002/anie.201607003).
- Chakraborty, S.; Hong, W.; Endres, K.; Xie, T.-Z.; Wojtas, L.; Moorefield, C.N.; Wesdemiotis, C.; Newkome, G.R. Terpyridine-based, flexible tripods: from a highly symmetric nanosphere to temperature-dependent, irreversible, 3D isomeric macromolecular nanocages. Am. Chem. Soc. 2017, 139, 3012-3020 (DOI: 10.1021/jacs.6b11784).
- He, Q.; Mao, J.; Wesdemiotis, C.; Quirk, R.P.; Foster, M.D. Synthesis and isomeric characterization of well-defined 8-shaped polystyrene using anionic polymerization, silicon chloride linking chemistry, and metathesis ring closure. Macromolecules 2017, 50, 5779-5789 (DOI: 10.1021/acs.macromol.7b01121).
- Sallam, S.; Luo, Y.; Becker, M.L.; Wesdemiotis, C. Multidimensional mass spectrometry characterization of isomeric biodegradable polyesters. J. Mass Spectrom. 2017, 23, 402-410 (DOI: 10.1177/1469066717711401).
- Xie, T.-Z.; Wu, X.; Endres, K.J.; Guo, Z.; Lu, X.; Li, J.; Manandhar, E.; Ludlow, J.M.; Moorefield, C.N.; Saunders, M.J.; Wesdemiotis, C.; Newkome, G.R. Supercharged, precise, megametallodendrimers via a single-step, quantitative, assembly process. Am. Chem. Soc. 2017, 139, 15652-15655 (DOI: 10.1021/jacs.7b10328).
- Alexander, N.E.; Swanson, J.P.; Joy, A.; Wesdemiotis, C. Sequence analysis of cyclic polyester copolymers using ion mobility tandem mass spectrometry. J. Mass Spectrom. 2018, 429, 151-157 (DOI: 10.1016/j.ijms.2017.07.019).
- Gerislioglu, S.; Adams, S.R.; Wesdemiotis, C. Characterization of singly and multiply PEGylated insulin isomers by reversed-phase ultra-performance liquid chromatography interfaced with ion mobility mass spectrometry. Chim. Acta 2018, 1004, 58-66 (DOI: 10.1016/j.aca.2017.12.009).
- Sallam, S.; Dolog, I.; Paik, B.A.; Jia, X.; Kiick, K.L.; Wesdemiotis, C. Sequence and conformational analysis of peptide-polymer bioconjugates by multidimensional mass spectrometry. Biomacromolecules 2018, 19, 1498-1507 (DOI: 10.1021/acs.biomac.7b01694).
- Hill, J.A.; Endres, K.J.; Mahmoudi, P.; Matsen, M.W.; Wesdemiotis, C.; Foster, M.D. Detection of surface enrichment driven by molecular weight disparity in virtually monodisperse polymers. ACS Macro Lett. 2018, 7, 487-492 (DOI: 10.1021/acsmacrolett.7b00993).
- Hill, J.A.; Endres, K.J.; Meyerhofer, J.; He, Q.; Wesdemiotis, C.; Foster, M.D. Subtle end group functionalization of polymer chains drives surface depletion of entire polymer chains. ACS Macro Lett. 2018, 7, 795-800 (DOI: 10.1021/acsmacrolett.8b00394).
- Endres, K.J.; Hill, J.A.; Lu, K.; Foster, M.D.; Wesdemiotis, C. Surface layer matrix-assisted laser desorption ionization mass spectrometry imaging: A surface imaging technique for molecular-level analysis of Synthetic material surfaces. Chem. 2018, 90, 13427-13433 (DOI: 10.1021/acs.analchem.8b03238).
- Endres, K.J.; Xie, T.-Z.; Chakraborty, S.; Hoopingarner, C.; Wesdemiotis, C. Monitoring metal-macromolecular assembly equilibria by ion mobility-mass spectrometry. Rapid Comm. 2019, 40, 1800667 (DOI: 10.1002/marc.201800667).
- Wei, B.; Gerislioglu, S.; Atakay, M.; Salih, B.; Wesdemiotis, C. Characterization of supramolecular peptide-polymer bioconjugates using multistage tandem mass spectrometry. J. Mass Spectrom. 2019, 436, 130-136 (DOI: 10.1016/j.ijms.2018.12.005).
- Mao, J.; Zhang, W.; Cheng, S.Z.D.; Wesdemiotis, C. Analysis of monodisperse, sequence-defined, and POSS-functionalized polyester copolymers by MALDI tandem mass spectrometry. J. Mass Spectrom. 2019, 25, 164-174 (DOI: 10.1177/1469066719828875).
- Snyder, S.R.; Wei, W.; Xiong, H.; Wesdemiotis, C. Sequencing side-chain liquid crystalline copolymers by matrix-assisted laser desorption/ionization tandem mass spectrometry. Polymers 2019, 11, 1118 (DOI: 10.3390/polym11071118).
- Mao, J.; Zhang, B.; Zhang, H.; Elupula, R.; Grayson, S.M.; Wesdemiotis, C. Elucidating branching topology and branch lengths in star-branched polymers by tandem mass spectrometry. Am. Soc. Mass Spectrom. 2019, 30, 1981-1991 (DOI: 10.1007/s13361-019-02260-0).
- Atakay, M.; Aksakal, F.; Bozkaya, U.; Salih, B.; Wesdemiotis, C. Conformational characterization of polyelectrolyte oligomers and their noncovalent complexes using ion mobility-mass spectrometry. Am. Soc. Mass Spectrom. 2020, 31(2), 441-449 (DOI: 10.1021/jasms.9b00135).
- Endres, K.J.; Berthelmes, K.; Winter, A.; Antolovich, R.; Schubert, U.S.; Wesdemiotis, C. Collision cross-section analysis of self-assembled metallomacrocycle isomers and isobars via ion mobility-mass spectrometry. Rapid Commun. Mass Spectrom. 2020, 34(S2), e8717 (DOI: 10.1002/rcm.8717).
- Payne, M.E.; Kareem, O.O.; Williams-Pavlantos, K.; Wesdemiotis, C.; Grayson, S.M. Mass spectrometry investigation into the oxidative degradation of poly(ethylene glycol). Degrad. Stabil. 2021, 183, 109388 (DOI: 10.1016/j.polymdegradstab.2020.109388).
- Snyder, S.R.; Wesdemiotis, C. Elucidation of low molecular weight polymers in vehicular engine deposits by multidimensional mass spectrometry. Energy Fuels 2021, 35(2), 1691-1700 (DOI: 10.1021/acs.energyfuels.0c02702).
- O’Neill, J.M.; Johnson, C.M.; Wesdemiotis, C. Multidimensional mass spectrometry of multicomponent nonionic surfactant blends. Chem. 2021, 93(35), 12090-12095 (DOI: 10.1021/acs.analchem.1c02551).
- Endres, K.J.; Dilla, R.A.; Becker, M.L.; Wesdemiotis, C. Poly(ethylene glycol) hydrogel crosslinking chemistries identified via atmospheric solids analysis probe mass spectrometry. Macromolecules 2021, 54(17), 7754-7764 (DOI: 10.1021/acs.macromol.1c00765).