REVIEW ARTICLE
Mucin-type O-glycosylation – putting the pieces together
Pia H. Jensen, Daniel Kolarich and Nicolle H. Packer
Department of Chemistry and Biomolecular Sciences, Faculty of Science, Biomolecular Frontiers Research Centre, Macquarie University,
Sydney, Australia
Introduction
Protein glycosylation is known to be involved in cellu-
lar targeting and secretion [1]. It can also help to regu-
late enzymatic activity, confer enhanced stability and
solubility to secreted proteins, and affect the function-
ality of proteins in the immune system. Moreover, gly-
coproteins participate in cell–cell and cell–matrix
interactions, and mediate complex developmental func-
tions [2]. Glycosylation is one of the major types of
post-translational modification that proteins can
undergo. In fact, 13 different monosaccharides and
eight amino acids have been reported across species to
be involved in glycoprotein linkages [3]. The two major
types of oligosaccharide attachment to the protein are
referred to as N-linked and O-linked glycosylation.
N-linked oligosaccharides are usually attached via a
GlcNAc linkage to Asn in the consensus sequence
NXT ⁄ S (C) (X „ P). O-linked oligosaccharides, how-
ever, can be variously attached to Ser or Thr via
O-linkages to fucose, Glc, mannose, xylose and other
sugars, as well as to the most commonly found mucin-
type O-linked a-GalNAc. Note that the single O-linked
b-GlcNAc attached to the hydroxyl group of Ser
and ⁄ or Thr, and has been found to be a cytoplasmic
signalling modification, similar to phosphorylation
Keywords

attachment site.
Abbreviations
CID, collision-induced dissociation; CR, charge-reduced; ECD, electron capture dissociation; ETD, electron transfer dissociation; GalNAc,
N-acetylgalactosamine; HexNAc, N-acetlyhexosamine; O-GlcNAc, O-linked GlcNAc.
FEBS Journal 277 (2010) 81–94 ª 2009 The Authors Journal compilation ª 2009 FEBS 81
[4,5]. We mention this linkage here because it may be
mistaken, by scientists new to the field, as a mucin-
type glycosylation, because of its equivalent mass
[N-acetlyhexosamine (HexNAc)].
The transfer of GalNAc from UDP-GalNAc to Ser
or Thr is catalysed by polypeptide N-acetyl-a-d-galac-
tosaminyltransferases [6–8]. These enzymes are sequen-
tially and functionally conserved across species [9,10],
as well as being differentially expressed over tissue and
time, suggesting complex and strict regulation. There
are up to 20 different known isoforms of polypeptide
N-acetyl-a-d-galactosaminyltransferases. They are dif-
ferentially expressed, and many have clear specificities
for the sites of attachment of the GalNAc to Ser ⁄ Thr.
This diversity determines the density and site occu-
pancy of the mucin-type O-glycosylation [11,12].
Attachment of the initial GalNAc occurs in the Golgi,
to the completely folded protein, and this starts the
action of numerous glycosyltransferases that result in
the extension of the GalNAc into numerous different
O-glycan structures. The enzymes responsible for this
diversification of the O-glycans are very specific in their
activity, and their functional importance has been
reviewed [13,14]; however, it is beyond the scope of this
minireview to discuss them in detail.

[29]), and are the targets of recognition by many
tumour-specific antibodies against glycans. The biolog-
ical significance of mucin-type O-glycosylation is, how-
ever, outside the scope of this review, and the
interested reader would be well advised to consult the
recent review by Tian and Ten Hagen [14].
This minireview will focus expressly on the analytical
technologies currently available for analysis of the major
mammalian types of mucin-type O-linked GalNAc-
linked glycosylation. The content is designed to give
newcomers to this field an introduction to what can be
done, and what is still challenging, in the analysis of
these specific, heterogeneous protein modifications.
What makes O-glycan analysis
challenging?
We believe that there are a variety of reasons why
O-linked protein glycosylation has been overlooked in
analysis as compared with N-linked glycans, as
follows.
First, mucin-type O-glycosylation lacks a known
amino acid consensus sequence. In contrast to N-gly-
cosylated sites, O-glycosylated sites do not reside in a
known amino acid sequence. Several prediction tools
have been developed and improved over time [30–33],
but none of them is very satisfying. It appears that the
lack of validated site glycosylation data is the biggest
barrier to developing a useful predictor.
Second, there is no enzyme for universal O-glycan
release from the protein. System-wide analysis of
mucin-type O-glycosylation remains a challenge, owing

glycoproteomics branch, which analyses both protein
and attached glycans, but is limited to studying one or
a few proteins. The authors stress the need to develop
real global glycoproteomic analysis tools to character-
ize both N-glycosylation and O-glycosylation on all
proteins of interest. This review attempts to give an
overview of the methods currently used in what is
arguably the last frontier of glycoanalysis – mucin-type
O-glycosylation.
Screening of intact O-glycoproteins –
what we can do
Lectins and antibodies are often used for screening
and comparing the glycosylation of large sample sets
of intact proteins. This may be performed either by
histology of tissue samples [41] or on arrays of
extracted proteins [42–44]. These types of analyses are
high-throughput as well as fairly reproducible, which
is useful when multiple proteins in multiple samples
are being compared [44]. They provide a broad profil-
ing that monitors changes in many glycans on many
proteins. It is important to keep in mind, however,
that little structural data can be obtained from lectin
studies alone [45]. Jacalin is generally regarded as an
O-glycan specific lectin, but has been shown to bind
N-glycosylated proteins as well [46]. Additionally, the
specificity of lectins can be complicated by their dif-
ferent binding affinities for other glycan structures,
which will also affect data interpretation [47]. Any
structural assumptions always need to be verified by
a complementary technique [42,43]. The same limita-

present in a sample, and can greatly assist in interpret-
ing complex glycopeptide data from the same protein.
There are several techniques being used at this time to
globally release O-linked oligosaccharides.
Fig. 1. The eight different reported core structures of mucin-type
O-glycans. The linkage positions are illustrated by the line connect-
ing the monosaccharides, and all linkages not labelled with a are
b-anomers. As illustrated, many of the cores have the same mass.
P. H. Jensen et al. Mucin-type O-glycosylation
FEBS Journal 277 (2010) 81–94 ª 2009 The Authors Journal compilation ª 2009 FEBS 83
O-glycan release
As there are no specific enzymes that release all
O-linked glycans, chemical release methods need to be
used. O-glycans can be released chemically from glyco-
proteins either in solution or from samples immobilized
on a poly(vinylidene difluoride) membrane. Reductive
b-elimination performed using sodium borohydride in
potassium or sodium hydroxide releases the O-glycans
and reduces them simultaneously. This reduction of the
terminal sugar protects them from peeling reactions
(degradation of the released glycans), and is the most
commonly used release method [52]. It is advisable to
treat glycoprotein samples with N-glycosidase F before
using this method, as N-linked glycans can also be par-
tially released by reductive b-elimination conditions,
and will complicate the subsequent interpretation.
Reductive b-elimination, however, does not allow for
subsequent fluorescent or colorimetric labelling (e.g.
with 2-aminobenzamide, 1-phenyl-3-methyl-5-pyrazo-
lone, or anthranilic acid) of the reducing terminus of

separated peaks can help to elucidate the structures.
Another chromatographic material commonly used in
the separation of glycans is primary amine-bonded sil-
ica [61,65,66], and if separation of neutral and acidic
glycans is desired, cation or anion exchange is a good
choice [54,67,68]. For separation of hydrazine released,
fluorescently labelled glycans, normal-phase chroma-
tography is often used [69]. The separation of labelled
as well as non-labelled O-glycans can be monitored
either on-line via a detector (i.e. fluorescence, UV, or
MS) or off-line (often larger scale), when fractions are
collected and analysed separately.
Detection of released O-glycans
MS has become one of the preferred methods for both
N-glycan and O-glycan analysis, owing to the sensitiv-
ity and relative ease of use. MS and MS ⁄ MS analysis
can be performed with both MALDI and ESI ioniza-
tion, and there are advantages and disadvantages of
both.
For MALDI-MS analysis, glycan samples are often
separated into neutral and acidic glycans, as the two
have widely differing ionization properties. Anionic
glycans do not respond well in positive ion mode
MALDI-MS, whereas neutral glycans do not ionize as
well in negative ion mode. Many laboratories perme-
thylate the hydroxyl groups on the released glycans
prior to MS analysis. Permethylation also methylates
the carboxyl group of sialic acid, and can be used as a
means of making all glycans neutral [70]. This
approach has the added advantages of increasing the

phy, as it accomplishes isomeric separation and the
simultaneous detection of both neutral and acidic gly-
cans using a single chromatographic separation with
negative ion mode ESI-MS detection [73–76]. Table 1
gives examples of the released mammalian O-mucin-
type glycan masses and compositions that are typically
detected with this approach. The masses listed are
designed to introduce the novice glycoproteomic mass
spectrometrist to masses that correspond to common
released, reduced O-glycans detected in negative ion
mode ESI-MS. It should be emphasized that each mass
may represent several different structures with the same
given composition. In most cases, extracted ion chro-
matograms of the O-glycans separated by the graphi-
tized carbon column will indicate whether more than
one structure is present, as the isobaric isomers will
elute at different retention times.
Alternative methods of LC-ESI-MS ⁄ MS have been
used by Royle et al. [77]; in these, normal-phase chro-
matographic separation of 2-aminobenzamide-labelled
O-glycans was achieved in positive ion mode. Graphi-
tized carbon LC-ESI-MS ⁄ MS has also been used to
separate isomers of permethylated oligosaccharide aldi-
tols [78], but this approach was found to be best for
the separation of released neutral O-glycans. However,
permethylated neutral and acidic O-glycan isomeric
alditols can be successfully separated and sequenced
with high sensitivity by reversed-phase LC-ESI-
MS ⁄ MS [79].
One of the major limitations of MS analysis of gly-

these domains are rich in Ser, Thr, and Pro, which are
not the amino acids cleaved by the most commonly
used proteases, such as trypsin, Lys-C, and chymotryp-
sin. In fact, it is thought that one of the major functions
of these domains and their glycans is to protect the pro-
tein from proteolytic degradation. Often, nonspecific
proteases have to be used, such as proteinase K [84] or
pronase, either free [85] or immobilized [40]. These
enzymes have been widely used in the analysis of
N-linked glycosylation, where they produce a small
amino acid tag with the intact glycans attached.
Pronase has also been used for O-glycopeptide analysis
[40], in which nonglycosylated peptides are completely
digested and the remaining O-glycans are tagged with
four to seven amino acids. One drawback to this
Table 1. Some masses and compositions of commonly identified
mucin-type released O-linked oligosaccharide alditols. Adapted from
Thomsson et al. [137].
Commonly identified
glycan masses
a
[M–H]
Possible composition (reduced
glycans, alditol form)
587.2 (Hex)
1
(HexNAc)
2
675.2 (Hex)
1

(NeuAc)
1
1041.4 (Hex)
2
(HexNAc)
2
(deoxyhexose)
2
1186.4 (Hex)
2
(HexNAc)
2
(deoxyhexose)
1
(NeuAc)
1
1187.5 (Hex)
2
(HexNAc)
2
(deoxyhexose)
3
1331.5 (Hex)
2
(HexNAc)
2
(NeuAc)
2
1332.5 (Hex)
2

several general glycopeptide enrichment techniques,
involving different chromatographic materials, such as
Sepharose [89], boronic acid [90–92], hydrophilic liquid
interaction chromatography [93–95], and graphite [84],
whereas titanium dioxide [96] has been applied specifi-
cally for the enrichment of sialylated glycopeptides.
Enrichment of glycopeptides by oxidative hydrazide
coupling of the sugars to a solid support [97,98]
destroys the glycan, so this approach cannot be used
for subsequent analysis of the oligosaccharide struc-
tures on the glycopeptide. Similarly, methods that trim
back glycans by partial deglycosylation (by successive
incubation with exoglycosidases such as neuramini-
dase, b-galactosidase and b-N-acetylhexosaminidase, or
by chemical cleavage), or that produce glycoproteins in
cell lines that have limited glycosylation machinery,
provide a simpler protein glycosylation profile for site
analysis [99,100], but do not give any information on
the true glycosylation at each site.
Site-specific assignment
The methods currently available for determination of
the glycan heterogeneity at specific sites of attachment
of mucin-type O-glycans still have limitations. With
N-glycans, where a site consensus sequence is known
and only one or two sites are present on a tryptic pep-
tide, it is relatively straightforward to determine the
actual site of attachment. With mucin-type O-glycosyla-
tion, there are often many Ser and Thr residues in close
proximity within the glycopeptide that, in theory, could
all be glycosylated. Therefore, sequencing of the peptide

Edman sequencing can be used in two different ways to
determine glycosylation sites. A regular protein Edman
sequencer will sequence through a glycosylated peptide
and leave a blank cycle for each glycosylated amino
acid. Sparrow et al. [111] have exploited this, and local-
ized six O-glycosylated sites out of 10 Ser and Thr resi-
dues in a peptide. Intact glycoamino acids do not elute
in the nonpolar solvents used in Edman chemistry, so
immobilizing the glycopeptide on a membrane prior to
sequencing was shown to allow for the use of polar
eluting solvents and detection of glycoamino acids in a
peptide sequence [101,112–116]. This promising tech-
nique is limited by the amount of sample needed
(pmol), the need for peptide purification, the need for
sialic acid removal and, more importantly, the current
limited availability of commercial protein sequencers.
ECD/ ETD-MS
Without the attached glycan
To date, most published work has used ECD ⁄ ETD to
determine the sites of protein O-phosphorylation
Mucin-type O-glycosylation P. H. Jensen et al.
86 FEBS Journal 277 (2010) 81–94 ª 2009 The Authors Journal compilation ª 2009 FEBS
[117,118]. Studies to determine the sites of O-glycosyla-
tion on a protein have usually reduced the complexity
by removing the glycans and tagging the Ser or Thr.
This yields information about the glycosylation sites,
but gives no information about the glycan heterogene-
ity at the different sites. For example, treatment with
sodium hydroxide removes O-glycans, leaving dehydro-
alanine in place of the modified Ser, and dehydrobu-

this fragmentation technique in the analysis of mucin-
type O-glycosylation. Mirgorodskaya et al. (1999) [105]
identified multiple O-glycan sites in several synthetic
peptides with ECD. Haselmann et al. (2001) later
assigned multiple O-linked sites occupied by both neu-
tral and acidic glycans on an MUC1 peptide with
known sequence [123]. Kjeldsen et al. (2003) [110]
located several O-glycosylated sites on bovine milk
protein PP3, the sequence and sites for which were
mostly known. Later, Renfrow et al. (2007) identified
several mucin-type glycosylation sites on an IgA pep-
tide after removal of acidic glycans. They experienced
some difficulties in ECD fragmentation around the
glycosylated sites, and speculated that it was the glycan
itself that obstructed fragmentation [124]. This was
previously also suggested by Hakansson et al. (2001)
[125]. Alternatively, they suggest that it may be the
structure of the gas-phase ion that inhibits fragmenta-
tion [126], owing to either the glycosylation or a high
level of Pro in the peptide. Recently, Sihlbom et al.
(2009) [127] analysed the site-specific glycosylation in
recombinant MUC1 by nanoLC-ECD-MS ⁄ MS. The
peptide analysed contained only one GalNAc per site,
and ECD successfully assigned one to five sites in the
known peptide. Even with a single GalNAc substitu-
ent, many different glycoforms of the peptide were
identified. The authors observed that low-abundance
glycoforms may have been missed, because the sensi-
tivity of the technique is quite low. ECD fragmenta-
tion is thus able to determine glycosylation sites and

together in ETD mode. ETD sequencing of a known
glycopeptide with one O-glycosylation site [133] and
on an O-GlcNAc-substituted glycopeptide with up to
eight charged ions (H
+
) [118,134] has been successful,
but this analysis also required the sequence of the
peptide to be known.
P. H. Jensen et al. Mucin-type O-glycosylation
FEBS Journal 277 (2010) 81–94 ª 2009 The Authors Journal compilation ª 2009 FEBS 87
The difficulty of site-specific analysis by ETD ⁄ ion
trap MS is shown in the analysis of multiply O-man-
nosylated peptides from human a-dystroglycan [135],
which demonstrates the huge heterogeneity that exists in
the glycosylation of these mucin-like domains. Recently,
Perdivara et al. (2009) [136] successfully performed
ETD on O-linked glycopeptides containing one and two
glycosylation sites with both neutral and acidic glycans
attached. This is the first study to actually perform
de novo site characterization of O-glycosylated peptides.
Conclusion
Commonly, either the analysis of the O-glycosylation on
a protein has been largely overlooked, or the glycans
have been removed, trimmed or desialylated to facilitate
analysis. We believe that if conclusions are to be drawn
about protein function, or if O-linked glycoprotein bio-
markers are to be discovered, we need to characterize
the complete O-linked glycoprotein, including the com-
position and structure of its O-glycans and the oligosac-
charide structural heterogeneity at each occupied amino

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