Electron Transfer Dissociation of Milk Oligosaccharides
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For structural identification of glycans, the classic collision-induced dissociation (CID) spectra are dominated by product ions that derived from glycosidic cleavages, which provide only sequence information. The peaks from cross-ring fragmentation are often absent or have very low abundances in such spectra. Electron transfer dissociation (ETD) is being applied to structural identification of carbohydrates for the first time, and results in some new and detailed information for glycan structural studies. A series of linear milk sugars was analyzed by a variety of fragmentation techniques such as MS/MS by CID and ETD, and MS3 by sequential CID/CID, CID/ETD, and ETD/CID. In CID spectra, the detected peaks were mainly generated via glycosidic cleavages. By comparison, ETD generated various types of abundant cross-ring cleavage ions. These complementary cross-ring cleavages clarified the different linkage types and branching patterns of the representative milk sugar samples. The utilization of different MS3 techniques made it possible to verify initial assignments and to detect the presence of multiple components in isobaric peaks. Fragment ion structures and pathways could be proposed to facilitate the interpretation of carbohydrate ETD spectra, and the main mechanisms were investigated. ETD should contribute substantially to confident structural analysis of a wide variety of oligosaccharides.
Key wordsElectron transfer dissociation Milk oligosaccharides Permethylated glycans Collision-induced dissociation Fragmentation O-18 stable isotope label
Carbohydrates participate in various biological processes. They are involved in diverse roles, such as molecular recognition, cell-cell and cell-matrix interactions, energy generation, and modification of protein conformations. Carbohydrates also serve as markers for many disease states. Increasing evidence shows that numerous biological functions are closely related to specific carbohydrate structures [1, 2, 3, 4]. However, compared with structural determinations of linear peptides or nucleic acids, elucidation of carbohydrate structures presents a much greater challenge, since many stereoisomeric structures can be formed from a very limited number of monosaccharides. Primary structural study requires not only composition and sequence analysis, but also the assignment of branching, linkage, and anomeric configuration . Due to its extreme complexity, carbohydrate structure analysis is still at an early stage of development.
Mass Spectrometry (MS) is one of the most important tools for the structural characterization of carbohydrates, as it offers high sensitivity and addresses the most frequent biological problems, particularly with respect to analytes of interest that are present as components of complex mixtures or in very small amounts. Tandem mass spectrometry (MS n ), including low- and high-energy collision-induced dissociation (CID), has proven to be a powerful means for carbohydrate structural identification [6, 7, 8]. Generally, glycans undergo two main types of cleavage upon CID: glycosidic cleavages resulting from the rupture between the two neighboring residues and cross-ring cleavages that involve the fragmentation of a sugar ring. The former provide information on composition and sequence, while the latter provide some details on the different linkage types. Internal fragments are generated upon the occurrence of two (or more) cleavages, either glycosidic or cross-ring, from more than one site in the molecule . They may also provide useful structural information.
Although low-energy CID does produce some cross-ring cleavages in oligosaccharides, the abundance of fragments resulting from cross-ring cleavages is relatively low in such spectra . By comparison, more structural information can be obtained from the cross-ring cleavages created by high energy CID . Recently, several novel fragmentation techniques have been applied to the analysis of carbohydrates. Adamson et al. and Zhao et al. have investigated the application of electron capture dissociation (ECD) to both linear and branched oligosaccharides by utilizing Fourier transform ion cyclotron resonance mass spectrometers (FT-ICR) [9, 10]. They showed that cross-ring cleavages were the dominant fragmentation pathway in ECD spectra. Whereas ECD is applied in the positive ion mode for characterization of neutral glycans, electron detachment dissociation (EDD) generates fragmentation in the negative mode for analyzing acidic glycans, and has been shown to be particularly useful for analysis of glycosaminoglycans (GAGs). Wolff et al. applied EDD to the analysis of GAGs and elucidated some mechanisms and pathways [11, 12]. Adamson and Hakansson also showed that EDD produces relatively abundant cross-ring cleavage ions for N-glycans and milk oligosaccharides .
High-energy CID is unavailable on many modern instruments. Other alternative fragmentation techniques, including ECD and EDD, are mostly limited to FT-ICR MS, but the cost and complexity of these instrument systems limit their availability. Electron transfer dissociation (ETD), a fragmentation technique very similar to ECD, was introduced by Syka et al. for peptide/protein fragmentation . In addition to its compatibility with high-end instrumentation, ETD can be carried out in quadrupole ion trap mass spectrometers, which are widely available. During ETD, multiply-charged ions react with an anion radical (e.g., fluoranthene), leading to the generation of both odd- and even-electron fragments and molecular species with lower charge states. In contrast to CID, which preferentially results in cleavage of the most labile bonds, ETD induces a gentle reaction pathway and initiates fragmentation of the precursor ions through transfer of an electron from the radical anion to the analyte and, hence, serves as a complement to CID . This technique has been successfully applied to investigations in proteomics and glycoproteomics, and has been shown to be especially useful for localizing post-translational modifications [16, 17], but so far, the use of ETD for structural studies of glycans has not been reported.
Ion trap instruments offer many significant advantages for glycan analysis. They have high sensitivity and the ability to select a particular parent ion, fragment it, detect the product ions, and then repeat the isolation/fragmentation process multiple times. In this way, they can provide additional information for addressing the difficult analytical problems presented by carbohydrate structures . Dissociation of a fragment ion may generate new types of product ions that cannot be observed in the first-stage dissociation of the original precursor ion [19, 20, 21, 22, 23].
Extensive studies have revealed that protonated oligosaccharides mainly produce glycosidic cleavages, yielding sequence but not linkage information [24, 25, 26]. Many reports have shown that fragmentation of different metal-adducted oligosaccharides (Li+, Na+, Mg2+, Ca2+, etc.) resulted in more cross-ring cleavages in low energy CID [25, 26, 27, 28]. Lebrilla and co-workers discussed the relationship between the metal size, sugar size, and fragment ion yield. Probably the metal ion binding to a ring oxygen localizes the oxygen’s electrons and prohibits glycosidic bond cleavages and it therefore induces abundant cross-ring cleavages under CID or IRMPD conditions [26, 29].
The derivatization of oligosaccharides by permethylation remains an important tool for detailed investigation carbohydrate structures. First, it decreases the intermolecular hydrogen bonding, thereby increasing the sample volatility and thus the intensity of the ion signals . Second, it stabilizes structures against the effects from the excess energy transferred during ionization procedures such as matrix-assisted laser desorption/ionization (MALDI) [19, 31]. Third, permethylation enhances the formation of diagnostic fragment ions and thereby increases the yield of useful structural information . Fourth, it can eliminate the negative charge associated with acidic residues and, thus, stabilize acidic residues in the positive mode; this feature is especially important for the analysis of sialylated glycans in which the carboxyl group is located on the same carbon as the glycosidic linkage .
Considering the limitations of classic fragmentation techniques in widely used instruments, we undertook an exploration of the potential for use of ETD, in conjunction with CID, for structural determinations on oligosaccharides. In the current study, a series of linear milk sugars—LNT, LNnT, LSTa, LSTb, and LSTc—has been investigated by ETD performed in the AmaZon ion trap (Bruker Daltonics, Billerica, MA, USA). Different metal salts were selected from alkali, alkaline earth, and transition groups, including Na+, Li+, Ca2+, Mg2+, and Zn2+, and tested to determine which metal adducts provided the most informative fragmentation behavior under ETD conditions. The metal iron (Fe) was not investigated as it cannot easily form complexes with oligosaccharides . In order to achieve unambiguous assignments of product ions, reduction and 18O labeling were used in parallel to provide two different types of modification at the reducing end of each milk sugar. The further utilization of MS3 techniques made it possible to verify many assignments and to distinguish isobaric peaks. A combination of these approaches was employed to investigate the major mechanisms in the fragmentation pathways and facilitated interpretation of the carbohydrate ETD spectra.
Lacto-N-tetraose (LNT, Galβ1→3GlcNAcβ1→3Galβ1→4Glc), lacto-N-neotetraose (LNnT, Galβ1→4GlcNAcβ1→3Galβ1→4Glc), LS Tetrasaccharide a (LSTa, Neu5Acα2→3Galβ1→3GlcNAcβ1→3Galβ1→4Glc), LS Tetrasaccharide b (LSTb, Galβ1→3(Neu5Acα2→6)GlcNAcβ1→3Galβ1→4Glc) and LS Tetrasaccharide c (LSTc, Neu5Acα2→6Galβ1→4GlcNAcβ1→3Galβ1→4Glc) were generous gifts from D. S. Newburg. All salts, including sodium chloride, lithium acetate, calcium chloride, magnesium sulfate, zinc acetate dihydrate, and other reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
2.2 Sample Preparation
18O-labeling and reduction were used in parallel to modify the reducing end of each milk sugar. For 18O-labeling, 2.7 mg of 2-aminopyridine was added into 1.0 mL of anhydrous MeOH to make the catalyst solution . Each dry, native oligosaccharide (1 μg) was dissolved in 20 μL of H 2 18 O (97%) and then 0.8 μL of AcOH and 1.5 μL of catalyst solution were added. The mixture was left at 40 °C for 16 h. The reducing ends of oligosaccharides were hydrolyzed and recyclized under the acidic conditions. H 2 18 O exchanged the anomeric hydroxyl group with 18OH [35, 36]. Then the 18O-labeled native samples were dried in a SpeedVac (Thermo-Fisher, Waltham, MA, USA) and were then ready for permethylation. The 18O-labeling experimental conditions were deliberately adjusted to achieve a 1:1 16O/18O ratio so that the spectra would display a 1:1 doublet for the 16O/18O species. For these derivatized mixtures, the fragment ions retaining the reducing end show both 16O and 18O product ions, while those retaining the non-reducing end do not: these differences validate the assignments of product ions. For simplicity and due to space constraints, the fragment ion peaks in these spectra are labeled only with the m/z values observed for the 18O-labeled species. The spectra of the pure 16O species appear in the Supplementary Material and may be used for comparison. To prepare the reduced glycans, a second set of the samples was treated according to the protocol reported by Costello et al. . Both the 18O-labeled and the reduced samples were subjected to permethylation before mass spectral analysis.
The native and derivatized oligosaccharides were permethylated using the procedure of Ciucanu and Kerek , as improved by Ciucanu and Costello . The permethylated oligosaccharide samples were each purified using a C-18 Ziptip (Millipore Corp., Billerica, MA, USA) and were desalted twice. Then the samples were dissolved in 50/50 methanol/water to a concentration of 0.5 pmol/μL. The final concentration of metal salts was 2–5 μM.
All experiments were performed with an AmaZon ETD Ion Trap mass spectrometer (Bruker Daltonics, Billerica, MA, USA). Samples were infused via the Apollo II electrospray ion source equipped with dual funnel transfer, at the flow rate of 100 μL/h. Enhanced resolution mode was used with the scan speed of 8100 m/z/s. The sample cations were transferred through the octopole and captured in the high capacity ion trap. Anion radicals of fluoranthene were generated in the negative chemical ionization source (nCI source), transferred into the ion trap, and then reacted with multiply-charged cations (i.e., ion/ion reactions). The parameter settings for positive-ion ESI-MS were as follows: capillary voltage, –4500 V; end plate offset, –500 V; nebulizer, 4 psi; dry gas, 2 L/min; dry gas temperature, 150 °C. The amplitude of the voltage for CID MS/MS was typically 0.3–0.8 V. For ETD MS/MS, the maximum accumulation time of fluoranthene anions was 20.0 ms. The “Remove” function was activated at 210.0 Da to remove excessive fluoranthene anions. The reaction time was typically 120–160 ms and the cutoff was 120–170 ms. The ICC ETD target was 200,000–300,000. The “Smart Decomposition” was set for two charge states. This function utilizes resonant excitation (but does not itself cause fragmentation) and performs a ramping of the voltage from 80% to 125% of the normal value for that m/z value, in order to overcome other attractive interactions (e.g., hydrogen bonds/noncovalent forces) between fragments . For MSn experiments, the fragmentation parameters had to be altered significantly, due to the different features of product ions. Mass resolution was 7000–10,000. Mass accuracy was 100–200 ppm above m/z 400 and 200–500 ppm below m/z 400. The software used for data processing and interpretation was Compass DataAnalysis (Bruker Daltonics).
3 Results and Discussion
Five (a) permethylated, (b) reduced and permethylated, or (c) both labeled with 18O at the reducing end and permethylated milk sugar samples, each cationized with Li+, Na+, Mg2+, Ca2+, or Zn2+ salts were subjected to electrospray ionization and fragmented in the AmaZon 3D ion trap. CID MS/MS, ETD MS/MS, CID/ETD MS3, and ETD/CID MS3 spectra were obtained. No [M + Zn]2+ ions were detected in the MS spectra; this result indicates that Zn2+ cannot form metal complexes with these sugars. In the ETD spectra of doubly-charged precursor ions [M + 2Li]2+ and [M + 2Na]2+, the corresponding charge-reduced species [M + 2Li]+∙, [M + 2Na]+∙, and the singly charged ions [M + Metal]+ were accompanied by few fragment ions. In contrast, both Mg2+ and Ca2+ adducts generated both the charged-reduced [M + Metal]+∙ and [M + Metal – H]+ and more types of fragment ions under ETD conditions. Consistent with the report of Harvey , the order of sensitivity was Ca2+ > Na+ > Mg2+. We observed that Ca2+ provided two to three times the sensitivity compared with Mg2+ (data not shown). In Ca2+ MS/MS spectra, we observed many glycosidic bond cleavages similar to Mg2+ spectra, but fewer cross-ring cleavages. Magnesium adducts provided a greater variety of fragment ions than did calcium adducts. After reduction or 18O-labeling on the reducing end and permethylation, the X, Y, and Z ions, which retained the intact reducing end exhibited a shift of 16 or 2 Da, respectively, relative to the permethylated native samples. In this way, isobaric peaks observed in the spectra of native oligosaccharides that might contain either the reducing or non-reducing ends of the glycans could be differentiated from one another on the basis of the occurrence of shifts in the spectra of the derivatives. Reduction and/or 18O-labeling also provided many clues that facilitated making definitive assignments for most fragment ions in the CID and ETD spectra. Where necessary, MS3 measurements were also carried out. The spectra of the reduced and permethylated samples adducted with magnesium salts provided the most valuable information that contributed to differentiation between isomers. Therefore, they will be discussed in detail in this paper. The CID data recorded for different derivatives and various cations exhibited high similarities with respect to the types of abundant ions, although they varied in the extent and types of the low abundance products that resulted from cross-ring cleavage. In order to avoid the repetitious presentation of similar information, the CID spectra of LNT and LSTa have been chosen to represent the typical fragmentation patterns for all CID spectra. Data obtained for the other glycans and for the derivatives not shown in the figures are available in the Supplementary Material. The nomenclature introduced by Domon and Costello has been used for designations of fragment ion assignments . The ions that result from glycosidic cleavages have been labeled in red; those that originate from cross-ring cleavages have been labeled in blue, and those from internal glycosidic cleavages, with or without additional loss of neutrals, in green. The peaks labeled in purple represent doubly charged ions.
In contrast, only singly-charged product ions were found in the ETD spectrum (Figure 2). Both [M + Mg]+∙ at m/z 943.5 and [M + Mg – H]+ at m/z 942.5, were observed in the ETD spectrum. As a indication that the gentle transfer process leads to relatively cool ions, the spectrum includes a cluster that corresponds to addition of water to the singly-charged molecular species. In this and all ETD mass spectra of the permethylated glycans, abundant fragment ion peaks are formed by elimination of 15 u (CH3∙) or 31 u (CH3O∙) from the otherwise intact charge-reduced radical, and from odd-electron forms of the Xn- and Zn-ions . In addition to the products resulting from glycosidic cleavages, a substantial population of products derived via cross-ring cleavages was present, and these contributed supporting evidence for assignment of the linkage positions. Internal fragments were also found in the ETD spectra. C3 and Z3 ions were isobaric and could not be distinguished under the experimental conditions. However, the ETD/CID MS3 spectrum obtained by selection of the ion at m/z 708.2 in the ETD MS/MS spectrum, followed by CID of this fragment, showed that there was a mixture of C3 and Z3 fragments (MS3 data in Supplementary Material). The abundant peak at m/z 550.2/551.2 was formed by loss of CH2CO or CH3CO∙ from 2,5X2. This type of fragment was found in all ETD spectra of glycans that had the [GlcNAc(1→3)Gal(1→4)Glc] moiety in each case. Subsequent losses of 15 u (CH3∙) or 31 u (CH3O∙) from the ion at m/z 550.2/551.2 were also observed. (It is quite possible that the CH3∙ or CH3O∙ loss occurs directly from [M + Mg] +∙ prior to the cross-ring cleavage and ketene loss.) The fragment that corresponds to the loss of a methyl radical from the 2,5X2' ion is isobaric with the 0,2X2' species and the contribution from each will need to be explored at high resolution. (The reader should note that the sequence fragment symbol followed by a prime (') is used herein as it has been employed for carbohydrates, i.e., to designate a species that has undergone loss of a neutral, and not as it has been used for peptide fragments where it signifies transfer of a hydrogen.) Extensive fragmentation occurred on the non-reducing end, whereas minimal fragmentation involved the reducing end. Comparison of the spectra of the native and 18O-labeled glycans affirmed that few product ions resulted from fragmentation at the reducing end. The bulky groups at positions C4, C5, and the anomeric carbon from Gal1 are all oriented in the same direction and, thus, form a suitable pocket to hold the magnesium cation. This could be one of factors that drive the occurrence of extensive ring cleavages.
Figure 3 shows the ETD spectrum of reduced and permethylated LNnT with Mg2+ adduction. All four kinds of fragment ions generated by glycosidic cleavages (B, C, Y, and Z), cross-ring cleavages (A and X) and internal cleavages, can be observed in this ETD spectrum. It should be noted that a number of [M + Mg – 71]+ or [M + Mg – 73]+ product ions were detected due to the elimination of the group NCH3Ac ± H∙ under the ETD conditions. Both B2(H) and B2(Mg) were observed, at m/z 464.2 and 488.2, respectively. The B2(H) ion originated from rupture of the glycosidic bond between GlcNAc2 and Gal3 and direct formation of an oxonium ion without attachment of a metal cation. B2(Mg), m/z 488.2, is a singly-charged even-electron species produced by electron transfer dissociation. Like all members of this series observed to date, it differs from the corresponding Bn(H) ion by 24 u and, thus, contains an extra 2H. (Simple replacement of 2H+ with Mg2+ would yield a mass shift of 22 u, since the most abundant isotope of Mg has the atomic mass 23.99.) The ion at m/z 708.2 was attributable to both C3 and Z3, and both assignments were also supported by its ETD/CID MS3 spectrum. It is worth noting that both Z3 and Z3∙ were observed in the ETD spectra of LNT and LNnT. The suggested structures for Z ions and B ions will be discussed later.
By comparison, various types of ions arising from cross-ring fragmentations, combined with the glycosidic fragment ions observed in the ETD spectrum allowed confident assignments on the sequences and linkages of the monosaccharide residues. Two sequential losses of 102 Da from the intact molecule suggested the presence of at least two 1,3 linkages, since this type of cleavage eliminates unsubstituted C4-C6. Within the sialic acid residue a number of cross-ring bond cleavages led to abundant fragments, e.g., m/z 1186.5, 1201.5, 1216.4, 1231.4, 1243.5. The reason is likely related to the three-dimensional structure of the sialic acid. The magnesium cation may prefer to bind with the multiple oxygen donors at the flexible chain extending from C5 and may, thus, facilitate extensive fragmentation within the sialic acid residue. Additionally, ∙NCH3Ac and ∙COOCH3 were both easily eliminated from the unit. As discussed above, losses of 71 or 73 Da correspond to elimination of NCH3Ac ± H∙ from the N-methyl, N-acetyl group (NMeAc).
3.4 ETD Fragmentation Mechanisms
In order to elucidate the mechanism(s) in the ETD pathway, metal coordination also needs to be considered. Linear peptides can undergo several types of coordination with metal ions. However, the possibilities for interactions of oligosaccharides and metals are broader, due to their complex structures . Metal coordination will change the configurations of carbohydrates, leading to multiple dissociation mechanisms . The groups of Lebrilla and Leary have studied [26, 29, 45] the binding sites of different metals to oligosaccharides utilizing the CID technique, and have thus provided valuable background information for the studies reported herein. Metal ions generally coordinate with several glycan oxygen atoms simultaneously [28, 29]. The most highly preferred binding position for alkali (e.g., Na+, Li+) and alkaline earth (e.g., Ca2+) metal ions is the pocket between two adjacent sugar rings in one oligosaccharide molecule; this means the metal can coordinate with both the oxygen in the glycosidic ring and with the oxygen in the C-6 hydroxyl group [26, 29]. The ionic radius of magnesium is small (0.65 Å) and it can easily fit within other sites around the oligosaccharide. This could be one of the reasons that a larger number of product ions were found in the ETD MS/MS spectra of glycan-Mg2+ adducts, than were seen in the ETD MS/MS spectra of glycan-Ca2+ adducts. The relatively bulky calcium ion may be restricted in its binding sites to oligosaccharides due to its larger ionic radius (0.99 Å); this property may cause less distortion of the original structure, stabilize the adduct and decrease the dissociation yield .
However, there could be other possible explanations for cross-ring cleavages, such as electron transfer with no dissociation followed by collision-induced dissociation (ETnoD-CID) when “Smart Decomposition” is applied. Electron transfer to the precursor ion is expected to decrease the overall activation barrier in the whole system. Then, the “Smart Decomposition” can rupture the weakest bonds on the glycan rings followed by CID-induced pericyclic retro-aldol and retro-ene mechanisms  with the charge retained on either the non-reducing or reducing end, leading to formation of A or X ions, respectively. CID, as either a first or second dissociation stage, can result in retro-Diels Alder fragmentations of the rings and to multiple neutral losses of methanol (32 Da), ketene (42 Da), ethanol (46 Da), and larger species from the permethylated glycans, as indicated on the spectra shown in the figures.
The utility of ETD has been demonstrated for characterizing a series of milk sugars and defining fragments that distinguish one from another. In this first phase of the research on the electron transfer dissociation of glycans, we utilized simple, linear, or minimally branched milk sugar isomers to investigate ETD pathways in carbohydrates. Generation of cross-ring cleavages was found to be the dominant ETD fragmentation pathway, in contrast to the preponderance of the products of glycosidic cleavages that has been observed in low- and high-energy CID. In ETD mass spectra, the presence of various product ions that originate from glycosidic cleavages, cross-ring cleavages, and internal cleavages allow unambiguous identification of the structures of oligosaccharides. For the glycans cationized by adduction with different metals, the extent of CID fragmentation is cation-dependent, but the patterns of fragment ions are often very similar. However, we have established that both the dissociation pathways and the efficiencies vary significantly as a function of cation adduct under ETD conditions. The dissociation behaviors depend on the type of metal adduct. Magnesium adduction generally yields the best cleavage efficiency and provides more useful information than does adduction with any of the other metals that have been evaluated to date. According to our initial results reported herein, multiply-charged glycans subjected to ETD undergo radical-driven fragmentation processes accompanied by complex hydrogen migrations and rearrangements. Our proposed ETD mechanisms are based on model studies. More investigations remain to be carried out, involving a broader range of glycans that include more structural features. The fragmentation-driving features present in the selected samples, e.g., the stereochemically favorable sites for metal adduction, and the amide substituents present in HexNAc and sialic acid residues, can also be found in many N- and O-linked glycans that can be released from glycoproteins or glycolipids. In summation, ETD shows considerable potential to become a very useful and powerful technique for detailed structural analysis of a wide variety of biological oligosaccharides.
The authors acknowledge support for this research by NIH-NCRR grant no. P41 RR010888. The authors are grateful to Bruker Daltonics for loan of the AmaZon ETD instrument and to Professor D. S. Newburg (Boston College) for his gift of the milk oligosaccharides. They thank Professor C. Lin and X. Yu for helpful discussions.
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