Analytica Chimica Acta 808 (2014) 44–55

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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca

Review

A review of electron-capture and electron-transfer dissociation tandem mass spectrometry in polymer chemistry Gene Hart-Smith ∗ NSW Systems Biology Initiative, School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, Australia

h i g h l i g h t s

g r a p h i c a l

a b s t r a c t

• ECD and ETD can produce unique and diagnostically useful polymer ion fragmentation data. • The operating principles of ECD and ETD are discussed in relation to other dissociation techniques. • Key characteristics of ECD and ETD spectra, as observed from biological analytes, are discussed. • ECD and ETD analyses are compared to CID analyses for different classes of synthetic polymer.

a r t i c l e

i n f o

Article history: Received 4 July 2013 Received in revised form 3 September 2013 Accepted 18 September 2013 Available online 26 September 2013 Keywords: Polymer chemistry Electrospray ionisation (ESI) Tandem mass spectrometry (MS/MS) Electron-capture dissociation (ECD) Electron-transfer dissociation (ETD) Collision-induced dissociation (CID)

a b s t r a c t Mass spectrometry (MS)-based studies of synthetic polymers often characterise detected polymer components using mass data alone. However when mass-based characterisations are ambiguous, tandem MS (MS/MS) offers a means by which additional analytical information may be collected. This review provides a synopsis of two particularly promising methods of dissociating polymer ions during MS/MS: electroncapture and electron-transfer dissociation (ECD and ETD, respectively). The article opens with a summary of the basic characteristics and operating principles of ECD and ETD, and relates these techniques to other methods of dissociating gas-phase ions, such as collision-induced dissociation (CID). Insights into ECDand ETD-based MS/MS, gained from studies into proteins and peptides, are then discussed in relation to polymer chemistry. Finally, ECD- and ETD-based studies into various classes of polymer are summarised; for each polymer class, ECD- and ETD-derived data are compared to CID-derived data. These discussions identify ECD and ETD as powerful means by which unique and diagnostically useful polymer ion fragmentation data may be generated, and techniques worthy of increased utilisation by the polymer chemistry community. © 2013 Elsevier B.V. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods of ion dissociation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Ion/electron and ion/ion interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Ion/electron interactions and ECD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Ion/ion interactions and ETD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Ion/neutral interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Ion/photon interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author. Tel.: +61 2 9385 3857. E-mail address: [email protected] 0003-2670/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aca.2013.09.033

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3.

4.

5.

ECD and ETD induced fragmentation pathways: Insights gained from protein and peptide ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Products of electron capture/transfer often do not dissociate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Unique bond cleavages are possible . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Labile bonds are preserved . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Non-covalent interactions can influence product ion spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ECD and ETD versus CID of polymer ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Poly(alkene glycols) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. Homopolymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.2. Block copolymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Poly(amides) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1. Hyperbranched poly(esteramides) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2. PAMAM dendrimers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Poly(acrylates) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Poly(esters) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Gene Hart-Smith received his Ph.D. from the University of New South Wales in 2010. His Ph.D. studies were concerned with the elucidation of free radical polymerisation reaction mechanisms, and utilised mass spectrometry as a primary tool. He has since applied mass spectrometric techniques towards the study of biological systems. His current research interests centre upon the examination of protein-protein interaction networks; he is particularly interested in the impacts of protein post-translational modifications on the dynamics of these networks. Gene Hart-Smith has published 17 peer-reviewed journal articles and 1 book chapter.

1. Introduction It has been over a century since Thomson published details of what could be described as the first mass spectrometer [1]. In the years since, numerous technological advances have been made in the measurement of the mass-to-charge ratios (m/z) of gas phase ions. Amongst the most influential of these technological advances have been the developments made in ‘soft’ ionisation, and in particular, matrix-assisted laser desorption/ionisation (MALDI) and electrospray ionisation (ESI). These techniques, which were established by Fenn [2], Karas and Hillenkamp [3,4] and Tanaka [5] in the 1980s, revolutionised the analysis of large molecules. For the first time they allowed macromolecules – such as proteins, peptides and synthetic polymers – to be ionised into the gas phase with minimal fragmentation, and therefore allowed these molecules to be subjected to in-depth mass spectrometric analysis. Polymer chemists have since made strong use of the analytical capabilities made possible by soft ionisation. Using ESI or MALDI, detailed insights have been gained into, for example, polymerisation initiation mechanisms, polymer molecular weight distributions, copolymer distributions and mechanisms of polymer degradation [6]. The majority of these MS-based investigations into polymer systems have been performed entirely using single-stage MS, which measures the m/z of intact ions. That is, it is often possible for the chemical formulae and structural compositions of detected polymer components to be inferred from their mass data alone. In other cases, however, further information is required before detected polymer components can be unambiguously characterised. This is especially true when analysing polymer samples produced using

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poorly understood synthetic procedures, in which the masses of the polymer components may not be easily predicted, or for samples in which isobaric components are generated. In such instances, tandem MS (MS/MS) may provide the additional information required for unambiguous characterisation of such polymer components [7]. During MS/MS, precursor ions – which may, for example, be the polymer components of interest – are induced to undergo chemical reactions that change their charge or mass; the ions that form from these reactions – product ions – are then mass analysed. In most applications of MS/MS, the precursor ions are induced to undergo unimolecular dissociations, the most common method of achieving this being collision-induced dissociation (CID) [8–10]. In polymer chemistry, particular attention has been devoted to CIDbased product ion scanning: the mass analysis of all CID-derived product ions produced from selected precursor ions. A broad range of polymer classes have now been studied using CID-based product ion scanning, and an in-depth review of CID-derived fragmentation of polymer ions was recently published by Wesdemiotis et al. [7]. Through careful interpretation of the precursor and product ion masses observed in CID-based product ion scans, unique characterisations of, for example, polymer architectures [11], end-group functionalities [12–15] and copolymer sequences [16,17] have been possible. Though CID-based product ion scanning has been the most heavily utilised method of performing MS/MS on polymer ions, numerous other MS/MS methods are available to polymer chemists. These include methods of dissociating gas-phase ions other than CID, and the use of scan types other than product ion scanning. These alternative methods have largely been developed in response to the needs of bioanalytical chemistry, a field in which MS/MS plays a crucial role. MS/MS techniques are, for example, central to much of the present research in proteomics, in which peptides are characterised from complex mixtures using datasets generated via liquid chromatography (LC)–MS/MS [18,19]. Many methods of performing quantitative biochemical analysis are also, for example, reliant upon MS/MS [20,21]. This heavy emphasis upon MS/MS in bioanalytical MS has driven the production of an array of commercially available MS instruments with various specialised MS/MS capabilities, few of which have yet to be significantly exploited in the field of polymer chemistry. For recent summaries of the various instruments and scan types available for MS/MS and multiple-stage MS (MSn ) in the context of polymer chemistry, interested readers are referred to publications by Hart-Smith and Barner-Kowollik [22], and Scionti and Wesdemiotis [23]. This review aims to provide a synopsis of two particularly promising alternative methods of dissociating polymer ions during MS/MS: electron-capture and electron-transfer dissociation (ECD

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and ETD, respectively). As elaborated upon below, ECD and ETD are related means of inducing radical-driven dissociation. They are capable of producing product ions that are complementary to those observed using CID, and can thus serve as an alternative means of generating diagnostically useful polymer ion fragmentation data. To place ECD and ETD in context, the review opens with a summary of the different methods by which gas-phase ions can be induced to undergo dissociation. Aspects of ECD- and ETD-induced fragmentation, as observed in studies into protein and peptide ions, are then covered; the implications of these observations on investigations into polymer ions are discussed. Finally, for the various polymer classes that have already been studied using ECD or ETD, observed fragmentation pathways are summarised and contrasted to those observed using CID. 2. Methods of ion dissociation The dissociation of gas-phase ions can be sorted into three categories: (1) ion/electron and ion/ion interactions, (2) ion/neutral interactions, and (3) ion/photon interactions [24]. ECD and ETD, which fall into the former category, are discussed below. To judge the relative usefulness of ECD and ETD as methods of fragmenting polymer ions, other means of achieving gas-phase ion dissociation – in particular, CID – must be considered. For this reason, summaries of ion/neutral interactions and ion/photon interactions are also provided. In assessing the usefulness of different MS/MS dissociation methods for a given polymer sample, the diagnostic utility of the product ions is of primary concern. However, several additional factors must also be considered: the efficiency of dissociation, the types of ions capable of undergoing dissociation, and the instrumentation capable of performing the dissociation. If it is desirable for the MS/MS analysis of the polymer sample to be interfaced with online LC, the speed of the dissociation technique is also crucial. Each of these factors is discussed for the different dissociation methods presented below. 2.1. Ion/electron and ion/ion interactions The ion/electron and ion/ion interactions upon which ECD and ETD are, respectively, based induce dissociations through the electronic excitation of ions. In contrast, CID and ion/photon interactions generate ion excitation that is predominantly vibrational in nature, as detailed in Sections 2.2 and 2.3. It is for this reason that unique sets of product ions are observed in ECD/ETD-based MS/MS. The specific means by which ECD and ETD operate, and the abovementioned considerations relating to their applicability to polymer samples, are elaborated upon below. 2.1.1. Ion/electron interactions and ECD The electron irradiation of ions in MS/MS was first attempted by Freiser et al. in the late 1970s [25]. It was, however, almost another two decades before the use of ion/electron interactions in MS/MS drew substantial attention. In 1998, Zubarev et al. first developed and applied ECD to multiply charged proteins [26]. ECD was found to be capable of inducing particularly extensive fragmentation of peptide and protein backbones, whilst often allowing labile post-translational modifications to remain intact (see Section 3). This addressed a critical issue facing the analysis of proteins and peptides, and prompted further development of this technique. During ECD, positively charged precursor ions are irradiated with low energy electrons (in the order of 0.1–10 V). Capture of an electron by the precursor ion produces a radical cation. As elaborated upon in Section 3, dissociation of such radical cations occurs without energy redistribution; dissociation of this nature does not necessarily favour cleavage of a molecule’s weakest bonds [26–28].

An example of the ECD process for a polymer ion is illustrated in reaction (1), where P = a polymer molecule; X = a cation; and n = 2 or above: •∗

[P + nX]n+ + e− → [P + nX](n−1)+

→ fragments

(1)

When evaluating the potential utility of ECD for a given polymer sample, polymer ion charge states are a particularly important consideration. This is because positively charged precursor ions of charge states ≥2 are required for products to be observed following ECD, as the capture of an electron by a singly charged precursor ion results in an uncharged species. As the probability of electron capture increases with ion charge [29], dissociation efficiencies also increase as the charge states of precursor ions increase. The efficiency of ECD is therefore maximised when polymer samples are ionised to form species with high charge states; this can generally only be achieved when using ESI. Furthermore, as heteroatoms (other than those in amine functional groups) tend to alkali cationise [30], and unsaturated hydrocarbons without heteroatoms tend to copper or silver cationise [31–33], addition of an appropriate metal salt during sample preparation for ESI may be necessary to produce multiply charged polymer ions suitable for ECD. The ability to perform ECD is also dictated by the mass analysis instrumentation being employed. In the past, ECD was limited to use on Fourier transform ion cyclotron resonance (FTICR) mass analysers. These instruments implement static magnetic fields during the trapping of precursor ions; under these conditions, the irradiation of precursor ions with an electron beam is relatively simple [26]. However, many other popular mass analysis devices rely upon the use radio frequency (RF) electrostatic fields (e.g. ion trap (IT) and quadrupole (Q) mass analysers); in these instruments, the efficient immersion of precursor ions with low energy electrons is technically challenging. It has since been shown that these challenges can be circumvented in IT [34] and Q-time-of-flight (Q-TOF) mass spectrometers [35,36]; however, ECD remains to be primarily associated with FTICR instruments. Finally, the speed by which contemporary ECD operates makes it an option for use in the LC–MS/MS analysis of polymer ions. In the early stages of the development of ECD, electrons were sourced from heated filaments; this produced low electron fluxes, and even under optimised experimental conditions it was necessary to perform ion irradiation in the order of seconds [29]. Further studies have, however, led to the contemporary method of producing electrons using indirectly heated dispenser cathodes [37,38]. The high electron fluxes that can be achieved using this technology have allowed ECD irradiation times to be reduced to the order of milliseconds. Despite these advances, the collection of high quality spectra using ECD often requires that multiple scans are accumulated, and remains slow relative to other dissociation methods; this can compromise the utility of ECD when it is applied to complex samples using LC–MS/MS (e.g. see ref. [39]). 2.1.2. Ion/ion interactions and ETD Several years after the invention of ECD, Coon et al. developed ETD, a similar dissociation method also based on electron transfer to multiply charged cations [40–42]. In this approach, negatively charged radical reagent ions are used to induce electron transfer; the dissociation pathways that follow this process are generally analogous to those observed in ECD. An example of the ETD process for a polymer ion is illustrated in reaction (2), where P = a polymer molecule; X = a cation; A−• = a radical anion; and n = 2 or above: [P + nX]n+ + A

−•

•∗

→ [P + nX](n−1)+

→ fragments

(2)

One of the key components in ETD is the production of reagent ions suitable for electron transfer. In ion/ion interactions, there is always a competition between proton transfer from a cation to an

G. Hart-Smith / Analytica Chimica Acta 808 (2014) 44–55

anion, and electron transfer from an anion to a cation [43,44]. ETD therefore relies on the production of radical anions with a propensity to induce electron transfer. Early implementations of ETD utilised anthracene radical anions for electron transfer; production of these anions was achieved via the introduction of anthracene (C14 H10 ) molecules into a chemical ionisation (CI) source [42]. Subsequent studies by Coon et al. tested several polycyclic aromatic hydrocarbon molecules as alternative reagents for ETD [40,41,45]. These studies have led to the current preference for fluoranthene (C16 H10 ) as a reagent molecule [45]. The efficiency of electron transfer from fluoranthene radical anions is, however, generally estimated to be in the order of ∼40% [46]; the search for alternative molecules with low electron affinity is therefore of ongoing interest to ETD. As with ECD, only polymer ions of charge states ≥2 can be analysed using ETD, whilst the efficiency of electron transfer also increases with increasing precursor ion charge state. ETD is therefore almost always applied in conjunction with ESI. With regards to the speed of dissociation, ETD is readily achieved in the order of milliseconds using commercially available instruments, and has found great utility in the LC–MS/MS analysis of complex samples (e.g. see ref. [47]). A particularly notable advantage that ETD has over ECD is that it is available on a relatively wide variety of instrument platforms. ETD was originally developed for use in IT mass analysers [42]; in these devices, the simultaneous storage of precursor and radical anions in overlapping regions of space is readily achieved. As IT mass analysers are often used as stand-alone instruments, and are also common components in hybrid mass spectrometers, the development of ETD has greatly expanded the scope of performing ECD-like fragmentation. The use of ETD in hybrid IT-orbitrap mass spectrometers [48] has, for example, had a large impact upon the field of proteomics [46,47]. ETD has also been developed for use in Q-TOF [49] and Q-FTICR instruments [50]. Continued improvements to the practicality and robustness of ETD, such as the front-end electrical discharge-based reagent ion source recently described by Earley et al. [51], bode well for this already relatively widely available tool. 2.2. Ion/neutral interactions Several forms of ion/neutral interaction can lead to gas-phase ion dissociation. These include low energy reactive collisions, collisions between ions and neutral species in excited electronic states, and collisions between ions and a surface [24]. Of most importance to MS/MS, however, is the ion/neutral interaction that leads to CID: the collision of precursor ions with ground-state neutral gaseous targets. This process causes translational energy to be transformed into internal energy, and unlike ECD and ETD, energy redistribution occurs after ion excitation [8–10,52,53]. That is, during CID the internal energy of the activated precursor ion is redistributed as vibrational energy; dissociation is therefore most likely to occur via cleavage of the precursor ion’s weakest bonds. An example of the CID process for a polymer ion is illustrated in reaction (3), where P = a polymer molecule; X = a cation; N = a ground-state neutral gaseous species; and n = 1 or above: [P + nX]n+ + N → [P + nX]n+∗ + N∗ → fragments

(3)

CID is generally classified as either low or high energy depending on the kinetic energy of the precursor ion. Low energy CID involves ion travel at kinetic energies in the order of 10−1 –102 eV; under these conditions, ion dissociation usually occurs after multiple collisions. In high energy CID, when kinetic energies in the keV range are employed, ion dissociations after few or single collisions can be expected. High energy CID is capable of depositing enough energy to cause electronic excitation of precursor ions, but particularly for

47

large molecules such as polymers, vibrational excitation remains more probable [24]. The ability to perform either low or high energy CID is dependent upon the mass spectrometric instrumentation being employed; however, virtually all tandem mass spectrometers are designed to be capable of performing some form of CID. CID also has the advantage of being capable of achieving high efficiency dissociations for many molecular ion types. For example, unlike ECD or ETD, singly charged polymer ions are amenable to CID. Furthermore, CID is a fast method of achieving dissociation (activation times of ≥10−3 s in IT and FTICR instruments, and 10−5 –10−4 s in “beam-type” tandemQ and Q-TOF instruments [54]), and is therefore well suited to LC–MS/MS experiments. These characteristics make CID a viable option for many MS/MS experiments, and for this reason, it is considered to be the default method of performing ion dissociation in MS/MS. Though CID is the most common method of generating ion/neutral interactions during MS/MS, surface-induced dissociation (SID) – which, like CID, tends to promote vibrational excitation of ions – has shown promise as an alternative technique. SID utilises ion/surface collisions as a means of activating ions; in this manner, high collision energies and short activation times are readily achieved. Relative to CID conducted under most conditions, SID generally produces narrow energy transfer distributions, which can be precisely varied [24]. These promising characteristics are, however, offset by the relative inaccessibility of SID, which has, to date, only been employed using specially modified sector, quadrupole, TOF or FTICR instruments [55–57]. 2.3. Ion/photon interactions Though ion/photon interactions have yet to be applied in MS/MS studies of synthetic polymers, these methods of dissociation – which involve the irradiation of precursor ions with photons – do hold promise for future research. From a practical standpoint, they are attractive in that no gases are required to enter the vacuum system of the mass spectrometer. From an analytical perspective, they are unique in that they offer wavelength-dependent information. That is, dissociation yields can provide insight into a precursor ion’s photon absorbance at a given wavelength [24]. Studies using these methods of MS/MS, which have so far focused on biological analytes [58–63], have commonly used infrared photons at a wavelength of 10.6 ␮m to induce dissociations. Precursor ions must absorb tens to hundreds of infrared photons before they obtain enough vibrational energy to dissociate. For this reason, this technique has been termed infrared multiphoton dissociation (IRMPD). Alternatively, UV photons at wavelengths from 157 to 355 nm have also been used upon samples that contain an absorbing chromophore; only single or very few such photons are generally required to achieve precursor ion dissociation [61–63]. A hypothetical example of ion/photon interaction-induced dissociation of a polymer ion is illustrated in reaction (4), where P = a polymer molecule; X = a cation; n = 1 or above; and m = the number of photons absorbed: [P + nX]n+ + mhv → [P + nX]n+∗ → fragments

(4)

As with CID, the vibrational excitation of precursor ions in ion/photon interactions can be performed on precursor ions of charge state 1 or above. With regards to instrumentation, IRMPD has been implemented on IT [64,65] and FTICR [66–68] mass spectrometers, while UV photodissociation has been achieved using IT [69,70] and beam-type [71,72] instruments. IRMPD is a relatively slow process, and irradiation must be performed in the order of 10−3 –1 s to achieve sufficient activation of precursor ions. UV photodissociation, on the other hand, is a much faster process; sufficient activation of precursor ions can be achieved in the order of

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10−15 –10−9 s, making the technique a promising one for LC–MS/MS analysis. Though ion/photon interactions do have attributes that bode well for their application to polymer systems, the vibrational excitations that they induce carry less potential than ECD or ETD to generate product ions that cannot also be observed using CID. As alternative dissociation techniques to CID, ECD and ETD are therefore more likely to provide unique fragmentation information than IRMPD or UV photodissociation. 3. ECD and ETD induced fragmentation pathways: Insights gained from protein and peptide ions ECD and ETD were developed primarily with the needs of bioanalytical MS in mind. The vast majority of the insights into the mechanisms by which ECD and ETD operate have therefore been obtained from studies into proteins and peptides. MS/MS analyses of these macromolecules, which consist of sequences of amino acid monomers bound by peptide bonds, often aim to induce backbone cleavage; the masses of the resultant product ions can then be used to deduce the precursor ion’s amino acid sequence. In relation to the analysis of synthetic polymers, ECD and ETDbased studies of proteins and peptides act as useful reference points. They point towards fundamental aspects of ECD and ETD induced fragmentation that can be generalised across a range of macromolecules, whether biological or synthetic. Findings into ECD and ETD-based MS/MS of proteins and peptides that are particularly pertinent to the analysis of synthetic polymers are summarised below. 3.1. Products of electron capture/transfer often do not dissociate When interpreting ECD or ETD-derived product ion spectra obtained from polymer ions, there is a strong possibility that spectra will be dominated by charge-reduced species (depending on the polymer ion’s chemical makeup). This phenomenon is commonly observed during ECD and ETD of proteins and peptides, in which the most abundant product ion is often the actual product of electron capture or transfer: [M + nH](n − 1)+• (where M = the protein or peptide, and n = 2 or above; unless specialised sample preparation steps are taken, ESI of proteins and peptides generally produce protonated gas-phase ions). That is, the precursor ion has gained an electron to form a charge-reduced species, but this newly formed radical cation has not undergone dissociation. Another major product ion that is often observed is a precursor ion that has lost a proton: [M + (n − 1)H](n − 1)+ (where M = the protein or peptide, and n = 2 or above) [73]. As with the radical cations

described above, these charge-reduced species are observed having not undergone dissociation. 3.2. Unique bond cleavages are possible In the analysis of polymer ions, the characteristic of ECD and ETD that is of perhaps the most interest is that unique bond cleavages are possible relative to techniques that rely upon vibrational excitation. The resulting ECD or ETD-derived product ion spectra therefore contain unique diagnostic potential. For example in the analysis of proteins and peptides, both ECD and ETD tend to favour dissociation via cleavage of amine backbone bonds, resulting in the production of c and z• , or c• and z, fragments (see Scheme 1, which illustrates peptide ion fragmentation nomenclature as proposed by Roepstorff and Fohlman [74], and modified by Johnson et al. [75]). These backbone fragments are not observed using other standard dissociation methods. For discussions into the mechanisms by which such ions form via ECD, interested readers are referred to a summary by Zubarev et al. [28]. In contrast, CID’s preference for low energy fragmentation pathways results in the cleavage of protein and peptide backbones at amide bonds [74,76–79], leading to the production of b and y ions (see Scheme 1). 3.3. Labile bonds are preserved Early studies into ECD identified it as a nonergodic technique [26]. That is, radical cations dissociate before they are stabilised by energy redistribution (vide supra); this results in cleavages that are much more random than those observed in CID. The nonergodic nature of ECD and ETD is of particular relevance to precursor ions that contain labile bonds. In polymer ions, such bonds are often, for example, contained within labile end-group functionalities ([12,13,15,80,81]. In proteins and peptides, examples of particularly labile bonds include amide bonds adjacent the amino acid proline [82], and bonds associated with labile posttranslational amino acid modifications (see e.g. [83–88]). When macromolecules containing such bonds are subjected to CID, these bonds are readily cleaved; the products of these cleavages dominate the CID analysis, and the resulting product ion spectra contain few signals. In contrast, ECD and ETD studies of proteins or peptides have demonstrated that the nonergodic nature of these techniques promotes the preservation of labile bonds. Labile post-translational modifications are, for example, frequently left intact, and extensive backbone cleavage is often observed, which can lead to the production of information-rich product ion spectra [46,89]. 3.4. Non-covalent interactions can influence product ion spectra Studies into proteins over 20 kDa have indicated that macromolecules of this size tend to experience particularly low

Scheme 1. Peptide ion fragmentation nomenclature as proposed by Roepstorff and Fohlman [74], and modified by Johnson et al. [75], illustrated on a singly-protonated peptide ion featuring four amino acid residues. Subscripts refer to the number of amino acid residues in the fragment. Fragments preferentially formed during CID are marked with open circles, and fragments preferentially formed during ECD and ETD are marked with filled circles.

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fragmentation efficiencies when ECD or ETD are employed. In these cases, product ion spectra consist almost entirely of charge-reduced species, and few if any backbone fragments are identified (see e.g. [29]). These observations have been explained by the presence of non-covalent interactions [90]; that is, products of dissociation interact to form higher order structures, the masses of which are no different from the undissociated charge-reduced species. This issue has also been observed in smaller proteins and peptides that experience tight folding [91,92]. Similar observations can be expected when attempting to apply ECD or ETD to large polymer ions, or polymer ions that are tightly folded. Activated ion techniques have been shown to be capable of circumventing this issue. These techniques involve applying ECD or ETD in conjunction with additional vibrational energy, which leads to the destruction of higher order structures. Infrared radiation and blackbody radiation have been used for this purpose [93,94], but

[R(OC2 H4 )x OR + nH]n+

49

specifically, these studies investigated the ECD-derived fragmentation of poly(ethylene glycol) (PEG) and poly(propylene glycol) (PPG) homopolymers [98,99], and PEG and PPG block copolymers (PEG-b-PPG) [100], using an FTICR instrument. 4.1.1. Homopolymers In their studies into poly(alkene glycol) homopolymers, Cerda et al. subjected the following precursor ions to both ECD and CID: multiply charged adducts of PEG, [H(OCH2 CH2 )x OH + nX]n+ ; multiply charged adducts of PEGMe2 , [CH3 (OCH2 CH2 )x OCH3 + nX]n+ ; and multiply charged adducts of PPG, [H(OCH2 CH(CH3 ))x OH + nY]n+ ; where X = H+ or Na+ , and Y = H+ , Na+ or NH4 + . These precursor ions do not have any particularly labile bonds, and contain only a limited number of possible sites of bond cleavage. Given these characteristics, it is not surprising that both ECD and CID were shown to trigger cleavages at ether bonds within the polymer backbones, as summarised in reaction (5) for protonated PEG.



(n−1)+

→ R(OC2 H4 )y − O∗ − (C2 H4 O)(x−y) R + (n − 1)H CID or ECD

[R(OC2 H4 )x OH + mH]m+ (A)

−→



(5)

(n−m−1)+

+ ∗(C2 H4 O)(x−y) R + (n − m − 1) H ∗

+

for CID = H ,

for ECD∗ = H

(B)

•

the most popular contemporary activated ion technique involves a short pulse of collisional activation [90,95–97]. Using these techniques, extensive c and z• ion formation has been detected for proteins and peptides that otherwise produce uninformative ECD or ETD-derived product ion spectra (e.g. [90,95]). 4. ECD and ETD versus CID of polymer ions There exist a plethora of monomeric repeat units, polymer architectures and end-group functionalities by which synthetic polymers can consist. Given this diversity of materials, generalisations regarding the specific diagnostic utilities of ECD and ETD for polymers cannot be made. Furthermore, in MS/MS experiments, the method of ion dissociation is not the only factor that will influence product ion spectra. The types of precursor ions that are generated for a particular polymer component (e.g. metal cation adducts, or protonated adducts) will also have a heavy influence on MS/MS data [24]. Given these factors, the usefulness of ECD or ETD versus CID must be evaluated on a case-by-case basis, and will be driven by the particular analytical questions being asked. Past ECD and ETD studies of polymer ions have, however, demonstrated the potential of these techniques to provide unique insights into the compositions of polymer components. For polymer classes that have already been subjected to ECD or ETD, these past studies also serve as a guide for the detailed interpretation of product ion spectra in future analyses. These previous studies are presented in detail below (grouped according to polymer class). The observations obtained from these studies, and in particular comparisons between ECD- or ETDderived product ions and CID-derived product ions, are discussed. 4.1. Poly(alkene glycols) The earliest ECD studies of synthetic polymers were conducted by Cerda et al. on poly(alkene glycols) [98–100]. More

Despite these identical sites of bond cleavage, the two dissociation techniques also showed important differences which may be seen in, for example, the PEG-derived product ion scans illustrated in Fig. 1. ECD was demonstrated to only produce the A ions of reaction (5), whereas both of the complementary A and B ions were produced using CID. The abundances of the A ions produced using ECD were also relatively even when compared to the A ions produced using CID. This can be explained by the nonergodic nature of ECD; that is, the ECD-derived product ions are directly dependent upon the site of electron capture, and there exist similar probabilities of electron capture by solvating oxygen atoms along the length of the polymer backbone. These particular features of the ECD analyses carried important implications towards the determination PEG and PPG charge solvation [99]. Some CID spectra of PEG (not illustrated) were also further complicated by the presence of multiple bond cleavages followed by rearrangements, leading to the formation of ions with an internal monomer loss: [CH3 (OCH2 CH2 )(x−1) OCH3 + (n−1)H](n−1)+ . Similar phenomena were not observed when using ECD, which generally produces only single bond cleavages [89]. It is also notable that these studies identified different mechanisms of ECD depending on the precursor ion type. It was shown that the A ions obtained from sodiated adducts lose neutralised Na atoms; for this to occur, an electron rearrangement is necessary. Similar electron rearrangements could not be identified in ECD of the protonated and ammonium adducts of PEG [98,99]. Another interesting observation from these studies was that, unlike proteins and peptides, the ECD spectra of PEG showed no stable charge-reduced radical cations. This was explained by the low electron affinities of the ether and hydroxyl functionalities of PEG. However, charge-reduced [CH3 (OCH2 CH2 )x OCH3 + (n−1)H](n−1)+ species were observed. 4.1.2. Block copolymers In their studies into block copolymers, Cerda et al. subjected the following ions to both ECD and CID analysis: [H(OCH2 CH(CH3 ))x (OCH2 CH2 )y OH + 2H]2+ [100]. It was noted that the abundances of these doubly charged ions were only in the order of ∼0.5%; high quality ECD spectra were, however, still obtained.

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Fig. 1. (a) CID and (b) ECD spectra of [H(OCH2 CH2 )44 OH + 3H]3+ . Asterisks refer to noise or background peaks. Ion designations are per reaction (5). Image taken with permission from Cerda et al. [99].

Interpretation of the product ions generated from the ECD experiments allowed detailed insights to be gained into the copolymer structures. It was, for example, found that all of the copolymers studied consisted of diblock structures, contrary to the triblock designation of the commercially obtained samples. Importantly, it was also found that CID of the same samples produced multiple bond cleavages and rearrangements analogous to those observed in the homopolymers described above [98,99]. The resulting product ions complicated the data interpretation, and precluded accurate determination of the monomeric compositions of the different

samples. These issues were bypassed in the comparatively simple ECD-derived data.

4.2. Poly(amides) In ECD or ETD studies of synthetic polymers, synthetic poly(amides) have drawn particular attention. This is because in ECD or ETD analyses of proteins and peptides, the electronic properties of amide bonds play a crucial role in determining dissociation

Scheme 2. The components of a PAMAM dendrimer. A generation 2 dendrimer with an ethylenediamine core is depicted. Image taken with permission from Kaczorowska and Cooper [103].

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pathways. Poly(amides) therefore represent model systems by which amide bond dissociations may be better understood. Driven in part by these reasons, several research groups have applied ECD to hyperbranched poly(esteramides) [101] or poly(amidoamine) (PAMAM) dendrimers [102–104]. Summaries of these investigations are presented below. 4.2.1. Hyperbranched poly(esteramides) Koster et al. applied both ECD and CID to doubly protonated hyperbranched poly(esteramide) ions; various types of poly(esteramide), each containing different monomeric functional groups, were studied [101]. This was the first investigation to demonstrate unique ECD-derived bond cleavages relative to CID in synthetic polymer ions. Specifically it was shown that for most of the poly(esteramides) that were studied, only amide bond cleavages could be observed following CID, with some samples also generating neutral losses of water. On the other hand, ECD produced spectra that were significantly more complex; cleavages of both ester and amide bonds were observed, leading to the formation of both odd- and even-electron ions. Product ions formed via consecutive cleavages induced by intramolecular H-shifts were also identified; similar ECD behaviour has also been observed in some peptides [105–107]. 4.2.2. PAMAM dendrimers PAMAM dendrimers can be classified according to three components: their core, branches, and surface groups. Successive polymer growth steps produce different generations of dendrimers, each marked by progressively higher masses, molecular sizes, and numbers of surface groups. These components of PAMAM dendrimers have been illustrated in Scheme 2. Such dendrimers were first studied using ECD by Lee et al. in 2006. These authors applied ECD (but not CID) to multiply protonated (4+ to 8+ charge states) generation 3 dendrimers with 1,12 diaminododecane cores and amidoethanol surface groups [102]. Scheme 3 shows, for a single monomer repeat unit, the possible cleavage sites along PAMAM backbones. Where relevant, possible fragments ions are named in an analogous manner to fragment ions derived from protein or peptide backbone cleavages. In the ECD experiments conducted by Lee et al., it was found that cleavages of the PAMAM bonds adjacent to tertiary amines, which produce S and E ions, were the most pronounced dissociation channel. In contrast to ECD of proteins and peptides, only minor quantities of c and z• ions were identified. Instead significant quantities of b• and y ions, which are generated from amide bond cleavages, were observed. These authors suggested that the unexpectedly pronounced b• and y ion formation could be explained by intramolecular charge-solvation between protonated quaternary amines and the amide nitrogens. Kaczorowska and Cooper extended these studies by investigating the effects of surface groups and structure (generation) on both ECD and CID of PAMAM dendrimers [103]. Multiply protonated (4+

51

to 6+ charge states) generation 1 or 2 dendrimers with ethylenediamine cores and amino or amidoethanol surface groups were subjected to the two dissociation methods. In agreement with the results obtained by Lee et al., Kaczorowska and Cooper also found S and E ions to be the most pronounced species in ECD spectra; significant b• and y ion formation, and only minor quantities of c and z• ions were identified. It was found that the different surface groups did not impact upon the dissociation channels, despite their different chemical properties. This led the authors to the conclusion that, for each of the surface groups studied, the mechanisms of ECD remain dependent on electron capture at tertiary amines and the presence of amide nitrogens on PAMAM backbones, and can be explained using the chargesolvation model proposed by Lee et al. [102]. Furthermore it was found that for each of the different generation dendrimers, backbone cleavage was most pronounced in the innermost generations. In combination, these dissociation characteristics led to structurally informative and simple to interpret ECD-derived spectra. In contrast to the ECD-derived spectra, Kaczorowska and Cooper found that the CID-derived spectra were significantly more complicated and difficult to interpret. Furthermore, unlike ECD, the CID experiments demonstrated dissocation channels that were heavily dependent upon the chemical properties of the dendrimer surface groups. For the dendrimers with amidoethanol surface groups, bond cleavages were generally found to occur on the outermost generations of the dendrimers; the observed fragments were products of neutral losses containing surface groups, in which rearrangements were found to be possible. Multiple bond cleavages were frequently observed, leading to product ions missing several surface groups, and most product ions were observed in multiple charge states. For dendrimers with amino surface groups, CID spectra were dominated by product ions formed as a result of neutral losses of water (in precursor ions of higher charge states) or NH3 (in precursor ions of lower charge states). In a follow-up to their abovementioned research, Kaczorowska and Cooper then studied metal ion complexes of PAMAM dendrimers [104]. Ag+ , Cu2+ , Zn2+ , Fe2+ and Fe3+ complexes of generation 2 PAMAM dendrimers were subjected to both ECD and CID. Precursor ions were of 5+ charge states (2–4 protons depending on the metal ion) and contained ethylenediamine cores and amidoethanol surface groups. These investigations were conducted to gain insight into the sites and coordination chemistries of the metal ions; they also provided an illustration of the importance of precursor ion type on ECD and CID fragmentation pathways. In several cases, the dissociation channels of the metal ion complexes were substantially different to those observed for the purely protonoted precursor ions described above. ECD of the Cu2+ and Fe3+ complexes, for example, resulted in pronounced a and x ion formation following electron capture by the metal ions (see Scheme 3). When these same metal ion complexes were subjected to CID, the most abundant product ions corresponded to species formed via cleavage of the ethylenediamine cores. It was hypothesised that core tertiary amines were involved in the coordination of both Cu2+ and Fe3+ ions, resulting in weakened core C–C bonds. In contrast, the product ion scans of the Ag+ , Zn2+ and Fe2+ complexes more closely resembled those obtained from their purely protonated counterparts. Importantly, ECD and CID resulted in distinct fragmentation pathways for each sample, and each technique provided unique and complementary information regarding the coordination chemistries of the metal ions. 4.3. Poly(acrylates)

Scheme 3. PAMAM dendrimer ion fragmentation nomenclature as proposed by Lee et al. [102], illustrated on a quadruply-protonated ion. n subscripts refer to the dendrimer generation number.

Recently, Miladinovi et al. published the first account of ECD of poly(acrylate) ions [108]. In this investigation, methyl

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Fig. 2. (a) CID and (b) ETD spectra of [HO(C3 H4 O2 )16 C2 H4 OCH3 + 2Na]2+ . Fragments marked by ‡ are doubly-charged. Ion designations are per the main text. Image taken with permission from Scionti and Wesdemiotis [109].

methacrylate (MMA) homopolymers (PMMA), random copolymers of butyl methacrylate (BMA) and MMA (MMA-r-BMA), and block copolymers of MMA and BMA (MMA-b-BMA) were analysed using both CID and ECD. More specifically, the following precursor ions were the subjects of analysis: for PMMA, [H(C5 H8 O2 )H + nM]n+ ; and for both MMA-r-BMA and MMA-b-BMA, [H(C5 H8 O2 )x (C5 H8 O2 )y H + nM]n+ ; where M = Li+ , Na+ or Cs+ . The CID spectra of the abovementioned precursor ions indicated that polymer backbone cleavages were a prevalent dissociation channel, whilst dissociations leading to neutral losses of CH3 OH and CH4 from side chains also occurred. Interestingly, evidence was also presented for the recombination of fragment ions in the gas phase, leading to product ions with higher charges or masses than the precursor ions. In comparison, the ECD spectra were far less complex. Following ECD of doubly charged precursor ions derived from the PMMA sample, only one fragment ion was observed: a singly-charged ion with the loss of a methyl group. The abundance of this fragment

ion was highest in the spectra obtained from Li+ adducts, and decreased as cation size increased. ECD of the equivalent triply charged precursor ions produced the same fragment in both its singly- and doubly-charged forms; it was subsequently determined that the singly-charged products formed via the reaction of an electron with a doubly-charged fragment. ECD of the copolymers produced similar results; product ions consisted entirely of species formed via methyl loss from a MMA group, or butyl loss from a BMA group. It was suggested that these ECD-derived fragmentation pathways could be used to rapidly identify side-groups in unknown poly(acrylate) samples. 4.4. Poly(esters) All of the abovementioned studies utilised ECD, rather than ETD, as a method for promoting radical-driven dissociations of polymer ions. The first ETD study on polymer ions was reported only recently by Scionti and Wesdemiotis, who studied poly(lactide)

Scheme 4. CID pathways of [HO(C3 H4 O2 )16 C2 H4 OCH3 + 2Na]2+ . R = CH2 CH2 OCH3 . Ion designations are per the main text. Image taken with permission from Scionti and Wesdemiotis [109].

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Scheme 5. ETD pathways of [HO(C3 H4 O2 )16 C2 H4 OCH3 + 2Na]2+ . R = CH2 CH2 OCH3 . Ion designations are per the main text. Image taken with permission from Scionti and Wesdemiotis [109].

(PLA) homopolymers, and poly(ethylene adipate) (PEA) and poly(butylene adipate) (PBA) copolymers using both ETD and CID [109]. In this study, the following precursor ions were the subjects of analysis: for PLA, [HO(C3 H4 O2 )16 C2 H4 OCH3 + 2Na]2+ ; for the PEA and PBA copolymers, [HO(C8 H12 O4 )9 C2 H4 OH ± 2Na]2± and [HO(C10 H16 O4 )8 C4 H8 OH ± 2Na]2± , respectively. As with the abovementioned ECD studies of polymer ions, entirely distinct dissociation pathways were observed in the CID and ETD experiments. For example, this can be seen in the sets of product ions in the two spectra illustrated in Fig. 2, which were both obtained from identical PLA precursor ions. The nomenclature proposed by Scionti and Wesdemiotis in the naming of the product ions are as follows: In = linear polymer chains, with the subscript representing the number of complete or partial repeat units; superscripts represent end-groups, with A = carboxylic acid functionality, H = hydroxy or hydroxyalkyl group, R = ester substituent, and V = vinyl or terminal alkene moiety. Almost all of the product ions in the PLA-derived CID spectrum (Fig. 2a) were found to be derived from (CO)O-alkyl bond cleavages following 1,5-H rearrangements at ester groups, as illustrated in Scheme 4a. In addition, it was determined that precursor ions were capable of losing one or two monomer repeat units via 1,4-proton transfers at the hydroxy terminated chain ends (Scheme 4b), leading to the formation of truncated versions of the precursor ion. Interpretation of the ETD-derived spectrum shown in Fig. 2b revealed dissociation mechanisms unlike those observed during CID. It was hypothesised that, as with CID, many of these product ions were derived from (CO)O-alkyl bond cleavages; however, in contrast to CID, the radical-driven cleavages of ETD resulted in radical ion (In RV• )and doubly sodiated singly-charged fragment (In AH* )  formation; the In RV product ions indicated that these species subsequently interact to form even electron species (see Scheme 5a). Two other product ion formation mechanisms were identified from these ETD experiments: a proton-sodium ion exchange in the In AH* products, leading to the formation of In AH ions (see Scheme 5a); and an intramolecular nucleophilic substitution at the (CO)–O bonds, leading to the formation of In HR* ions (see Scheme 5b). In their analyses of the PEA and PBA copolymer precursor ions, Scionti and Wesdemiotis identified dissociation mechanisms that were analogous to those described above. It was, however, noted

that during CID, these copolymers readily underwent sequential 1,5-H rearrangements; these sequential dissociations generated internal fragment ion series that obscured the identities of the original end-groups. Similar sequential dissociations were not observed during ETD, leading to product ion spectra that were relatively simple to interpret. 5. Conclusions and outlook The benefits of nonergodic radical-induced dissociation in bioanalytical MS are well appreciated. In this field, ECD and ETD are now established as powerful methods by which unique structural information may be obtained from precursor ions during MS/MS. The potential held by ECD and ETD has yet to be realised to this extent in the field of polymer chemistry. However, the studies on synthetic polymers presented in this review have already confirmed that these techniques are readily capable of generating product ions with unique diagnostic potential. In the majority of instances, ECD/ETD promote unique bond cleavages relative to CID, leading to the formation of ECD or ETD-specific product ions. Even when ECD/ETD and CID promote the same bond cleavages (e.g. in poly(alkene glycols) [98–100] or poly(esters) [109]), the dissimilar mechanisms of dissociation, and the respective nonergodic and ergodic natures of these techniques, result in spectra that carry distinct sets of information. Furthermore, in many of the polymer systems that have been studied using both ECD/ETD and CID, it has been observed that ECD/ETD produce product ion scans that are relatively simple to interpret; this is largely due to the fact that these techniques tend to induce only single bond cleavages, whereas multiple or sequential bond cleavages are commonplace in CID experiments. The universality of CID, its ease of implementation, and its broad-scale applicability ensure that it is unlikely to be supplanted as the standard method of dissociating polymer ions during MS/MS. The abovementioned characteristics of ECD and ETD, however, indicate that these methods can provide unique and complementary insights into the components of polymer samples. Given that ECD or ETD are now available on a wide variety of ESI-interfaced instrument platforms, these techniques are now primed to be more fully exploited by polymer chemists.

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Acknowledgements G.H.-S. gratefully acknowledges Professor Christopher BarnerKowollik, the late Professor Michael Guilhaus and Associate Professor Mark Raftery for their many educational discussions on mass spectrometry. G.H.-S. also extends his thanks to Professor Marc Wilkins for his support. G.H.-S. acknowledges funding from the Australian Research Council (ARC) and receipt of an Australian Postdoctoral Fellowship (APD).

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