Fragmentation of N-Linked Carbohydrates with Q-Trap and Tof-Tof Mass Spectrometers
Summary
1. N-Linked carbohydrates from well-characterized glycoproteins were examined
by nanospray ionization with Q-Trap mass spectrometer and by MALDI with
a Tof-Tof instrument.
2. The neutral carbohydrates produced mainly [M + Na]+ ions on the Q-Trap
instrument. Sialylated glycans produced [M – nH]n- ions.
3. Fragmentation spectra obtained from the [M + Na]+ ions in both the quadrupole
collision cell and the quadrupole ion trap were very similar and did not show
the limited mass range experienced by 3D-ion traps.
David J. Harvey1; Jane Thomas-Oates2; Jerry Thomas2; Dietmar Waidelich3, Laura Main4, Christof Lenz3, Martin Hornshaw4
1University of Oxford, Oxford, UK; 2University of York, York, UK; 3Applied Biosystems, Darmstadt, Germany, 4Applied Biosystems, Warrington, UK.
Results and Discussion
Q-Trap
Nanospray spectra of the N-linked glycans with sodium acetate added to the
solvent produced mainly [M + Na]+ and [M + 2Na]2+ ions (Figure 1). As with
spectra recorded by electrospray, the signal strength of the [M + Na]+ ions fell
with increasing mass to a much greater extent than that observed by MALDI.
The signals from the doubly charged ions, on the other hand, did not show
such a pronounced fall.
MS3 Spectra
MS3 spectra were obtained with excellent signal:noise ratios from the quadrupole ion
trap without the mass-range limitations imposed by 3D-ion traps. Figures 3 and 4
show two examples from fragments of the glycan whose MS2 spectrum is shown in
Figure 1.
Negative ion spectra
Sialylated glycans produced strong negative ion spectra due to proton abstraction,
mainly from the sialic acids. Most ions were multiply charged because of the ease
with which the acidic protons could be extracted (Figure 5).
Tof-Tof High energy spectra
Spectra were obtained with the Tof-Tof instrument using MALDI to produce [M +
Na]+ ions with little sign of fragmentation as illustrated by N-linked glycans from
chicken egg white glycoproteins (mainly ovalbumin, Figure 7)
Figure 9 shows the high-energy CID spectrum of a biantennary glycan; The 1,5X ions
are produced from each sugar ring and define the sequence and branching. The
branch point is indicated by the large mass unit gap between the 1,5X2 and 1,5X3 ions.
4. Strong MS3 spectra were obtained from the quadrupole trap from fragment ions
produced in the first collision cell.
6. Fragmentation of the negative ions from sialylated carbohydrates gave mainly
singly charged fragment ions produced by more specific processes that
could be rationalized by initial abstraction of protons from various hydroxyl
groups.
Strong spectra were produced by MALDI with the Tof-Tof instrument with [M
+ Na]+ ions being the major products. Fragmentation, with argon in the
collision cell, gave high-energy-type spectra that contained abundant crossring cleavage products, particularly X-type fragments. The strong 1,5X ions
were formed from most of the constituent sugar rings and defined the
sequence and branching of the glycans.
7. The Tof-Tof instrument produced [M + Na]+ ions by MALDI from the
carbohydrates with little sign of in-source fragmentation.
8. Unlike the low-energy spectra produced by Q-Tof instruments, the spectra were
dominated by ions produced by cross-ring cleavages.
Figure 5: Negative ion nanospray spectrum of sialylated N-linked glycans
from bovine fetuin. Open diamond = galactose, star = Neu5Ac
9. The major cross-ring cleavage ions were X-type with the 1,5A ions produced
from all constituent sugar rings; these ions are normally absent from lowenergy spectra.
Figure 1: Positive ion nanospray spectrum of N-linked glycans from
chicken egg glycoproteins (mainly ovalbumin) recorded with the Q-Trap
mass spectrometer. Open circle – mannose, filled square = GlcNAc.
Figure 3: MS3 spectrum of the ion at m/z 1239.4 (B3 plus B4/Y3γ and B4/Y4β) from
the spectrum shown in Figure 2.
Introduction
Na]+
Li]+
Fragmentation of the [M +
and [M +
ions from N-linked carbohydrates
by collision-induced dissociation (CID) produces mainly glycosidic fragments
yielding sequence and some branching information, together with some weaker
cross-ring fragmentation providing information on linkage. Modern
instrumentation can provide additional dimensions to these analyses, such as
the ability to perform successive stages of fragmentation, the so-called MSn
technique, and to record CID spectra over a wide range of energies. This paper
investigates the utility of both of these techniques to reveal details of the
structures of N-linked carbohydrates using a Q-Trap for MSn experiments and a
Tof-Tof instrument to provide the high-energy collisions necessary to accentuate
the abundance of the cross-ring fragments.
Neutral N-glycans ionized by nanospray gave both singly and doubly charged
ions but with some drop in sensitivity at higher masses. The major ions in the
presence of sodium acetate added to the electrospray solvent were [M +
Na]+. Fragment ions were obtained in both the collision cell and quadrupole
ion trap and were found to be very similar. The major fragments were B- and
Y-type glycosidic cleavages with small amounts of cross-ring fragments
defining linkage. MS3 fragmentation spectra were obtained with a good
signal:noise ratio in the quadrupole ion trap from fragments generated in the
collision cell. These spectra did not show the limited mass range experienced
by 3D-ion traps. They showed that many of the glycosidic fragments in the
positive ion spectra were formed by several pathways.
Sialylated glycans gave strong negative ion spectra and formed multiply
charged ions of general formula [M – nH]n- with proton loss mainly from the
acidic groups. Fragmentation gave mainly singly charged ions and was much
more specific than fragmentation of the positive ions because fragmentation
was initiated by proton loss from individual acid and hydroxy groups. Several
specific, diagnostically useful fragment ions were formed.
5. MS3 Spectra of the N-linked glycans showed that most of the fragment ions
derived from [M + Na]+ precursors were produced by multiple pathways.
10. Other ions not seen in low-energy spectra were C-2 fragments and low-mass
ions containing hydrogen rather than sodium as the ionizing adduct.
Conclusions
Fragmentation of the [M + Na]+ ions in the collision cell produced spectra that
were very similar to low-resolution spectra recorded with other instruments.
Spectra were dominated by ions produced by glycosidic B- and Y-type cleavages
(Domon and Costello [1] nomenclature) with only weak cross-ring cleavage
products defining linkage (Figure 2). Many of the ions appeared to be products of
several fragmentation pathways, such as losses of GlcNAc from the B4 ion that
could originate from one of three positions. Consequently, additional information
is required in order to gain unambiguous structural data. Such information can be
acquired by, for example, MSn scans, negative ion fragmentation spectra or by
producing fragmentation under higher energy conditions.
It is apparent by the similarity in the fragment ions between fragmentation of the
B4 ion, for example and the molecular ion that multiple pathways produce many of
the fragments. However, the signal:noise ratio was improved over that of the MS2
spectrum allowing the weaker cross-ring cleavage ions, such at that at m/z 599.2,
to be observed clearly.
Fragmentation of these multiply charged ions gave mainly singly charged fragment
ions due to loss of charged sialic acid residues. The spectra differed from the
positive ion spectra in that they contained mainly cross-ring fragments and C-type
glycosidic ions rather than the B- and Y-fragments that were typical of the positive
ion spectra. Production of the fragment ions could be rationalized by proton
abstraction from various acid and hydroxyl groups. Most fragment ions were,
therefore produced by only one pathway. Consequently, many ions were diagnostic
for specific structural features. Thus, formation of the 2,4A ions (Figure 6) could by
rationalized by initial proton abstraction from a 3-OH group (Scheme 1).
Figure 7: MALDI Spectrum of N-glycans obtained with the Tof-Tof mass
spectrometer
The high energy CID spectra of the N-linked glycans (Figure 8a) contained more
cross-ring cleavage fragments than the corresponding low energy spectra (Figure
8b), as reported by others [2,3]. In particular, X-type cleavage ions were abundant.
The 1,5X ions provided information on chain sequence and branching. The ion
labelled D contains four mannose residues consisting of the three from the 6antenna and the core mannose. It was, thus, specific for the composition of the 6antenna.
Figure 9: Positive ion high-energy CID spectrum of the [M + Na]+ ion from
the complex N-linked glycan (Gal)2(Man)3(GlcNAc)4(Fuc)1 recorded with a
Tof/Tof mass spectrometer, Open diamond with spot = fucose.
These results demonstrate the additional information that can be obtained
from fragmentation of N-linked glycans using features available with the new
instrumentation that was not obtainable from simple single-stage
fragmentation under low energy conditions. The MS3 experiments allow
mechanistic studies to be performed on the diagnostic fragments and
fragmentation studies to be carried out on small fragments cleaved from the
parent molecules. The use of negative ions gives very specific fragmentation
and details of the fine structure of the glycans. The use of high energy to
fragment the compounds generate very abundant cross-ring fragments that
provide important information of sequence, branching and linkage.
References
Figure 10 shows the spectrum of a truncated complex N-linked glycan from
chicken egg white glycoproteins (see Figure 7). 1,5X ions define the sequence
and branching. The bisecting GlcNAc residue is revealed by the ion formed
by loss of 221 mass units (GlcNAc) from Ion D as observed earlier for highenergy spectra recorded with a magnetic sector mass spectrometer [4].
1.
2.
3.
Methods
4.
Materials
N-linked glycans were released from the well-characterised glycoproteins
ribonuclease B, porcine thyroglobulin, chicken ovalbumin, bovine fetuin, and
human α1-acid glycoprotein with hydrazine.
Mass spectrometry
Q-Trap
MS, MS2 and MS3 spectra experiments were performed on a 4000 Q-Trap
(Applied Biosystems/MDS Sciex) instrument equipped with a nanospray ion
source (800 V) and medium nanospray needles (Protana). Samples were
sprayed from water:methanol (1:1, v:v). Positive ion spectra were obtained with
added sodium acetate. Nitrogen was used as both curtain and collision gas.
MS2 spectra were acquired from N-linked glycans at 1000 amu/sec. MS2 and
MS3 collision and excitation energies were set to be appropriate for the mass of
the compounds. Q1 resolution was low to allow transmission of the isotopic
envelope from each ion. The declustering potential (30) was low to prevent insource decay.
Domon, B.; Costello, C. E. A systematic nomenclature for carbohydrate
fragmentations in FAB-MS/MS spectra of glycoconjugates
Glycoconjugate J. 1988, 5, 397-409.
Mechref, Y.; Novotny, M. V.; Krishnan, C. Structural characterization of
oligosaccharides using MALDI-TOF/TOF tandem mass spectrometry
Anal. Chem. 2003, 75, 4895-4903.
Stephens, E.; Maslen, S. L.; Green, L. G.; Williams, D. H.
Fragmentation characteristics of neutral N-linked glycans using a
MALDI-TOF/TOF tandem mass spectrometer Anal. Chem. 2004, 76,
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Harvey, D. J.; Bateman, R. H.; Green, M. R. High-energy collisioninduced fragmentation of complex oligosaccharides ionized by matrixassisted laser desorption/ionization mass spectrometry J. Mass
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Acknowledgements
Figure 6: Negative ion MS2 spectrum of the disialylated biantennary glycan from
bovine fetuin (m/z 1110.5, [M – 2H]2-).
I thank Professor R. A. Dwek, Director of the Oxford Glycobiology
Institute for help and encouragement and the Wellcome Trust for a grant
to purchase the Q-Tof mass spectrometer.
A copy of this poster is on the Glycobiology web site at:
Tof-Tof
High energy CID was performed with a 4700 Tof-Tof system (Applied
Biosystems) with MALDI (DHB) sample introduction and argon as the collision
gas. One and 2 KeV collision spectra were recorded.
Figure 2: Positive ion CID spectrum of a hybrid N-linked glycan obtained
from the collision cell of the Q-Trap mass spectrometer.
Figure 4: MS3 spectrum of the ion at m/z 833.3 (B3Y3γY4β) from the spectrum
shown in Figure 2.
Scheme 1: Fragmentation producing 2,4A ions in negative ion spectra.
Figure 8: (a) Positive ion high-energy CID spectrum of the [M + Na]+ ion
from the high-mannose N-linked glycan (Man)5(GlcNAc)2 recorded with a
Tof-Tof mass spectrometer; (b) low energy spectrum recorded with a Q-Tof.
Figure 10: Positive ion high-energy CID spectrum of the [M + Na]+ ion from
the truncated complex N-linked glycan (Man)5(GlcNAc)2 recorded with a
Tof-Tof mass spectrometer
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