Andrew S.Weiskopf and Paul Vouros, Northeastern
University, Boston, MA
David J. Harvey, University of Oxford, Oxford, UK
The data presented here can be acquired using the Thermo Finnigan LCQ
Series of ion trap mass spectrometers.
This application report provides a brief overview of
protein oligosaccharides that present a unique analytical
challenge among the families of biomolecules. Peptides
and oligonucleotides are linear oligomers, so sequence
information is all that is necessary for complete primary
structure characterization. Oligosaccharides, on
the other hand, are branched structures, with their constituent
monosaccharides linked together by a variety
of glycosidic bonds. As a result, while six amino acids
can yield only 720 different peptides, six unique monosaccharides
can be arranged to form many millions of
oligosaccharides. Though much is understood about
carbohydrate biosynthesis to eliminate most of these
isomers from consideration, the permutations that
remain still provide a formidable analytical problem.
Electrospray ionization tandem mass spectrometry
(ESI-MS/MS) has become a valuable technique in
Permethylation of the
oligosaccharide before analysis more effectively directs
fragmentation of the ion into various familiar series of product ions: glycosidic-bond fragments which indicate
monosaccharide sequence and branching, and crossring
fragments which identify the linkages between
monosaccharides. However, interpretation is complicated
by the relatively low abundance of these crossring
fragments, and cleavage of especially labile bonds
can suppress the formation of other fragments.
capabilities of the LCQ Series present a
solution to these problems encountered with
MS/MS. The removal of monosaccharide and disaccharide
substituents by successive stages of MSn
provides a facile method for obtaining sequence information,
but can produce ion species which themselves
yield fragments of greater variety and higher relative
intensity. As a result, the LCQ Series reveals structural
details otherwise lost by traditional tandem MS, and
provides a new approach to the mass spectrometric
characterization of protein glycans.2Goal
can be used in oligosaccharide analysis to determine
monosaccharide sequence and composition, carbohydrate branching, and monosaccharide
The ability of MSn
to perform separate characterizations of
two isobaric oligosaccharides, even when infused into the LCQ Series as
an unseparated mixture.
That early stages of MSn
can remove labile substituents, producing
an ion substrate which yields previously unattainable information when subjected
to further stages of MSn
Figure 1a. Two HexNAc5Hex5 glycans released from chicken ovalbumin.
Figure 1b. GlcNAc8Man3 oligosaccharide from chicken ovalbumin.
Oligosaccharides were released from hen ovalbumin by hydrazinolysis and
were used as an unseparated mixture. The oligosaccharides were then dissolved
in a slurry of sodium hydroxide and dimethyl sulfoxide, permethylated with
methyl iodide, and extracted into chloroform per Ciucanu and Kerek.3
Ionization method: ESI
Sample introduction: direct infusion, 3-5 L/min
with LCQ syrin
Sample conditions: 50 ng/L total mixture in
50:50 MeOH/water, 1 mM sodium acetate
Capillary temperature: 200C
Needle voltage: +4.5-5 kV
Sheath gas: nitrogen, 30 unit
Collision energies: 60-65 units, except where noted
Figure 2. MS2 of m/z 1169.5, the doubly-sodiated molecular ion of N5H5-I
Figure 1a shows two oligosaccharides from the ovalbumin carbohydrate mixture.
Since galactose and mannose are both hexoses, and therefore diastereomers
of one another, both permethylated oligosaccharides have the same composition
) and molecular weight (2293 Da). With
these two oligosaccharides infused together, MS2
of their doubly-sodiated
molecular ions at m/z 1169.5 (Figure 2) produces fragments corresponding
to both N5
-I and N5
with each oligosaccharide effectively contaminating the spectrum of the
The fragment at m/z 937.7 corresponds to loss of a Hex-HexNAc disaccharide
antenna, which is a structural feature only found on N5
Therefore, selecting the m/z 937.7 ion and subjecting it to MS3
(Figure 3a) yields structural information on N55
even as N5
-II is continuously infused into the
instrument. Favorable cleavage sites lie adjacent to GlcNAc residues, particularly
terminal GlcNAcs, resulting in the preferred fragmentation of glycosidic
bonds (noted by B and Y labels) and suppression of cross-ring fragments
(A and X-type ions).4
By performing MSn
sup> on a fragment
which has already undergone these losses in a previous stage of MS (Figure
3b, m/z 1333.7), cross-ring fragment products emerge, revealing the nature
of saccharide linkages in the molecule. Similar results can be achieved
for N5H5-II by MSn of the ion at m/z 1606.8 from MS2.
Figure 3a-b. MS3 and MS4 for isolation and characterization of glycan
The oligosaccharide in Figure 1b is a heavily GlcNAcylated oligosaccharide
of ovalbumin. The MS2 spectrum of the doubly-sodiated ion (Figure
4) is dominated by ions corresponding to the loss of non-reducing terminal
GlcNAc sugars. No other information is obtainable due to the sup-pression
of other bond cleavages. By reducing the collision energies to 35 units
and using MS3-6, one can not only count terminal GlcNAc residues
in a facile manner, but can produce a species upon MS7 which
is no longer susceptible to suppression effects (Figure 5). MS8
of this ion subsequently reveals the locations of the previously attached
GlcNAc residues, indicating three acetylaminosugars on one mannose antenna,
a fourth and fifth on the other arm, and a sixth residing on the bisected
mannose of the core (Figure 6).
Figure 4. MS2 of the ovalbumin glycan GlcNAc8Man3.
The use of higher collision energies to remove two
or three residues per stage of MSn can also produce
the m/z 1087.8 ion in fewer steps. These alternate
MSn experiments yield the same structural information
by MS5 or MS6, suggesting that perhaps
l integrity of the m/z 1087.8 ion is
maintained regardless of its pathway of formation.
Figure 5. MS2-7 for stepwise removal of terminal GlcNAc residues and formation of the more stable GlcNAc2Man3(OH)6 core substrate, m/z 1087.7.
Figure 6. Determination of GlcNAc8Man3 glycan branching patterns by MS6.
Electrospray ion trap mass spectrometry is a
particularly effective method for detailed characterization
of protein oligosaccharides. The MSn
capabilities of the LCQ Series overcome the
significant limitations often encountered with
traditional triple quadrupole techniques. By
exploiting unique saccharide losses, MSn enables
the isolation and analysis of individual glycan species, even in the presence of an isobaric
contaminant. The removal of especially labile
substituents in early stages of MSn produces stable
ion substrates which, upon further MSn analysis,
yield spectra that are far more structurally informative
than those obtained by MS/MS.
1. Reinhold, V.N., Reinhold, B.B., Chan, S., Methods Enzymol., 271, 377, (1996).
2. Weiskopf, A.S.; Vouros, P.; Harvey, D.J. Rapid Commun. Mass Spectrom., 11, 1493,(1997).
3. Ciucanu, I.; Kerek, F. Carbohydr. Res., 131, 209, (1984).
4. Nomenclature system from Domon, B.; Costello C.E. Glycoconj, J. 5, 397, (1988).
Page: All 1 2 3 4 5
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