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Ion Mobility Mass Spectrometry for Ion Recovery and Clean-Up of MS and MS/MS Spectra Obtained from Low Abundance Viral Samples

  • David J. Harvey
  • Max Crispin
  • Camille Bonomelli
  • Jim H. Scrivens
Research Article

Abstract

Many samples of complex mixtures of N-glycans released from small amounts of material, such as glycoproteins from viruses, present problems for mass spectrometric analysis because of the presence of contaminating material that is difficult to remove by conventional methods without involving sample loss. This study describes the use of ion mobility for extraction of glycan profiles from such samples and for obtaining clean CID spectra when targeted m/z values capture additional ions from those of the target compound. N-glycans were released enzymatically from within SDS-PAGE gels, from the representative recombinant glycoprotein, gp120 of the human immunodeficiency virus, and examined by direct infusion electrospray in negative mode followed by ion mobility with a Waters Synapt G2 mass spectrometer (Waters MS-Technologies, Manchester, UK). Clean profiles of singly, doubly, and triply charged N-glycans were obtained from samples in cases where the raw electrospray spectra displayed only a few glycan ions as the result of low sample concentration or the presence of contamination. Ion mobility also enabled uncontaminated CID spectra to be obtained from glycans when their molecular ions displayed coincidence with ions from fragments or multiply charged ions with similar m/z values. This technique proved to be invaluable for removing extraneous ions from many CID spectra. The presence of such ions often produces spectra that are difficult to interpret. Most CID spectra, even those from abundant glycan constituents, benefited from such clean-up, showing that the extra dimension provided by ion mobility was invaluable for studies of this type.

Graphical Abstract

Keywords

N-glycans Ion mobility CID Contamination 

Introduction

Mixtures of N-Glycans released from glycoproteins frequently contain contaminants such as polymers, residual buffers, and salts that inhibit ionization of the target compounds or produce ions that mask those from the glycans. This situation is frequently encountered with the small amounts of material that are released in-gel from sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)-separated glycoproteins from, for example, viral glycoproteins [1, 2, 3, 4]. Such contamination is often difficult to remove by traditional wet chemistry methods without sample loss. Pre-mass spectral fractionation such as, for example, the use of HPLC in LC/MS systems can reduce this problem considerably, but contamination issues remain problematic with direct infusion techniques or those using MALDI ionization. Furthermore, electrospray spectra are characterized by the production of multiply charged ions, often in several charge states from a single compound, and fragmentation can occur in the ion source or other regions of the instrument, further complicating the spectra. Under these conditions, ions selected for collision induced dissociation (CID) frequently include contributions from other ions with equivalent m/z values arising from these mechanisms, often leading to ambiguous spectra or, in the worst case, rendering the spectra uninterpretable.

We have been investigating the use of ion mobility to address these problems in situations where chromatographic inlets to the mass spectrometer are not available. Ion mobility can be regarded as an orthogonal technique to chromatography with the advantage that separations can be performed in milliseconds rather than the tens of minutes normally required for chromatography and one which offers the possibility of reducing sample losses because of the reduced need for pre-mass spectrometric clean-up. In parallel to glucose units that can be used to assign retention data to HPLC peaks, collisional cross sections can be used for glycans. These cross sections can be measured directly with drift tube instruments or indirectly with traveling-wave instruments using suitable callibrants [5, 6, 7, 8]. Furthermore, with nanospray infusion, the operator can select specific ions for CID studies rather than relying on data-dependent acquisition to select precursor ions for CID studies. Unfortunately, mass spectrometry suffers from the disadvantage of being unable to separate isomers and other isobaric species, a task that can often be performed by chromatography. Nevertheless, instruments fitted with ion mobility analyzers have the potential to redress this disparity because ion mobility separation relies on the shape of the molecules as well as mass and charge.

We [3, 9, 10, 11] and other investigators have already shown that in the absence of the extra dimension provided by mass spectrometers fitted with chromatographic inlet systems, ion mobility can be used to advantage to extract ions of interest from samples containing small amounts of material and to display a relatively clean glycan profile [9, 10]. Several investigators [12, 13, 14, 15, 16, 17, 18] have shown that lipids, nucleotides, peptides, and glycans fall on different mobility:m/z bands and can, thus, be separated. By use of this method, for example, singly charged [M + Na]+ N-glycan ions have been separated from mixtures containing various additional adducts and fragments [14]. Vakhrushev et al. [19] separated singly- and doubly charged sialylated N-glycan-derived fragment ions with overlapping isotope clusters [m/z 1199.93 (doubly charged) and 1200.42] in negative ion mode and demonstrated separation of the deprotonated glycan ions from Neu5Ac2-Hex5-HexNAc3 (m/z 1008.8, doubly charged) and Neu5Ac3-dHex-Hex6HexNAc5 (m/z 1007.3, triply charged) [20].

Ion mobility also has the ability to separate isomers, particularly of small carbohydrates [17, 21, 22, 23, 24, 25, 26, 27, 28, 29]. Fenn and McLean [30] showed that some isomeric oligosaccharides from milk could be separated, and Williams et al. [31] separated Man3GlcNAc3 isomers from ovalbumin and assigned structures based on negative ion fragmentation data. Plasencia et al. [32] have observed three peaks from the [M + Na2]2+ ion of the permethylated glycan of composition Hex5HexNAc4 from the same source. These peaks were assigned structures based on molecular modeling simulations but the structures were not confirmed by fragmentation. Yamaguchi et al. [33] partially separated the two isomers of the monogalactosylated biantennary glycans from IgG as 2-aminopyridine derivatives with a Waters Synapt G2 traveling wave instrument (nitrogen drift gas) and, again, modeled these compounds to assign structures. Isomers of Man7GlcNAc2 ionized as the [M + Na2]2+ ion have been partially separated; in this case the isomers were detected by monitoring selected fragment ions [34]. Both et al. [35] have investigated the use of fragment ions generated in the trap region of a Synapt mass spectrometer (before mobility separation) for differentiating between various isomeric disaccharides. Monosaccharide fragments generated in this manner exhibited similar arrival time distributions (ATDs). Also of interest is the energy-resolved technique introduced by Hoffmann et al. [36], which also involved trap fragmentation. Four isomeric trisaccharides with slightly different ATDs were investigated and were shown to fragment at different collision energies allowing selective suppression of their signals to improve resolution. Larger isomeric glycans have also been resolved; thus Rashid et al. [37] have separated α-1,4-linked maltooligosaccharides with a degree of polymerisation of up to 35 from α-1,6-linked dextran and α-1,4/1,6-linked pullulan. Conformers of maltohexaose were also separated. 3-Amino-quinoline-labeled partially methyl-esterified α-(1,4)-linked galacturonic acid oligosaccharides have also given asymmetrical ATDs attributable to isomers [38]. Complex ATDs for several doubly and triply sodium adducted permethylated N-glycans released from human tissues have been observed [26], suggesting isomer separation, but the species producing each of the glycan constituents were not determined.

The additional separating power of ion mobility has already found application in medicine and pharmaceutics. Investigators from Clemmer’s group, for example, have used it for differentiating various disease states, particularly cancer, by examination of N-glycans from serum glycoproteins [26, 39, 40]. Olivova et al. [41] have used traveling-wave ion mobility spectrometry (TWIMS) to separate the heavy and light chains of IgG1. These glycoproteins have similar m/z values but different charge states. The analysis allowed the glycoforms of the heavy chain containing differently galactosylated biantennary glycans to be resolved. Several drugs, present in relatively low concentration in various pharmaceutical formulations, have been analyzed directly using the power of ion mobility to extract the ions from the drugs following ionization by desorption electrospray [42], and Eckers et al. [43] have used the technique to detect impurities in formulated drug products. Damen et al. [44] have similarly examined lot-to-lot heterogeneity in the N-glycosylation profile of the therapeutic monoclonal antibody Trastuzumab. Further applications can be found in reference [45].

The object of the present work is to evaluate the potential of ion mobility for the structural identification of N-glycans released from glycoproteins when only small amounts are available. Glycans released from the recombinant human immunodeficiency virus-derived glycoprotein, gp120, was used as a representative example. Negative ion methods were used because this ionization technique has been shown to provide more structurally specific fragment ions than the parallel positive ion techniques [46, 47, 48, 49, 50].

Materials and Methods

Materials

Recombinant HIV-1 gp120JRCSFBG505, bearing a C-terminal poly-His tag, was expressed transiently from human embryonic kidney 293 T cells as described previously [51, 52] and purified by immobilized metal affinity chromatography using a HisTrap HP column (GE Healthcare, Amersham, UK), and concentrated. N-linked glycans were released in-gel using the method originally described by Küster et al. [53] with the enzyme protein N-glycosidase F (PNGase F, EC 3.5.1.52) following separation by SDS-PAGE, extraction by sonication in water, and purification with a porous graphitised carbon column (Thermo Scientific, Runcorn, UK). Methanol was obtained from BDH Ltd. (Poole, UK) and ammonium phosphate was from Aldrich Chemical Co. Ltd. (Poole, UK). Dextran from Leuconostoc mesenteroides was obtained from Fluka (Poole, UK).

Sample Preparation for Mass Spectrometry

Following release from the glycoproteins, the glycans (1 μL samples of aqueous solution) were cleaned with a Nafion 117 membrane as described by Börnsen et al. [54]. They were diluted with water (4 μL), and methanol (6 μL) plus 1 μL of an aqueous solution of ammonium phosphate (0.05 M, to maximize formation of [M + H2PO4] ions, (the ions usually encountered from biological samples)). Samples were then centrifuged at 10,000 rpm (9503 × g) for 1 min to sediment any particulates.

Electrospray, Ion Mobility Mass Spectrometry

Traveling wave ion mobility experiments were carried out with a Waters Synapt G2 traveling wave ion mobility mass spectrometer (TWIMS) (Waters MS-Technologies, Manchester, UK) fitted with an electrospray (ESI) ion source. Samples were infused through Waters thin-wall nanospray capillaries. Ion source conditions were: ESI capillary voltage 1.2 kV, cone voltage 100 V, ion source temperature 80°C. The T-wave velocity and peak height voltages were 450 m/s and 40 V, respectively. The T-wave mobility cell contained nitrogen and was operated at a pressure of 0.55 mbar. Fragmentation was performed after mobility separation in the transfer cell with argon as the collision gas. The instrument was externally mass calibrated with sialylated N-glycans released from bovine fetuin [55] and the ion mobility cell was calibrated with dextran from Leuconostoc mesenteroides. Data acquisition and processing were carried out using the Waters Driftscope (ver. 2.1) software and MassLynx (ver. 4.0). CID spectra were interpreted according to previously published data [46, 47, 48, 49, 50] and further confirmation of structure was made by comparing collision cross sections with those of reference glycans [7]. The scheme devised by Domon and Costello [56] was used to name the fragment ions.

Results and Discussion

MS Spectra

As an example of the use of ion mobility to extract glycan ions from spectra derived from low abundance samples following an in-gel release with PNGase F [53], N-Glycans were released in-gel from the glycoprotein gp120, and two typical ESI spectra are shown in Figure 1b and c. The spectrum in panel b is of a reasonably concentrated sample but that in panel c is of a sample with low concentrations of glycans (released in-gel from about 5 μg of glycoprotein) and with a high background that obscures most of the glycan ions. However, the glycan ions from the latter sample could be separated by ion mobility as shown in the m/z:drift time plot in Figure 1a (DriftScope display). The circled region labeled 1 contains the singly charged ions, mainly as phosphate adducts. These ions were selected and exported to MassLynx to give the spectrum shown in Figure 1d. Most of the noise has been removed, allowing some minor constituents to be seen. Some of these glycans have been characterized in earlier publications [9, 10, 11, 53] and the structures of all but the minor constituents were confirmed by negative ion CID using fragmentation in the transfer cell of the Synapt instrument (after mobility separation) [46, 47, 48, 49, 50] and cross section measurements [7]. The main diagnostic ions in the negative ion CID spectra can be summarized as follows. Location of the core fucose was reflected in the 2,4A ion from the reducing-terminal GlcNAc residue (see Figure 2 for an example); the two triantennary glycans were identified by the presence of a fragment at m/z 831 (3-branched isomer) and m/z 1053 [D ion (formed by loss of the chitobiose core and 3-antenna)], 1035 and 1017 (6-branched isomer) [57]; bisected glycans gave a prominent fragment formed by loss of GlcNAc from the D-ion. Sialic acid linkages were identified by the method reported by Wheeler and Harvey [58]. Identified glycans are listed in Table 1, with their structures in Scheme 1. Many of the glycans were detected in different ionic states (i.e., various combinations of deprotonated molecules, sodium salts and phosphate adducts).
Figure 1

(a) DriftScope display (m/z:drift time) for N-glycans from gp120. Regions enclosed by ovals are singly charged ions (1), doubly charged ions (2), and triply charged ions (3). (b) Negative ion ESI spectrum of N-glycans released from the glycoprotein gp120. (c) Negative ion ESI spectrum of low concentrations of N-glycans released from the glycoprotein gp120. (d) Mobility-extracted singly charged ions from Sample 2 (from region 1 of panel a). (e) Mobility-extracted doubly charged ions from Sample 2 (from region 2 of panel a). (f) Mobility-extracted triply charged ions from Sample 2 (from region 3 of panel a). Conventions for the structural diagrams and fragment ion labeling in this and the other figures are as in the legend to Scheme 1. Ions are labeled with the most abundant peak of the isotope cluster. Where two isomers of the triantennary glycans are detected, only one is shown because of lack of space. All detected isomers are shown in Scheme 1 and Table 1

Figure 2

(a) Negative ion CID spectrum (transfer cell) of the ion at m/z 1721 from the sample of gp120 glycans whose ESI spectrum is shown in Figure 1d. The inset shows the ATD profile (red trace) and two diagnostic ions from each of the two main constituents. The horizontal lines show the regions that were extracted to produce the spectra shown in panels b and c. (b) Mobility-extracted CID spectrum of the phosphate adduct of the biantennary glycans Gal1Man3GlcNAc4Fuc1 (29, 30, region b from the inset above). (c) Mobility-extracted CID spectrum of the CID spectrum of the 2,4A6 fragment ion (m/z 1720) from the high mannose glycan Man9GlcNAc2 (3, region c from the inset above). (d) Mobility-extracted CID spectrum of the phosphate adduct of the biantennary glycans Gal1Man3GlcNAc4Fuc1 (29, 30) from the spectrum shown in Figure 1b (high concentration sample). (e) Mobility-extracted CID spectrum of the CID spectrum of the 2,4A6 fragment ion (m/z 1720) from the high mannose glycan Man9GlcNAc2 (3) from the spectrum shown in Figure 1b

Table 1

Masses, Compositions, Occurrence, and Structures of the Measured Ions from gp120

Mass1

m/z 2

Charge

Ion3

Sample

Composition

Structure4

1

2

Hex

HexNAc

Fuc

Neu5Ac

910.3

1007.3

1

a

+

+

3

2

0

0

1

1072.4

1169.4

1

a

+

+

4

2

0

0

2

1234.4

1331.4

1

a

+

+

5

2

0

0

3

1239.5

1356.4

1

a

+

+

3

3

1

0

4

1275.5

1372.5

1

a

+

+

4

3

0

0

5

1316.5

1413.5

1

a

+

3

4

0

0

6

1396.5

1493.5

1

a

+

+

6

2

0

0

7

1421.5

1518.5

1

a

+

4

3

1

0

8, 9

1437.5

1534.5

1

a

+

+

5

3

0

0

10, 11

1727.6

1

b

+

+

1

12

1462.5

1559.5

1

a

+

+

3

4

1

0

13

1478.5

1575.5

1

a

+

+

4

4

0

0

14, 15

1768.6

1

b

+

+

1

16, 17

1519.6

1616.5

1

a

+

3

5

0

0

18

929.8

1

c

+

1558.5

1655.5

1

a

+

+

7

2

0

0

19, 20

 

876.2

2

c

+

+

1583.6

1680.6

1

a

+

+

5

3

1

0

21, 22

1873.7

1

b

+

+

1

23

1599.6

1696.5

1

a

+

+

6

3

0

0

24

1889.7

1

b

+

+

1

25

1640.6

1737.6

1

a

+

+

5

4

0

0

26

1930.8

1

b

+

+

1

27

1110.4

2

f

+

+

2

28

1624.6

1721.6

1

a

+

+

4

4

1

0

29, 30

1914.7

1

b

+

+

1

31, 32

1665.6

1762.6

1

a

+

+

3

5

1

0

33

1720.6

1817.6

1

a

+

+

8

2

0

0

34

957.3

2

c

+

+

1745.6

1842.6

1

a

+

+

6

3

1

0

35

2035.8

1

b

+

1

36

1066.3

2

d

+

+

1077.2

2

e

+

+

1786.7

1883.6

1

a

+

+

5

4

1

0

37

2076.7

1

b

+

+

1

38

1086.9

2

d

+

+

1183.4

2

f

+

+

2

39

1243.4

2

e

+

+

1827.7

1924.6

1

a

+

4

5

1

0

40, 41

2117.8

1

b

+

+

1

42, 43

1868.7

1965.7

1

a

+

+

3

6

1

0

- 5

1882.6

1979.6

1

a

+

+

9

2

0

0

44

1038.3

2

c

+

+

1989.7

2086.8

1

a

+

5

5

1

0

45

2279.8

1

b

+

1

46, 47

1188.4

2

d

+

+

1285.0

2

f

+

2

48

2005.7

1196.4

2

d

+

+

6

5

0

1

49, 50

1207.4

2

e

+

+

1218.3

2

g

+

+

1292.9

2

f

+

+

2

51, 52

1303.9

2

e

+

+

1438.5

2

f

+

3

53, 54

958.7

3

j

+

2030.8

2127.7

1

a

+

4

6

1

0

- 5

2320.8

1

b

+

+

1

- 5

2151.8

2248.8

1

a

+

+

6

5

1

0

55, 56

2441.9

1

b

+

+

1

57, 58

1269.4

2

d

+

+

1329.4

2

h

+

+

1366.0

2

f

+

+

2

59, 60

1426.0

2

e

+

+

1523.0

2

i

+

+

3

61, 62

1007.4

3

j

+

+

2192.8

2289.8

1

a

+

5

6

1

0

- 5

2482.9

1

b

+

1

- 5

2354.9

1371.1

2

d

+

+

6

6

1

1

- 5

1467.5

2

f

+

+

2

- 5

2370.9

1379.0

2

d

+

+

7

6

0

1

63

1475.5

2

f

+

+

2

64

1621.0

2

f

+

+

3

65

2516.9

1452.0

2

d

+

+

7

6

1

1

66

1512.0

2

h

+

+

1548.5

2

f

+

+

2

67

1608.5

2

e

+

+

1064.7

3

k

+

+

1694.1

2

f

+

+

3

68

1705.1

2

i

+

+

1754.1

2

e

+

+

1765.1

2

g

+

+

1129.1

3

j

+

+

1839.6

2

f

+

+

4

69

1850.6

2

i

+

+

1226.1

3

j

+

+

1233.4

3

l

+

2882.0

1634.6

2

d

+

+

8

7

1

1

70

1731.1

2

f

+

2

71

1876.6

2

f

+

3

72

1250.8

3

j

+

1347.8

3

j

1

+

4

73

3247.2

1372.5

3

j

1

+

9

8

1

3

74

a Monoisotopic mass of the neutral glycan.

b m/z of measured ion.

c Ionic composition: a, [M + H2PO4]; b, [M – H]; c, [M + (H2PO4)2]2–; d, [M – H + H2PO4]2–; e [M – H2 + Na + H2PO4]2–; f, [M – H2]2–, g, [M – H3 + Na2 + H2PO4]2–; h, [M – H2 + Na + (H2PO4)2]2–; i, [M – H3 + Na]2–; j, [M – H3]3–; k, [M – H2 + H2PO4]3–; l, [M – H4 + Na]3–.

d As in Scheme 1.

e Insufficient fragmentation data to assign structures (probably bisected triantennary or tetra-antennary glycans lacking various galactose residues).

Scheme 1

Structures of the glycans identified in the gp120 samples. Symbols for the structures are: Open image in new window = GlcNAc, Open image in new window = mannose, Open image in new window = galactose, Open image in new window = fucose, Open image in new window = Neu5Ac (sialic acid). The angles of the lines joining the symbols denote the linkage positions: | = 2-link, / = 3-link, - = 4-link and \ = 6-link. Alpha bonds are shown with dashed lines and beta bonds with full lines. Further details are given in the article by Harvey et al. [66]. Fragment ions are named according to the system proposed by Domon and Costello [56]

The spectrum in Figure 1e is of the doubly charged ions extracted from region 2 of the DriftScope display (Figure 1a) from the low abundance sample, and Figure 1f is that of the triply charged ions. The glycans in the doubly and triply charged spectra were the larger high-mannose compounds as diphosphate adducts and di-, tri-, and tetra-sialylated di-, tri-, and tetra-antennary complex glycans as deprotonated ions. Ions at the m/z 1250.8 and 1347.8, not seen in earlier studies, corresponded to tri- and tetra-sialylated glycans with poly-N-acetyllactosamine extensions to their antennae (Compounds 73 and 74, Scheme 1). This analysis, therefore, allowed glycan ions that were obscured by the contamination to be visualized and, consequently, they could be fragmented in the transfer cell. However, because of the presence of the contaminating ions, in some cases, species other than the targeted glycan ions were also selected, leading to extraneous ions in the CID spectra making them difficult to interpret, particularly when the target ions were present in low abundance. Ion mobility was then used again to remove these contaminating ions as described below.

CID Spectra

Structural Information from Glycans of Low Abundance Whose Masses Coincide with Those of Other Compounds

It is clear from Figure 1a that selection for CID of the molecular ions from most of the singly charged glycans would also include several other ions from the background and from regions 2 and 3. Such a spectrum is shown in Figure 2a. The target glycan is of a mixture of the monogalactosylated biantennary glycans 29 and 30 (Scheme 1) of m/z 1721 (phosphate adducts) from the low abundance sample. The mass of the parent ions are the same as that of the first isotope peak from the 2,4A6 fragment ion of the high-mannose glycan Man9GlcNAc2 (44), and ions from both sources are present in the spectrum. After ion mobility separation, the arrival time distribution (ATD) profile of this ion is asymmetric (inset to Figure 2a), and when spectra were extracted from each side of this peak, as shown by the horizontal lines in the inset, relatively clean spectra of the target glycans, Gal1Man3GlcNAc4Fuc1 (Figure 2b) and of the fragment ion (Figure 2c) were obtained. In spectrum 2b, the ions at m/z 424 (Gal-GlcNAc-CH = CH2-O) and 262 (GlcNAc-CH = CH2-O) define the compositions of the two antennae and the distribution of the galactose residue between the antennae is defined by the D (loss of the 3-antenna and chitobiose core) and D-18 ions at m/z 526/508 (no galactose on the 6-antenna) and m/z 688/670 (galactose on the 6-antenna). Corresponding spectra from the other, more abundant sample (Figure 1b) where the Gal1Man3GlcNAc4Fuc1 glycan (29, 30) was relatively more abundant is shown in Figure 2d and e so that these features can be seen more clearly. However, it can be seen that even when the concentration of glycans such as 29 and 30 are very low and coincident with ions from other sources, it can still be possible to extract structural information using ion mobility and negative ion fragmentation.

Even with the more abundant glycans, the CID spectra still benefitted from ion mobility clean-up, as shown in Figure 3, where ion mobility has effectively removed contamination ions in the low mass region. Panel a shows the total ESI spectrum of the high-mannose glycan Man9GlcNAc2 (44), and panel b shows the mobility-extracted fragment ions from the region of the large peak in the inset to panel c (100–150 ms region). The lower panel of this inset shows the DriftScope image with the ATD profile (labeled b) in the upper panel. The main spectrum in panel c of Figure 3 is of the extracted contaminating ions (region from 0–80 ms of the DriftScope display).
Figure 3

(a) Negative ion CID spectrum (transfer fragmentation) of the ion at m/z 1979 corresponding to the phosphate adduct of the high mannose glycan, Man9GlcNAc2 (44). (b) Mobility extracted CID spectrum from the boxed region of the DriftScope plot shown below in panel c. (c) Extracted contaminating ions. The inset shows the m/z:drift time (DriftScope) display with the boxed area containing the fragment ions from Man9GlcNAc2 (3). Section b of the inset shows the corresponding ATD plot

Use of Ion Mobility to Separate Isobaric Compounds

Figure 4 shows the CID spectrum of the glycan(s) of mass 1534.5 from the high abundance sample having the composition Hex5GlcNAc3. Fragment ions are inconsistent with the presence of a single compound: those at m/z 647 (D), 629 (D-18), 575 (0,3A3), and 545 (2,4A3) indicate the presence of a compound with three mannose residues in the 6-antenna (compound 50), whereas the ion at m/z 424, which contains a Gal-GlcNAc moiety, leaves only two remaining mannose residues for the 6-antenna (compound 11), assuming that the molecules contained the common trimannosyl-chitobiose core. That these two compounds are present is consistent with the bimodal ATD peak (inset to Figure 4a, red trace) and their presence was confirmed by extracting the spectra from each side of the peak to give the spectra shown in Figure 4b and c. ATD plots of the mass-different diagnostic fragment ions [25, 27, 34, 59, 60, 61, 62] for these two compounds (blue and green traces for selected ions from compounds 11 and 10, respectively) maximized separately under the two peaks of the total ATD profile, further confirm the presence of these two compounds. This technique can also be used to identify the presence of isomeric high-mannose isomers and will be the subject of a future communication.
Figure 4

(a, inset) ATD plot (red trace) of the ion at m/z 1534 from gp120 together with selected diagnostic fragment ions (blue and green traces) for the two glycans shown in panels b and c respectively. (a) CID spectrum of the ion at m/z 1534. (b) Mobility extracted CID spectrum (transfer region) of the phosphate adduct of the hybrid glycan 10. (c) Mobility extracted CID spectrum (transfer region) of the phosphate adduct of the hybrid glycan 11

Use of Ion Mobility to Extract Spectra of Multiple Glycans When CID Selects Ions of Different Composition but with the Same m/z Value

A common problem with acquisition of CID spectra from constituents in mixtures, particularly when they are present at low abundance, is the presence of several species exhibiting the same m/z value, all of which contribute fragment ions to the CID spectrum. Even the spectra of relatively abundant species can also be affected by this problem. For example, the CID spectrum of the ion at m/z 1007 (Figure 5a) from the high abundance HIV sample, targeted for the phosphate adduct (singly charged) of the glycan Man3GlcNAc2 (1), contains many additional ions, particularly above the mass of the parent ion, making the spectrum difficult to interpret. The Driftscope analysis (Figure 6) shows the presence of at least three compounds that fragment in the transfer region of the instrument (regions labeled 1–3 in Figure 6) and several compounds that have fragmented prior to mobility separation (regions 4, 5, and 6). The spectra of these regions are shown in Figure 5b–g, respectively. A clean spectrum of the target compound, Man3GlcNAc2 (1), was extracted from region 1 and is shown in Spectrum 5b. Spectrum 5d from region 3 is from the triply charged ion from the glycan of composition Hex6HexNAc5dHex1Neu5Ac3 (61, 62). The major fragment ion at m/z 290 in this spectrum is the B1 ion consisting of sialic acid, and the singly charged ion at m/z 2441.8 is formed by loss of two sialic acids. The ion at m/z 2340.8 is the corresponding 0,2A7 ion, typical for compounds of this type. The rather poor spectrum is probably the result of the collision energy (transfer voltage 66.7 V) being set to be appropriate for the targeted singly charged ion of Man3GlcNAc2 (Spectrum 5b) and which is too high from optimal fragmentation of the triply charged species and has caused extensive loss of two sialic acids. A voltage of 55 V would be more appropriate for the full spectrum to be recovered as demonstrated by a spectrum from a parallel sample (inset to Figure 5d) recorded with this voltage. The virtual absence of the doubly charged ions at m/z 1366 and 1315, representing loss of one sialic acid and formation of the corresponding 0,2A7 ion after ion selection and before mobility separation, produces the spectrum in region 5 (Figure 5f). The spectrum in region 6 (Figure 5g) is that from the loss of two sialic acids (m/z 2241). Losses of sialic acid by fragmentation within the instrument and, presumably, also in the ion source region questions the quantitative relationship between the various sialylated species shown in the MS spectra (Figure 1d and e) and suggests that stabilization of the sialic acids by methylation [63, 64] or amidation [65] or analysis by HPLC should be employed to obtain this information.
Figure 5

(a) Negative ion CID spectrum of the ion at m/z 1007 from gp120. (b) Mobility-extracted CID spectrum (singly charged) of the trimannosyl-chitobiose glycan 1 from region 1 of the drift time:m/z (DriftScope) plot shown in Figure 6. Spectra of the regions 2–6 of the DriftScope plot are shown in panels (c)(g), respectively. The inset to panel (d) shows the CID spectrum of the tri-sialylated triantennary glycans 61 , 62 recorded with a collision cell voltage of 55 V

Figure 6

(a) DriftsScope display (m/z:drift time) of transfer region CID fragments from m/z 1007 following mobility separation. (b) Corresponding ATD plot. The peak from the Man3GlcNAc2 glycan (1) is labeled

The spectrum in Figure 5c (region 2) is of a doubly charged ion. The fragments are singly charged with prominent peaks at m/z 1558, 1498, and 1355 corresponding to the 2,4A6, B5, and 2,4A5 ions from Man8GlcNAc2 (34). A triphosphate adduct (doubly charged) from this compound would have a mass of m/z 1006.25, which would fall within the 4 m/z mass window used to select m/z 1007 for fragmentation. The unstable nature of this species is possibly reflected by the prominent ion at m/z 1817 (singly charged) in Figure 5e from region 4 of Figure 6, which corresponds to the monophosphate adduct of this high-mannose glycan.

Conclusion

From the above results, it can be seen that ion mobility can be used in a number of ways to aid structural determination of N-glycans, particularly when they occur at low concentration or in contaminated samples. Extraction of ions in different charge states can often eliminate contamination such as polyethylene glycol [10] and can reveal the presence of minor compounds whose molecular ions are masked by ions from major compounds in different charge states but with different m/z values.

CID spectra can also be improved. Simply selecting an ion for CID analysis from complex mixtures can lead to contaminated spectra. Much of this contamination can be removed by the additional stage of ion mobility. Clearly, there are other methods, such as HPLC, of cleaning samples prior to CID but, in the absence of such techniques, for example when MALDI is used as the ionization technique, ion mobility provides a simple method for achieving similar results. Less contamination would be introduced into the MS/MS spectra if the ion selection window is reduced to a width of only 1 Da. However, we have frequently found with samples such as the ones analyzed here, it is advantageous to use a wider window so that isotopic peaks are displayed in order to aid identification of other adducts of the glycans, such as the chloride adduct. Although we routinely add phosphate to the sample in an attempt to form only the phosphate adducts, the final spectrum will depend to a large extent on the original composition of the sample. Furthermore, using a narrower mass selection window often compromises sensitivity, and maximum sensitivity is normally required in the analysis of glycans derived from gel-separated glycoproteins. Our philosophy is to use a minimum of pre-MS clean-up in order to minimize sample losses and to use the mass spectrometer at its full potential. The extra dimension provided by ion mobility is invaluable in this context.

Thirdly, the presence of isobaric and isomeric compounds can be revealed by plotting the ATD profiles of diagnostic fragment ions. These plots frequently reveal slightly different drift times from the constituents in cases where the overall ATD profile only suggests their presence by slight asymmetry or broadening. Negative ion spectra are particularly appropriate because they usually contain diagnostic ions that differ in mass between the isobaric compounds.

Finally, although not discussed here, ion mobility data can be described by cross section values that provide an additional physical property that can assist compound identification and which can be incorporated into databases for more automated analyses of complex glycan profiles. Clearly, therefore, ion mobility provides extra dimensions of analysis and should help considerably in the future analyses of N-glycans, particularly when sample amounts are limited.

Notes

Acknowledgments

The authors thank Professor Raymond Dwek, FRS, for his continuing help and support and the Oxford Glycobiology Bequest for funding. This work was supported by a grant from an International AIDS Vaccine Initiative Neutralizing Antibody Center CAVD grant (Glycan characterization and Outer Domain glycoform design) and the Scripps CHAVI-ID (1UM1AI100663).

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Copyright information

© American Society for Mass Spectrometry 2015

Authors and Affiliations

  • David J. Harvey
    • 1
    • 2
  • Max Crispin
    • 1
  • Camille Bonomelli
    • 1
  • Jim H. Scrivens
    • 2
  1. 1.Oxford Glycobiology Institute, Department of BiochemistryUniversity of OxfordOxfordUK
  2. 2.Department of Biological SciencesUniversity of WarwickCoventryUK

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