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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

electron transfer dissociation (ETD) ⁄ electron
capture dissociation (ECD); glycopeptides;
MS; mucin oligosaccharides; O-glycosylation;
released glycans; site specificity
Correspondence
N. Packer, Department of Chemistry and
Biomolecular Sciences, Faculty of Science,
Biomolecular Frontiers Research Centre,
Macquarie University, Building E8C, Room
307, Sydney, NSW, 2109, Australia
Fax: +61 2 9850 8313
Tel: +61 2 98508176
E-mail:
Website: />academics/npacker.html
(Received 12 June 2009, revised 3
September 2009, accepted 11 September
2009)
doi:10.1111/j.1742-4658.2009.07429.x
The O-glycosylation of Ser and Thr by N-acetylgalactosamine-linked
(mucin-type) oligosaccharides is often overlooked in protein analysis. Three
characteristics make O-linked glycosylation more difficult to analyse than
N-linked glycosylation, namely: (a) no amino acid consensus sequence is
known; (b) there is no universal enzyme for the release of O-glycans from
the protein backbone; and (c) the density and number of occupied sites
may be very high. For significant biological conclusions to be drawn, the
complete picture of O-linked glycosylation on a protein needs to be deter-
mined. This review specifically addresses the analytical approaches that
have been used, and the challenges remaining, in the characterization of
both the composition and structure of mucin-type O-glycans, and the
determination of the occupancy and heterogeneity at each amino acid

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.

O-glycans are known to be associated with many
known, and many yet to be defined, critical biological
functions. Alteration of mucin-type O-glycosylation
pathways in animal models leads to diverse effects,
ranging from embryonic death to developmental
defects and disease. Mutations or other factors that
specifically change or inhibit O-linked glycosylation of
proteins have been associated with a variety of differ-
ent diseases, such as familial tumoral calcinosis (hyper-
phosphataemia leading to the development of calcified
masses in soft tissues) [15,16], Tn syndrome (haemoly-
sis of a subset of haematopoietic cells, leading to
thrombocytopenia and haemolytic anaemia) [17,18],
IgA nephropathy [19–21], high-density lipoprotein
metabolism [22,23], and tumour formation and meta-
stasis [24–26]. Additionally, it has been associated
with altered immune response, mostly due to altered
adhesive properties resulting in decreased rolling on
P-selectins, E-selectins, and L-selectins [27].
Changes in O-glycosylation specifically on the high
molecular weight mucin glycoproteins have been impli-
cated in processes as varied as inflammatory responses,
angiogenesis, autoimmunity, and cancer. The mucins
are highly O-glycosylated proteins found in secretions
and mucous membranes and characterized by repeat
sequence domains that have a high frequency of Ser
and Thr residues carrying a large number of glycans in
very close proximity [28]. The mucins and their glyco-
sylation have been implicated in many types of cancers
(e.g. aberrant glycosylation of MUC1 in breast cancer

[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

to structural heterogeneity and the lack of specific
enzymatic tools comparable to N-glycosidase F or
N-glycosidase A. A general endo-N-acetylgalactosaminy l-
transferase activity has been reported [34], but the
commercially available O-glycanase has a specificity
restricted to the disaccharide sequence Gal–GalNAc
only [35], and therefore resistance to O-glycanase
should not be taken as evidence for the lack of
O-linked saccharide chains.
The third reason concerns glycan heterogeneity on
glycosylation sites. Mucin-type O-glycosylation is very
heterogeneous, and there is no general detection or
isolation method to accommodate this [36]. Several
attempts to metabolically incorporate tags on the
glycans have been successful [37,38], but have been
limited to cell culture and animal studies. Note that it
has been shown that the O-glycosylation pattern of
insect cell lines changes with alterations in culture
media [39].
Mucin-type O-glycosylation P. H. Jensen et al.
82 FEBS Journal 277 (2010) 81–94 ª 2009 The Authors Journal compilation ª 2009 FEBS
The different glycoproteomic approaches to the
characterization of glycoproteins were recently
described by Dodds et al. [40]. They divided the field
into three major parts: (a) the proteocentric branch,
which uses glycosylation as a means of enriching a
subset of glycoproteins, only to cleave off the glycan
in order to identify the proteins; (b) the glycocentric
branch, which looks only at the glycans released from
a protein or subset of proteins; and (c) the reductionist

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-

tions apply for different antibody-binding profiles,
particularly if the epitope is composed of peptide plus
glycan. Nonspecific binding of antibodies is also com-
mon [42]. It is surprising to note that the exact struc-
tural epitope recognized by the widely used diagnostic
commercial antibody against the O-glycosylated
cancer antigen CA125 (MUC-16, marker of ovarian
cancer) is not known.
MS may also be used to determine the overall glyco-
sylation profile of an intact purified glycoprotein
[48–50]. This can provide a general picture of the
different glycoforms on the protein, but yields no site
information. However, such a profile is difficult to
obtain with a highly O-glycosylated mucin-type pro-
tein, owing to its extensive glycan heterogeneity and
very large mass.
Released O-glycan analysis – what we
can do
Mucin-type O-glycans are built from eight core struc-
tures, many with the same monosaccharide residues in
different linkages (Fig. 1) [51]. Most commonly, core 1
and core 2 glycans are found in humans. Core 1 gly-
cans are small glycans that are often terminated with
sialic acid, whereas core 2 glycans have the potential
to be elaborated into larger glycans. Many of the core
structures have the same mass, and linkage analysis is
usually needed to differentiate them. The glycomic
approach of releasing and characterizing the total com-
plement of O-glycans from proteins provides informa-
tion about the heterogeneity of the glycan species

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

O-glycans, as is used for N-glycan detection and quan-
titation [53–56]. b-Elimination using hydrazine has
been explored widely in an attempt to release O-glycans
and retain the reducing end, without too much peeling
of the glycans [57–59]. A nonreductive b-elimination
method has also been described [60], and an alternative
method of releasing the glycans by b-elimination in a
mix of tetrahydroborate and tetradeuterioborate incor-
porates a deuterium label in the reduced terminus for
comparative quantitation [61]. Another approach,
using the addition of a chemical tag during b-elimina-
tion and Michael addition, yields side reactions and is
not specific for mucin-type O-glycans [62]. These label-
ling approaches are particularly useful for the fluores-
cent quantification of the released O-glycans. It is,
however, the belief of the authors that techniques
involving derivatization of the reducing terminus of
eliminated O-glycans have the potential to produce
artefacts, destroy oligosaccharide modifications, and
decrease sample yield, and that their use should there-
fore be kept to a minimum.
Separation of released O-glycans
Several different chromatographic materials have been
used to separate released, reduced O-glycans. Graphi-
tized carbon has the remarkable capacity to separate
different structural isomers of glycans that have the
same composition [61,63,64]. This separation is based
on size, linkages and ⁄ or branching, and allows a quick
comparison of a large set of samples. Exoglycosidase
digestions of the sample and ⁄ or tandem MS of the

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

mass of the smaller O-glycans and stabilizing the sialic
acids against loss for MALDI analysis, as well as
directing the fragmentation of the glycans in MS ⁄ MS.
Disadvantages are the increased sample manipulation
and the possible loss of any modifications that may be
present on the glycans, such as acetylation, sulfation,
and phosphorylation, owing to the conditions of deriv-
atization.
Dihydroxybenzoic acid is the most commonly used
matrix, and has been used in both negative and posi-
tive ion mode MALDI-MS [54,65,67,68,71]. Other
studies have used 3-aminoquinoline [68], dihydroxyace-
tophenone [61] and ammonium citrate [54] matrix in
the analysis of acidic glycans in negative ion mode.
MALDI-MS is often used as a global glycan profiling
technique, but unless the isomers are fractionated
off-line, the approach does not give information on
the possible compositional isomers, as they have the
same m ⁄ z. In general, O-linked glycans are smaller and
more diverse structures than N-linked glycans, and in
MALDI-MS, where the matrix produces a lot of noise
Mucin-type O-glycosylation P. H. Jensen et al.
84 FEBS Journal 277 (2010) 81–94 ª 2009 The Authors Journal compilation ª 2009 FEBS
in the low-mass range, detection of the smaller O-gly-
cans may be difficult.
Released O-glycans can also be analysed by ESI-MS
and MS⁄ MS, and this can result in specific diagnostic
ions for specific structures [72]. This MS is often cou-
pled with on-line LC separation. The authors favour
this approach, using graphitized carbon chromatogra-

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-

can samples is that different component monosaccha-
rides have the same mass. Hexoses such as Glc,
galactose and mannose all have the same mass, and it
is still only possible to determine the monosaccharide
composition by acid hydrolysis of the oligosaccharides
and separation by high-performance anion exchange
chromatography with pulsed amperometric detection
[80], with GC-MS [81], or by labelling the hydrolysed
monosaccharide residues with different UV [82] or
fluorescent tags [53–56]. Similarly, although MS ⁄ MS
can give some information on specific glycan linkages,
obtaining this information usually requires further
experimentation with specific exoglycosidase digestion
[69], linkage analysis by GC-MS [83], or NMR
[65,66,68,80].
O-glycopeptide analysis – the
remaining challenge
The important cornerstone of glycoproteomics is
assigning macroheterogeneity and microheterogeneity,
i.e. assigning both the glycosylation sites and the dif-
ferent glycoforms present on each site. Obtaining the
whole picture is still the major challenge in the analysis
of mucin-type O-glycosylation.
Obtaining the glycopeptide
Glycopeptides with O-linked glycans on a single site are
easier to analyse than large N-glycosylated peptides, as
they usually have smaller, less heterogeneous glycan
structures attached. Mucin-like domains, however, are
much more difficult, as they have numerous O-linked
sites in very close proximity. As mentioned before,

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

(HexNAc)
1
(NeuAc)
1
733.3 (Hex)
1
(HexNAc)
2
(deoxyhexose)
1
749.3 (Hex)
2
(HexNAc)
2
895.3 (Hex)
2
(HexNAc)
2
(Deoxyhexose)
1
966.3 (Hex)
1
(HexNAc)
1
(NeuAc)
2
1040.4 (Hex)
2
(HexNAc)
2

(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

(HexNAc)
2
(deoxyhexose)
2
(NeuAc)
1
a
The masses are those of reduced glycans detected in negative
mode carbon LC-ESI-MS.
P. H. Jensen et al. Mucin-type O-glycosylation
FEBS Journal 277 (2010) 81–94 ª 2009 The Authors Journal compilation ª 2009 FEBS 85
approach is that there is very heterogeneous cleavage of
the amino acid backbone, and when this heterogeneity
is added to the diversity of the attached glycans, it
becomes difficult to interpret the mass spectra.
Mirgorodskaya et al. [86] have used partial acid
hydrolysis to successfully identify O-glycosylation sites
in synthetic glycopeptides. They found that peptide
bonds N-terminal to Asp, Ser and, occasionally, Thr
and Gly were especially labile. They obtained good
sequence coverage of most of the peptide, but signifi-
cant hydrolysis of glycosidic bonds was also observed.
Additionally, this method only works with known
sequences of purified peptides, and cannot be applied
to complex mixtures [87].
Enrichment of the glycopeptides after digestion of
the protein improves their detection, as they are usu-
ally less abundant than the nonglycosylated peptides in
a digest, owing to glycan heterogeneity, and are also
suppressed in the ionization process [88]. There are

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

backbone with the glycans still attached is a prerequisite
for unambiguous assignment and characterization of
the heterogeneity of the occupied glycosylation sites. Ed-
man sequencing was, for a long time, the only technique
that allowed sequencing through glycopeptides to reveal
the glycosylation sites, and, if performed on solid phase,
gave partial information on the glycans attached [101].
MS has now emerged as the basic detector for pep-
tide characterization. In the commonly used methods
of collision-induced dissociation (CID) and IR multiph-
oton dissociation fragmentation, glycans are detached
from the amino acids by vibrational excitation, which
mainly results in glycosidic fragmentation and some
cross-ring cleavages. Although these data give some
information on the branching and composition of the
O-glycans on the peptide [102–104], there is hardly any
fragmentation of the peptide backbone, and so no
amino acid sequence information or glycan site identifi-
cation is obtained [105]. In the last decade, new MS
fragmentation techniques have emerged for potential
use in the determination of mucin-type O-glycosylation
sites, namely electron capture dissociation (ECD)
[106,107] and electron transfer dissociation (ETD)
[108,109]. ECD and ETD usually maintain labile modi-
fications, owing to the high rate of amide bond cleav-
age and the moderate amount of excess energy [110].
This leads to fragmentation of the peptide backbone
with the modification still intact, opening the possibility
of determining sites with the glycan still attached.
Edman sequencing

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-

tyric acid in place of the modified Thr [119], and the
sites of glycosylation are determined by the change in
the resulting mass of the peptide. The same effect can
be obtained with ammonia treatment, which needs less
clean-up prior to analysis [120]. Variants of this
method using different chemistries for better detection
of deglycosylated Ser or Thr residues have been used
[62,121]. The drawbacks to this approach can be non-
specific dehydration of unmodified Ser and Thr resi-
dues, and the inability to determine whether the Ser
and ⁄ or Thr residues were modified by glycans or by
other groups such as phosphate, which are b-elimi-
nated in the same way. Czeszak et al. [122] have
reported an improved method for site determination,
using dimethylamine-catalysed b-elimination of the gly-
cosylated site and employing a fixed-charge derivatiza-
tion of the N-terminus of the peptide with a
phosphonium group. With CID-MS⁄ MS, the fixed
charge greatly improved the peptide fragmentation,
leading to good sequence coverage and site identifica-
tion [87]. The same laboratory has used the fixed-
charge approach on synthetic peptides with a single
GalNAc attached [122]. These methods all help to
determine the sites of O-glycosylation, but have the
limitation of ‘throwing away’ the glycan structure and
heterogeneity information.
With the attached glycan
Since the introduction of ECD ⁄ FT ion cyclotron reso-
nance MS fragmentation in 1998 by Zubarev et al.
[107], several studies have been published on the use of

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

some glycan heterogeneity, especially if the peptide
sequence is known. De novo sequencing and assign-
ment is still difficult to achieve by this method.
ETD ⁄ ion trap MS is the newest type of fragmenta-
tion [108,128] to show promising results in mucin-type
O-glycosylation site analysis. A limited number of
studies have been performed so far. Wu et al. (2007)
[109] performed a thorough study on O-glycopeptides
with ETD fragmentation, and found that isolation
and fragmentation of the charge-reduced (CR) species
by CID (CR-CID) yielded additional product ions
(c and z), particularly for larger m ⁄ z peptide ions
(> 1000). A related method used supplemental activa-
tion to enhance fragmentation of all ETD ⁄ ECD frag-
ment ions [129]. According to Wu et al. (2007) [109]
CR-CID of a single isolated CR species generates
spectra that are cleaner and easier to interpret than a
general hit with supplemental activation. Other studies
support the finding of limited fragmentation informa-
tion being obtained from ETD of precursor ions with
m ⁄ z values larger than 1000 [129,130]. In addition, the
low-resolution data from ion traps makes charge state
assignments of both precursor and fragment ions diffi-
cult. The newer OrbiTrap technology offers higher-
resolution scanning in conjunction with ETD fragmen-
tation. In general, the ETD ⁄ linear trap is useful for
detection of ions if speed and sensitivity is desired,
whereas the ETD ⁄ OrbiTrap can be used if resolution
and accuracy is the aim [131,132]. As yet, it is not
possible to have speed, sensitivity and high resolution

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

acid site. Most of the tools are now available to deter-
mine the compositions and structures of the attached
O-glycans and to identify some of the sites that may be
occupied by them. The development of ECD ⁄ ETD-MS
fragmentation may provide the final step in determining
the diversity and extent of glycosylation at each site.
The success of this new technique will depend on good
sample preparation and new software development to
help in interpreting the complex spectra that result.
Acknowledgements
P. H. Jensen was supported by the Danish Agency for
Science, Technology and Innovation (grant 272-07-
0066). D. Kolarich was supported by an Erwin Schro
¨
-
dinger Fellowship from the Austrian Science Fund
(grant J2661) and Macquarie University.
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