Collision Cross Sections and Ion Mobility Separation of Fragment Ions from Complex N-Glycans
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Ion mobility mass spectrometry (IM-MS) holds great potential for structural glycobiology, in particular in its ability to resolve glycan isomers. Generally, IM-MS has largely been applied to intact glycoconjugate ions with reports focusing on the separation of different adduct types. Here, we explore IM separation and report the collision cross section (CCS) of complex type N-glycans and their fragments in negative ion mode following collision-induced dissociation (CID). CCSs of isomeric fragment ions were found, in some cases, to reveal structural details that were not present in CID spectra themselves. Many fragment ions were confirmed as possessing multiple structure, details of which could be obtained by comparing their drift time profiles to different glycans. By using fragmentation both before and after mobility separation, information was gathered on the fragmentation pathways producing some of the ions. These results help demonstrate the utility of IM and will contribute to the growing use of IM-MS for glycomics.
KeywordsIon mobility Complex N-glycans Collision cross section Glycomics
Ion mobility-mass spectrometry (IM-MS) is emerging as a powerful tool to interrogate glycoconjugates and is capable of differentiating isomeric structures [1, 2, 3, 4, 5]. IM-MS can improve glycan detection by its ability to separate glycans from many contaminating compounds whose ions display different collisional cross sections (CCSs) [6, 7] and fall on different CCS-m/z trend lines. Most examples of the technique reported to date demonstrate separation of glycans arising from varied adduct formation [5, 8, 9, 10, 11], ion polarity [10, 12, 13], methylation [14, 15], and reduction . The separation of gas-phase ions by IM is driven by the size, shape, and charge of the analyte as it passes through a neutral gas, commonly nitrogen or helium, under the influence of a weak electric field. Properties that alter the three-dimensional structure of a glycan, such as fluorescent labelling, permethylation or the radius of a cation/anion adduct will, therefore, influence the drift time and the resulting arrival time distribution (ATD). The drift time is directly related to the CCS of a given carbohydrate ion, which is an intrinsic molecular property thus making its use for structural analysis universally applicable.
Glycan CCS values can be measured directly by drift tube (DT) instruments or by traveling wave (TW) instruments following calibration with compounds of known CCS values, such as dextran , analogous to glucose units in HPLC glycan analyses . Glycan structural analysis by mass spectrometry is most easily accomplished by negative ion collision-induced dissociation (CID), which generates multiple cross-ring cleavage products diagnostic of specific structural features such as antenna branching and location of fucose residues [19, 20, 21]. Recently, IM separation of positive fragment ions has proven effective in discriminating anomeric and linkage information [22, 23, 24, 25] as well as differentiating isomeric Lewis and blood group species . Accordingly, combining IM and CID for glycomics holds tremendous potential, and here, we explore IM properties of complex type N-glycan standards and corresponding fragments as negative ions. We identify unique drift time properties of complex glycan fragments and show that isomeric structures can be identified by CID products in cases where identification by their parent ion precursors is difficult.
Materials and Sample Preparation
Complex type N-glycan standards were purchased from Dextra Laboratories (Reading UK). Biantennary glycans containing fucose attached to their antennae were released with hydrazine from glycoproteins present in human parotid glands as described earlier . Hybrid glycans (Glc1Man4GlcNAc3 and Gal1Man5GlcNAc3) were from the HIV glycoprotein, gp120 that had been produced in the presence of the α-mannosidase II inhibitor, swainsonine. The triantennary glycan, A3G3 (see Fig. 9 for structure and a description of the terminology used to describe glycan structures), branched on the 3-antenna was from bovine fetuin (Sigma) following desialylation by heating with 2% acetic acid for 30 min at 80 °C and the corresponding compound containing fucose attached to the 4-branch of the 3-antenna was obtained likewise from human α1-acid glycoprotein (Sigma-Aldrich) . The A2 glycan was released from chicken ovalbumin by hydrazinolysis followed by re-acetylation.
Samples were diluted with HPLC-grade water to a final concentration of 150 μM prior to use. One microliter from the stock solution was added to 8 μl 1:1 methanol/water (HPLC grade) and a trace amount of 100 μM ammonium phosphate solution to generate phosphate adducts (the adducts most commonly observed on samples from biological sources). Sample solutions were stored at − 20 °C between analyses.
Ion Mobility-Mass Spectrometry
Traveling wave (TW) IM-MS measurements were performed on a Synapt G2Si instrument (Waters, Manchester, UK). For each sample analysis, 2 μl of N-glycan stock material was ionized by nano-electrospray ionization (nano-ESI) from gold-coated borosilicate glass capillaries prepared in-house . Instrument settings were as follows: capillary voltage 0.8–1.0 kV, sample cone 150 V, extraction cone 150 V, cone gas 40 l/h, source temperature 80 °C, trap collision voltage 4–160 V, transfer collision voltage 4 V, trap DC bias 60 V, IMS wave velocity, 450 m/s, IMS wave height 40 V, trap gas flow 2 ml/min, and IMS gas flow 80 ml/min. The vacuum pressures were as follows: backing = 3.3 mbar, source = 8.4e−3 mbar, trap = 2.4e−2, IMS = 2.8 mbar, and transfer = 2.7e−2. For investigating the origin of fragment ions from parent and primary fragments (later referred to as trap/transfer experiments), the first collision voltage (in the trap) was adjusted to a value where all relevant higher mass fragments were detected followed by a ramped secondary collision voltage (in the transfer) slowly over the range 30 to 110 V (higher voltages resulted in loss of the ion beam). Single ion plots of the target ions were then extracted from the accumulated data to identify their precursors. Data was acquired and processed with MassLynx v4.1 and Driftscope version 2.8 software (Waters, Manchester, UK). The nomenclature used to describe the fragment ions is that devised by Domon and Costello  with the addition of the use of the subscript R (for reducing terminus) to describe fragments of the reducing-terminal GlcNAc residue and R-1 to describe fragments from the other core GlcNAc. This nomenclature simplifies description of the fragmentation processes because it avoids the subscript number changing with different antenna chain lengths. An ion, named ion D, formed by loss of the chitobiose core and 3-antenna defines the composition of the 6-antenna.
TW-IM-MS and Collision Cross Section Estimation
Results and Discussion
IM separation of one hybrid and 13 complex type N-glycans were assessed using a traveling wave ion mobility instrument with nitrogen as the drift gas. The glycans included bi-, tri-, and tetra-antennary structures with core fucose (n = 6), bisecting N-acetylglucosamine (GlcNAc) (n = 3), and terminal sialic acid residues (N-acetylneuraminic acid; Neu5Ac) (n = 4). Collision cross sections of neutral (non-sialylated) glycans were measured as phosphate adducts due to the observation that phosphate, along with several other anions such as chloride and bromide, acts to stabilize neutral glycans during negative ionization and are the most common adducts found in glycans found in biological samples. Furthermore phosphate adducts do not have multiple isotopes, as is the case with chloride or bromide adducts  which may complicate interpretation of IM results. Sialylated N-glycan parent ions were measured as deprotonated species due to proton loss from carboxyl groups (pKa = 2.6) in solution. However, all CID products of both sialylated glycans and phosphate anion adducts were deprotonated ions following loss of a proton or H3PO4, respectively, as opposed to positive mode analysis where the cation adduct is generally retained with the product ions. Therefore, all CID IM measurements were performed on [M-H]− ions in this report.
General Correlations Between CCS Values and Structure
Information from Fragment Ions
To complicate matters further regarding contributions to the structures forming m/z 688, the trap/transfer spectrum of the first of the biantennary peaks suggested additional formation from the cross-ring 2,4AR-1 ion. Such an ion must have lost an equivalent mass (60 units) from another part of the molecule as that gained from the cross-ring fragment, leaving a residue of 102 mass units. Fragmentation of this ion showed that, indeed, the major fragment represented loss of 102 U, supporting such a double cross-ring structure. To conclude, therefore, the second of the two peaks from the biantennary glycans appeared to be ion i from the 6-antenna (the D ion), but the first peak appeared to be a complex mixture of structures, precluding the identification of a peak specific to the 3-antenna.
Another ion showing isomeric separation was the cross-ring fragment m/z 748 (Man3GlcNAc1 plus -O-CH=CH-O−). Five structures (m–q, Scheme 1) are possible from complex glycans lacking galactose and another four (not shown) if galactose is present. Three ATD peaks (Fig. 7, bottom) were found from glycans lacking galactose. The biantennary glycan F(6)A2 yielded a single peak. Man3GlcNAc2, which can only yield ion m, gave a single ATD similar to that from F(6)A2. This peak corresponds to structures n and/or o. The un-galactosylated triantennary glycan, A3, also yielded this peak but, in addition, produced a second which, presumably, could be assigned structure p. It was not present in the spectrum of the fucosylated glycan from human α1-acid glycoprotein which contains the fucose attached to this GlcNAc residue, supporting this structure. The non-glycosylated, tetra-antennary glycan produced peak 2. Antennae containing galactose could not be differentiated by this technique. These results show that some of the ions at m/z 748 have different cross sections but that it was only the un-galactosylated triantennary glycan that appeared to give a unique structure.
These results confirm that fragmentation of branched structures is very heterogeneous due to neutral losses from different arms. Additionally, this is complicated by the possibility of cross-ring fragmentation of either GlcNAcs on the chitobiose core. Furthermore, we do not account for possible gas-phase conformers which is exceedingly difficult to assign especially for deprotonated glycan ions as discussed above. It is equally possible that deprotonated fragments with the same structure adopt different conformers which would lead to alternate drift times and account for the observations described here. Therefore, although negative ion analysis is desirable when performing MS/MS alone for its propensity to generate cross-ring fragmentation, the presence of larger fragments complicates interpreting IM ATD data and efforts should focus on investigating smaller fragment ions.
The combination of CID applied pre-IM yields considerable glycan structural data and through interpreting the spectra of pure, synthetic glycan standards, we can start to understand the gas-phase behavior and, therefore, the potential of IM for glycomics. Here, we have described IM separation of complex type N-glycans as both intact glycan phosphate adducts and of fragments following CID in negative ion mode. These results show that (1) some isomeric structures can be identified by the drift time properties of CID products in cases where IM separation of parent ions fails and (2) the presence of various product ion structures is represented by multiple ATD peaks.
W.S. and M.C. gratefully acknowledge a research grant from Against Breast Cancer (www.againstbreastcancer.org; UK Charity 1121258).
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