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Tandem mass spectrometry entails isolation of a designated m/z range followed by ion activation, dissociation, and analysis of the resulting product ions [367]. The analyte ions or precursor ions can be measured intact first, followed by ion activation by one of the methods described in the following subsections, and mass analysis of the resulting fragment ions.

Peptide fragments follow a nomenclature adapted from what was first proposed by Roepstorff and Fohlman [444]. Fragment ions containing the N-terminus are named a- , b- or c-type based on the position, as shown in Figure 8. Similarly, product ions containing the C-terminus are named x-, y- or z-type. The numbering denotes the bond cleaved counting from the N or the C-terminus.

Figure 8: Nomenclature for peptide backbone fragments generated from gas phase ions

Similar to peptide fragmentation, a fragment nomenclature system for glycans was introduced by Domon and Costello [129]. Analogous to peptide fragmentation, fragments containing either end of the oligosaccharide (reducing or non-reducing end) are categorized as A-, B- and C-type (non-reducing end) or X-, Y- and Z-type (reducing

end) with a numbering system that indicates the position of the bond or residue at which cleavage occurred, as shown in Figure 9. While, B-, Y-, C- and Z-type ions are all generated by glycosidic bond fragmentation, A- and X-type ions are cross-ring fragments and therefore assigned with additional two numbers that denote the two bonds in a pyranose ring that were cleaved. The bonds are numbered from 0 to 5, starting with the O-C1 bond. For branched oligosaccharides, Greek letters (α, β, γ) are used to annotate the branches or antennae. The largest branch is designated as α, the next largest as β and so on for subsequent branches in decreasing order of size. If primary branches are further branched, superscript primes are used with Greek letters to indicate the order of branching, such as α’, β’ and α’’, β’’ for primary and secondary branches, respectively.

Figure 9: Domon and Costello nomenclature for glycan fragmentation

Reproduced from [129], with permission. Copyright © 1988, Glycoconjugate Journal

1.2.5.1 Collisionally-activated dissociation or Collision-induced dissociation (CAD or CID)

Collisionally-activated dissociation activates precursor ions by exciting them in a dissociation cell that is filled with a neutral gas such as nitrogen, argon or helium.

Repeated collisions lead to vibrational excitation of the precursor ions and the bonds dissociate in the order of their decreasing fragility, leading to generation of fragment or product ions. CAD is the most commonly used dissociation mode on modern mass spectrometers. It allows quick and efficient fragmentation that enables fast spectral acquisition and deeper coverage in LC-MS/MS experiments compared to other dissociation modes. Collisional activation can be broadly categorized into resonant- excitation and beam-type. Resonant-excitation dissociation is achieved in ion trapping instruments, where product ions cool rapidly, thus limiting the extent of dissociation. Beam-type CAD product ions, on the other hand, undergo further collisions with gas, resulting in a higher extent of dissociation [261,365].

Collisional dissociation was the first dissociation method applied for the purposeof peptide sequencing and PTM identification by mass spectrometry; it is still widely used today for peptide and PTM identification (211–213). Collisional-dissociation of peptides primarily yields b- and y-type ions, which are useful for peptide sequencing. One disadvantage of using CAD is that any PTMs that are linked to the peptide backbone with a bond more fragile than the peptide amide bond (e.g. sulfation, phosphorylation, O- GlcNAc, etc.) may be lost in this process and only the unmodified peptide backbone can be determined from the tandem MS [415]. Another disadvantage is that, for large proteins, collisional activation only produces fragments near the N- and C-terminus while the central part of the protein remains poorly covered [76].

Glycan fragmentation by collisional-dissociation primarily yields glycosidic bond fragments, with cross-ring fragments in lower abundances [324]. More comprehensive

cross-ring fragmentation can be achieved by manipulating ion charge states, using metal ion adduction, or inducing high-energy (kV) dissociation, at the cost of higher analysis complexity.

1.2.5.2 Electron-activated dissociation (ExD)

Electron-activated dissociation is a broad term used to refer to a number of dissociation modes including electron capture dissociation (ECD), hot electron capture dissociation (hECD), electron transfer dissociation (ETD), negative electron transfer dissociation (NETD), electron detachment dissociation (EDD) and electronic excitation dissociation (EED) [33,97,378,488,589,636].

The ETD process combines ion-ion chemistry with tandem mass spectrometry [513]. In ETD, multiply charged analyte ions are reacted with radical anions, generated in a chemical ionization source, to transfer electrons to the precursor cations, and results in the formation of an odd-electron hypervalent species that dissociates by a non-ergodic process. ETD reactions are typically performed in analyzers that allow trapping of the precursor ions with reagent radical anions, such as those from anthracene and fluoranthene. Ion trap instruments only allow resonant-excitation of the precursors and suffer from an m/z range cutoff that limits acquisition in the low m/z range, depending on the RF trapping field magnitude. Newer instrument and dissociation cell designs now enable ETD and beam-type collisional dissociation to be performed in the same multipurpose dissociation cell [446]. This enables combination of collisional and electron-based dissociation for better sequence coverage on large proteins and glycans [161,331].

Negative electron transfer dissociation (NETD) is a variant of ETD used for negative mode analysis, where reagent cation radicals are reacted with precursor anions to induce dissociation. It is an extremely useful fragmentation mode for generating complementary information on analytes that do not ionize efficiently or fragment too extensively in the positive mode due to their acidic nature, including acidic glycans and ribonucleotides, as well as acidic peptides [257,366,442,453,576].

Electron capture dissociation (ECD) is achieved by directing a beam of low- energy electrons into a region containing multiply-charged analyte cations. While the exact mechanism of peptide fragmentation by ECD is still under debate, the overall reaction is driven by electron capture by the analyte polycations, followed by charge reduction and generation of an excited radical species that rapidly decays by bond cleavage to generate the fragment ions. Peptide fragmentation by ECD and ETD predominantly produces N-Cα bond cleavage. As a result, ECD and ETD primarily generate c- and z-type ions; some secondary fragmentation from side chain losses may be seen and are more common in ECD than ETD [553,637].

ETD and ECD generate more complete coverage for large proteins and informative cross-ring fragments for glycans [76,208,255,327,630,637]. Because these dissociation modes do not involve vibrational excitation, fragile PTMs often remain intact during ECD/ETD and their linkage site can be identified in the product ion spectra [370,380,567,637].

Increasing the electron energy during ECD fragmentation can lead to extensive secondary fragmentation, including amino acid side chain losses from z• ions and

formation of w ions. Additionally, b- and y-type ions may be seen. This type of fragmentation is termed hotECD (hECD) and has been shown to be useful in discriminating isomeric amino-acids such as norvaline from valine and leucine from isoleucine [298,299,568,606].

Further increasing the electron energy above what is needed for hECD can lead to a completely different dissociation process known as Electronic Excitation Dissociation (EED) [605]. For glycans, EED results from formation of a distonic ion due to electron detachment from an oxygen atom. This is followed by electron recapture to generate a diradical that rapidly undergoes fragmentation. The advantage of using EED is the generation of extensive fragmentation without charge-reduction, which minimizes the precursor charge state dependence that ETD and ECD suffer from. EED also produces significantly more structural information than ECD and ETD and is compatible with analysis of glycans ionized with a wide range of charge carriers.

Electron Detachment Dissociation (EDD) is the negative mode complement of ECD that utilizes higher-energy electrons (>10 eV). EDD is initiated by electron detachment from a multiply charged analyte anion, which produces an anionic radical that subsequently undergoes radical-induced fragmentation to generate a variety of product ions [63]. Similar to NETD, this fragmentation mode has been shown to be extremely useful in analysis of acidic analytes, including proteins, peptides and acidic glycans such as GAGs, without the loss of labile groups or side chains [3,492,574,575].

Until recently, most electron-based activation methods could only be implemented in FTICR mass spectrometers because a strong magnetic field is required to

contain the electrons for activation of precursor ions. The application and dissemination of these methods was therefore limited due to the expensive FTICR instrumentation whose relatively lower scan speed and lower sensitivity hindered their effective implementation during high-throughput LC-scale experiments; however, Voinov et al. have recently demonstrated electron activated dissociation of biomolecules on QQQ and Q-TOF instruments fitted with a radio-frequency-free electromagnetostatic cell [550,552,553].

1.2.5.3 Photodissociation

Photodissociation is an alternative method for ion activation and fragmentation, which offers higher selectivity than collisional and electron-based dissociation methods. This is because bond cleavages are specific to the wavelength of light used and the absorption spectrum of particular bonds. Two commonly used photodissociation modes are classified by the wavelength ranges they operate in: IRMPD (Infrared multiphoton dissociation) and UVPD (Ultraviolet photodissociation). While photodissociation is not widely available on commercial MS instruments, a number of academic research groups have used it for biopolymer characterization. IRMPD is often combined with CAD or ExD for supplemental activation, to achieve more extensive fragmentation. Photodissociation is typically implemented in instruments where ions can be trapped for some period of time, so that they can be exposed to photon irradiation. Without making major modifications to the mass spectrometer, a window is added to the instrument to allow introduction of a laser beam. A quartz window may be used for transmission of near UV light (e.g. 355 nm). UVPD (e.g., 193 nm) requires the use of a LiF or MgF2

window, IRMPD by CO2 (10.6 µm) typically uses a ZnSe window. Photodissociation requires the analyte ion to have a suitable chromophore to allow photon absorption and ion excitation. Ly and Julian have summarized the use of UV lasers of different wavelengths in tandem MS [341]. In cases where the laser is not directly absorbed by the analyte, a chromophore may be attached covalently or non-covalently. IRMPD is typically used in conjunction with other dissociation modes such as ETD and facilitates better dissociation by pre-activating the ion. This involves unfolding of the precursor ion by irradiation leading to electron acceptor sites getting exposed for better ExD fragmentation. Many research groups have explored the use of UVPD and IRMPD in glycomics and proteomics, some of which are discussed in the following sections [124,125,303,318,344,442,446,480,492].