COVER STORY

COMPLEX
CARBOHYDRATES
Improvements in methods, standards, and software are still needed to
move MASS SPEC ANALYSIS of carbohydrates into the mainstream
CELIA HENRY ARNAUD, C&EN WASHINGTON

CARBOHYDRATES ARE UBIQUITOUS.

They densely carpet cell surfaces. They
are the main component of the extracel-
lular matrix that surrounds cells. They
are involved in many aspects of cell-cell
communication.
Glycans, a type of carbohydrate, are
“the first thing a signaling molecule
encounters when it contacts a
cell,” says Robert J. Linhardt,
a biochemist at Rensselaer
Polytechnic Institute who
studies carbohydrate
structure and func-
tion. “They’re the
first thing a virus
encounters when
it infects a cell.” As
many as 80% of hu-
man proteins are thought
to be decorated with carbo-
hydrates, he notes.
Despite the importance of car-
bohydrates, characterization methods
for them have lagged behind those for their
fellow biopolymers, nucleic acids, and pro-
teins. In mass spectrometric analyses of
glycosylated proteins, scientists often strip
the carbohydrates away and focus on the
protein. Even when they try to zero in on
the carbohydrates, researchers often must
be satisfied with incomplete information
that tells them composition but not how
those carbohydrates are put together.
COMPLEX MIXTURE The structure of N-linked glycans, a complex mixture of
COU RTESY OF CARLITO LEBRILLA

Researchers are coming to realize that

they can no longer ignore carbohydrates.
And if they want to determine the relation-
ship between structure and function, they
need more than a cartoon understanding
of carbohydrate structure.
Mass spectrometry—alone and in com-
bination with liquid chromatography—is
making headway in providing that de-
tailed understanding. Improved analytical
methods are providing information on
carbohydrate structure, including pin-
oligosaccharides made from only a few types of building blocks, can be determined by
deconstructing them in mass spectrometry. Starting from the left, an oligosaccharide
is broken down into smaller pieces by removing one monosaccharide at a time. The
overlaid structure shows a fully fucosylated and sialylated tetraantennary glycan.
pointing isomers and defining linkages interpretation accessible to nonexperts.
between sugars. But two hurdles stand Many of the challenges in analyzing
in the way of their broader adoption: the carbohydrates stem from how the biopoly-
lack of standards that can make such mers are made. Nucleic acids and proteins
analyses truly quantitative and the lack of are encoded by templates. That means
software that can make data analysis and that the universe of possible sequences

CEN.ACS.ORG 12 JUNE 9, 2014

is already known and circumscribed. As a
result, the sequence of a gene reveals the
sequence of the protein it encodes. Carbo-
hydrates have no such template.
Carbohydrate synthesis starts in the
endoplasmic reticulum, a cellular structure
studded with ribosomes where proteins are
made. Branched chains of sugars are assem-
bled on lipid-linked precursors and then
transferred to protein nitrogen atoms dur-
ing translation. These N-linked glycopro-
teins are transferred to another organelle,
the Golgi, where some sugars are trimmed
away and many others are added. Also in
the Golgi, other complex carbohydrates are
attached to protein oxygen atoms.
“What happens in the endoplasmic re-
ticulum is universal. All cells have the same
machinery,” says Yehia Mechref, an analyt-
ical chemistry professor at Texas Tech Uni-
versity who studies carbohydrates. “What
happens in the Golgi is cell-specific.” Each
cell has its own set of Golgi enzymes that
trim and customize the sugars.
“The Golgi is the least-understood or-
ganelle,” Linhardt says. “We don’t know
the control points in the Golgi that actually
determine carbohydrate structure. We do
know that it’s responsive to the envi-
ronment and the organism’s needs at
the time.”
The resulting carbohydrates are a
complex mixture. By some estimates, these
variations add up to thousands of possible
glycans. They can contain a range of mono-
saccharides, many of which are isomers
of one another. They can have different
branching patterns and dif-
possible combination to be produced,” Or-
lando says. “If you’re looking at human gly-
cans, there are only certain building blocks
available.” That limits the possible glycan
masses that could be encountered.
Carlito B. Lebrilla, a mass spectrometrist
at the University of California, Davis, agrees.
From a chemical perspective, there are of-
ten many more combinatorial possibilities
than biology actually uses. “If you look at it
from the chemical point of view, you could
make all these different combinations, but I
think biology is simpler than that,” he says.
TO ILLUSTRATE he points to immuno-
globulin G, a type of heavily glycosylated
antibody. It has approximately 70 different
glycan compositions. Each of those com-
positions might exist as about five isomers
for a total of about 350 types. The problem
is not identifying an unlimited number of
compositions and isomers, he notes—it’s
determining which glycans are attached to
particular sites.
Vernon N. Reinhold, head of the Gly-
comics Center at the University of New
Hampshire, advocates multistage tandem
mass spectrometry, so-called MSn, as the
key to determining carbohydrate sequence
and structure. He has spent more than a
decade developing and trying to convince
other mass spectrometrists of the benefits
of his approach.
The more common approach of doing
MS/MS is “totally inadequate for detail-
ing glycans,” even when coupled with LC,
Reinhold asserts. “You need to look at
every linkage and every branch, as well as
consider the stereochemistry,” he says.
Reinhold believes that “high resolution
is of little value when trying to understand
the structural isomers of linkage and
branching.” Lower resolution ion trap
mass analyzers are readily available, cost-
effective, and extremely powerful for un-
raveling structural detail, he says.
To analyze glycans, Reinhold blocks all
free hydroxyls with methyl groups, infuses
the protected glycans into the instrument
by electrospray, and selectively isolates
each glycan for activation and disassembly.
Reinhold’s team has developed a library of
the resulting fragments. Although multiple
disassembly stages are possible, it’s rare that
they need to go further than MS5, he says.
Reinhold’s method works for the N- and
O-linked glycans of proteins and lipids and
the free glycans of milk. It doesn’t work so
well for ionic glycans such as heavily sul-
fated glycosaminoglycans (GAGs). GAGs,
such as the anticoagulant heparin, are lin-
ear polysaccharides made of repeating di-
saccharide units. The disaccharides consist
of an amino sugar and a uronic sugar.
Sulfation makes GAGs chemically
challenging to deal with in a mass spec-
trometer, says Joseph Zaia, a professor of
biochemistry and associate director of the
Center for Biomedical Mass Spectrometry
at Boston University School of Medicine.
“Sulfates are very acidic. Inside a mass
spectrometer they can be labile unless you
take a lot of care. We’re still not able to pre-
cisely measure the mass of intact GAGs.”
But some headway is
G LYCOCONJUGATE J. 1988, DOI: 10.1007/BF0 10 49915

ferent linkages between the being made. I. Jonathan

sugars. They can be linked BREAKING UP The ions formed when oligosaccharides fragment in Amster, a mass spectrome-
to proteins in different a mass spectrometer are named according to which bonds are cleaved trist at the University of
ways. These subtle struc- and whether they are numbered from the reducing or nonreducing end Georgia, has teamed up
tural differences can have of the oligosaccharide. X, Y, and Z fragment ions are numbered from with Rensselaer’s Linhardt
profound effects on glycan the reducing end of the oligosaccharide. A, B, and C fragment ions are to develop methods for
function. And these subtle numbered from the nonreducing end. A and X ions involve cleavage sequencing GAGs.
differences make carbohy- across a sugar ring, whereas B, C, Y, and Z ions involve cleavage of the “GAGs have some regu-
drates a challenge to ana- bonds between the sugar rings. larity to their structure,”
lyze by mass spectrometry. Amster says. “It’s the mod-
But the situation might Nonreducing end Glycosidic bonds Reducing end ifications that are really
not be so dire, according to Y3 Y1 challenging. Most meth-
n,mX Z3 1,5X Z1 n,mX
Ron Orlando, a mass spec- 3 1 0 ods developed for protein
trometrist at the Complex HO O HO HO O HO analysis can’t be directly
Carbohydrate Research 5 adapted for getting at sites
O 4 O 0 O O
Center at the University OH O OH O OH O OH OH of sulfation.”
of Georgia. Carbohydrates OH 3 1 Amster and Linhardt
2
may not have a template, OH NHAc OH NHAc successfully sequenced the
but they are made via a de- n,mA B1 0,2A B3 n,mA GAG portion of the sim-
1 3 4
fined biosynthetic pathway. C1 C3 plest proteoglycan, a mol-
“The biosynthetic path- Ac = acetyl ecule called bikunin, which
way doesn’t allow for every is found in plasma and

CEN.ACS.ORG 13 JUNE 9, 2014


serves as a proteinase inhibitor. The math
suggested that there were hundreds of mil-
lions of possibilities for bikunin sulfation.
They were surprised at what they found:
Bikunin’s GAGs are far more regular than
they anticipated.
Amster attributes their success in se-
quencing GAGs to choosing the right frag-
mentation method. “We used some of our
more complicated electron-based methods
for sequencing, but they weren’t working,”
Amster says. “We tried the simple peptide
method of collisional dissociation. That
cracked these glycans apart.”
Improvements in fragmentation meth-
ods are also helping scientists sequence
other glycans. Especially important are
methods that result in so-called cross-ring
cleavages—cleavages within a particular
sugar ring—instead of just cleavages be-
tween rings.
“Cleavage between rings will tell you
how residues are lined up with respect to
one another on a branching structure, but
you don’t know the position of the linkages
unless you get fragmentation within the
sugar rings themselves,” says Catherine
COVER STORY
“What happens in the Golgi is cell-specific.”
E. Costello, professor of biochemistry and
director of Boston University’s Center for
Biomedical Mass Spectrometry. For gener-
ating cross-ring cleavages, electron-based
dissociation methods are particularly ef-
fective and provide extensive linkage infor-
mation in a single step, she says.
Zaia is particularly optimistic about
a specific activation method. “Electron
transfer dissociation in the negative mode
is particularly useful for glycosaminogly-
cans,” he says. “But not all commercially
available instruments will do electron dis-
sociation in the negative mode.”
Although mass spectrometry of glycans
continues to improve, many researchers
prefer to combine mass spec with separa-
tion methods. “I would rather separate
and simplify my mass spec challenge than
ignore separations and just make it a really
complicated mass spec problem,” Geor-
gia’s Orlando says.
Pauline M. Rudd, head of glycoscience
research at the National Institute for Bio-
CEN.ACS.ORG 14 JUNE 9, 2014
processing Research & Training, in Dublin,
Ireland, has been a pioneer in coupling
chromatography with mass spec for glycan
analysis. She combines enzyme digestion
with chromatography and mass spectrom-
etry. The enzymes, which remove terminal
sugars, are monosaccharide- and linkage-
specific. The enzymes plus the chromato-
graphic retention times are enough to work
out the structure of most glycans, Rudd
says. She then uses mass spec as a confir-
matory method.
Texas Tech’s Mechref is likewise devel-
oping methods to define glycan structures
on the basis of their chromatographic
retention times and their mass-to-charge
ratios. His goal is to develop glycomics
methods that use the same setup as a pro-
teomics analysis. Lack of quantitative stan-
dards that can be used to define retention
times is slowing the process down.
“People use glycans derived from
glycoproteins rather than using glycan
standards,” he says. “Synthesis of such
standards is not trivial. When you talk to quences are not nearly as useful, he says. improvements will make the technology
people doing carbohydrate synthesis, they One of the problems is that a particular accessible to more scientists. “We’d like
always say, ‘Yes, we could do it.’ But when glycan can be represented differently in to develop technologies that would be ap-
you ask about the time frame, they say it different databases. “There’s work to get to plicable by anyone at a university with a big
will take years.” a single nomenclature that everyone would core facility,” Linhardt says. “You’ve got to
Standards that could be used to opti- use,” Costello says. In the meantime, push the boundaries of what instruments
mize instrument conditions for glycan there’s a need to translate the existing no- can do, but at the same time, you’ve got
analysis could make such analyses more menclatures from one database to another. to make it accessible to the biologists and
popular. “The tuning conditions under Glycomics experts hope that continued chemists who want to do this work.” ◾
which most instruments are set up are op-
timized for peptide and protein analysis,”
Costello says.
Standard mixtures could help research-
ers determine the best parameters for
glycan analysis on particular instruments.
Researchers “can learn to set these things
up and get good glycan data, which would
be better than they would get if they ran Redefining
it under standard peptide analysis condi-
tions,” she says. UHPLC & UHPLC/MS
BUT PERHAPS the biggest hurdle to more
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software that is more adapted to glycan
analysis,” Costello says. The lack of good
data interpretation tools means that analy-
ses take much longer than desired.
Zaia’s group has written software to
interpret glycan mass spectra. “We wrote
our software GlycReSoft to assign glycan
compositions and abundances,” he says.
The first version of the software was de-
veloped for LC/MS profiling of GAGs. It
incorporates masses, retention times, and
abundances. A second version of the soft-
ware incorporates MS/MS mass spectra and
is being expanded to other types of glycans.
Improved data analysis methods could
lead to much-needed glycan spectral data-
bases. “Although there have been years of
activity, we do not have an accurate data-
base that we can take MS/MS data and just
search against,” Mechref says. Proteomics
researchers have long benefited from data- © 2014 All trademarks are the property of
Thermo Fisher Scientific Inc. and its subsidiaries.
bases of protein sequences.
Existing databases of carbohydrate se-

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