Mucin-type O-glycosylation - putting the pieces together

REVIEW ARTICLE

Mucin-type O-glycosylation – putting the pieces togetherPia 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: [email protected]

Website: http://www.chem.mq.edu.au/

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 analysischallenging?

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-acetylgalactosaminyl-

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 wecan 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


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



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 – theremaining 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 massesa [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




1186.4 (Hex)2 (HexNAc)2 (deoxyhexose)1 (NeuAc)1



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.



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



[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.



The difficulty of site-specific analysis by ETD ⁄ iontrap 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.

References

1 Gagneux P & Varki A (1999) Evolutionary consider-

ations in relating oligosaccharide diversity to biological

function. Glycobiology 9, 747–755.

2 Varki A & Lowe JB (1999) Biological roles of glycans.

In Essentials of Glycobiology, 2nd edn. (Varki A,

Cummings R, Esko J, Freeze H, Hart G & Marth J

eds), pp. 57–68. Cold Spring Harbor Laboratory Press,

Woodbury, NY.

3 Spiro RG (2002) Protein glycosylation: nature, distri-

bution, enzymatic formation, and disease implications

of glycopeptide bonds. Glycobiology 12, 43R–56.

4 Copeland RJ, Bullen JW & Hart GW (2008) Cross-

talk between GlcNAcylation and phosphorylation:

roles in insulin resistance and glucose toxicity. Am J

Physiol Endocrinol Metab 295, E17–E28.

5 Wells L, Vosseller K & Hart GW (2001) Glycosylation

of nucleocytoplasmic proteins: signal transduction and

O-GlcNAc. Science 291, 2376–2378.

6 Hang HC & Bertozzi CR (2005) The chemistry and

biology of mucin-type O-linked glycosylation. Bioorg

Med Chem 13, 5021–5034.

7 Van den Steen P, Rudd PM, Dwek RA & Opdenakker

G (1998) Concepts and principles of O-linked glycosyl-

ation. Crit Rev Biochem Mol Biol 33, 151–208.

8 Hanisch FG (2001) O-glycosylation of the mucin type.

Biol Chem 382, 143–149.

9 Schwientek T, Bennett EP, Flores C, Thacker J,

Hollmann M, Reis CA, Behrens J, Mandel U, Keck B,

Schafer MA et al. (2002) Functional conservation

of subfamilies of putative UDP-N-acetylgalactos-

amine:polypeptide N-acetylgalactosaminyltransferases

in Drosophila, Caenorhabditis elegans, and mammals.

One subfamily composed of l(2)35Aa is essential in

Drosophila. J Biol Chem 277, 22623–22638.

10 Ten Hagen KG, Tran DT, Gerken TA, Stein DS &

Zhang Z (2003) Functional characterization and

expression analysis of members of the UDP-Gal-

NAc:polypeptide N-acetylgalactosaminyltransferase

family from Drosophila melanogaster. J Biol Chem

278, 35039–35048.

11 Clausen H & Bennett EP (1996) A family of UDP-

GalNAc: polypeptide N-acetylgalactosaminyl-transfer-

ases control the initiation of mucin-type O-linked

glycosylation. Glycobiology 6, 635–646.

12 Ten Hagen KG, Fritz TA & Tabak LA (2003) All in

the family: the UDP-GalNAc:polypeptide N-acety-

lgalactosaminyltransferases. Glycobiology 13, 1R–16R.

13 Tarp MA & Clausen H (2008) Mucin-type O-glycosyl-

ation and its potential use in drug and vaccine devel-

opment. Biochim Biophys Acta 1780, 546–563.

14 Tian E & Ten Hagen KG (2009) Recent insights into

the biological roles of mucin-type O-glycosylation.

Glycoconj J 26, 325–334.

15 Ichikawa S, Guigonis V, Imel EA, Courouble M,

Heissat S, Henley JD, Sorenson AH, Petit B,

Lienhardt A & Econs MJ (2007) Novel GALNT3

mutations causing hyperostosis–hyperphosphatemia

syndrome result in low intact fibroblast growth factor

23 concentrations. J Clin Endocrinol Metab 92, 1943–

1947.

16 Kato K, Jeanneau C, Tarp MA, Benet-Pages A,

Lorenz-Depiereux B, Bennett EP, Mandel U, Strom

TM & Clausen H (2006) Polypeptide GalNAc-transfer-

ase T3 and familial tumoral calcinosis. Secretion of

fibroblast growth factor 23 requires O-glycosylation.

J Biol Chem 281, 18370–18377.



17 Ju T & Cummings RD (2002) A unique molecular

chaperone Cosmc required for activity of the mamma-

lian core 1 beta 3-galactosyltransferase. Proc Natl

Acad Sci USA 99, 16613–16618.

18 Ju T & Cummings RD (2005) Protein glycosylation:

chaperone mutation in Tn syndrome. Nature 437,

1252.

19 Allen AC, Bailey EM, Brenchley PE, Buck KS,

Barratt J & Feehally J (2001) Mesangial IgA1 in IgA

nephropathy exhibits aberrant O-glycosylation:

observations in three patients. Kidney Int 60, 969–973.

20 Hiki Y, Kokubo T, Iwase H, Masaki Y, Sano T,

Tanaka A, Toma K, Hotta K & Kobayashi Y (1999)

Underglycosylation of IgA1 hinge plays a certain role

for its glomerular deposition in IgA nephropathy.

J Am Soc Nephrol 10, 760–769.

21 Hiki Y, Tanaka A, Kokubo T, Iwase H, Nishikido J,

Hotta K & Kobayashi Y (1998) Analyses of IgA1

hinge glycopeptides in IgA nephropathy by

matrix-assisted laser desorption ⁄ ionization time-of-

flight mass spectrometry. J Am Soc Nephrol 9,

577–582.

22 Kathiresan S, Melander O, Guiducci C, Surti A, Burtt

NP, Rieder MJ, Cooper GM, Roos C, Voight BF,

Havulinna AS et al. (2008) Six new loci associated

with blood low-density lipoprotein cholesterol,

high-density lipoprotein cholesterol or triglycerides in

humans. Nat Genet 40, 189–197.

23 Willer CJ, Sanna S, Jackson AU, Scuteri A,

Bonnycastle LL, Clarke R, Heath SC, Timpson NJ,

Najjar SS, Stringham HM et al. (2008) Newly identi-

fied loci that influence lipid concentrations and risk of

coronary artery disease. Nat Genet 40, 161–169.

24 Kim YJ & Varki A (1997) Perspectives on the signifi-

cance of altered glycosylation of glycoproteins in

cancer. Glycoconj J 14, 569–576.

25 Ono M & Hakomori S (2004) Glycosylation defining

cancer cell motility and invasiveness. Glycoconj J 20,

71–78.

26 Springer GF (1997) Immunoreactive T and Tn

epitopes in cancer diagnosis, prognosis, and immuno-

therapy. J Mol Med 75, 594–602.

27 Sperandio M (2006) Selectins and glycosyltransferases

in leukocyte rolling. FEBS J 273, 4377–4389.

28 Baldus SE, Engelmann K & Hanisch FG (2004)

MUC1 and the MUCs: a family of human mucins

with impact in cancer biology. Crit Rev Clin Lab Sci

41, 189–231.

29 Mall AS (2008) Analysis of mucins: role in laboratory

diagnosis. J Clin Pathol 61, 1018–1024.

30 Chen YZ, Tang YR, Sheng ZY & Zhang Z (2008)

Prediction of mucin-type O-glycosylation sites in

mammalian proteins using the composition of k-spaced

amino acid pairs. BMC Bioinformatics 9, 101,

doi:10.1186/1471-2105-9-101.

31 Hansen JE, Lund O, Nielsen JO, Hansen JE & Brunak

S (1996) O-GLYCBASE: a revised database of O-gly-

cosylated proteins. Nucleic Acids Res 24, 248–252.

32 Hansen JE, Lund O, Tolstrup N, Gooley AA,

Williams KL & Brunak S (1998) NetOglyc: prediction

of mucin type O-glycosylation sites based on sequence

context and surface accessibility. Glycoconj J 15,

115–130.

33 Julenius K, Molgaard A, Gupta R & Brunak S (2005)

Prediction, conservation analysis, and structural char-

acterization of mammalian mucin-type O-glycosylation

sites. Glycobiology 15, 153–164.

34 Iwase H & Hotta K (1993) Release of O-linked

glycoprotein glycans by endo-alpha-N-acetylgalacto-

saminidase. In Methods in molecular biology: glyco-

protein analysis in biomedicine, vol. 14, pp. 151–159.

Humana Press, Totawa, NJ.

35 Dwek RA, Edge CJ, Harvey DJ, Wormald MR &

Parekh RB (1993) Analysis of glycoprotein-associated

oligosaccharides. Annu Rev Biochem 62, 65–100.

36 Sharon N & Lis H (2004) History of lectins: from

hemagglutinins to biological recognition molecules.

Glycobiology 14, 53R–62R.

37 Agard NJ, Baskin JM, Prescher JA, Lo A & Bertozzi

CR (2006) A comparative study of bioorthogonal

reactions with azides. ACS Chem Biol 1, 644–648.

38 Hang HC, Yu C, Kato DL & Bertozzi CR (2003) A

metabolic labeling approach toward proteomic

analysis of mucin-type O-linked glycosylation. Proc

Natl Acad Sci USA 100, 14846–14851.

39 Lopez M, Tetaert D, Juliant S, Gazon M, Cerutti M,

Verbert A & Delannoy P (1999) O-glycosylation

potential of lepidopteran insect cell lines. Biochim

Biophys Acta 1427, 49–61.

40 Dodds ED, Seipert RR, Clowers BH, German JB &

Lebrilla CB (2009) Analytical performance of

immobilized pronase for glycopeptide footprinting and

implications for surpassing reductionist glycoproteo-

mics. J Proteome Res 8, 502–512.

41 Mayoral MA, Mayoral C, Meneses A, Villalvazo L,

Guzman A, Espinosa B, Ochoa JL, Zenteno E &

Guevara J (2008) Identification of galectin-3 and

mucin-type O-glycans in breast cancer and its

metastasis to brain. Cancer Invest 26, 615–623.

42 Li C, Simeone DM, Brenner DE, Anderson MA,

Shedden KA, Ruffin MT & Lubman DM (2009)

Pancreatic cancer serum detection using a lectin ⁄glyco-antibody array method. J Proteome Res 8,

483–492.

43 Ueda K, Fukase Y, Katagiri T, Ishikawa N, Irie S,

Sato TA, Ito H, Nakayama H, Miyagi Y, Tsuchiya E

et al. (2009) Targeted serum glycoproteomics for the

discovery of lung cancer-associated glycosylation

disorders using lectin-coupled ProteinChip arrays.

Proteomics 9, 2182–2192.



44 Wu YM, Nowack D, Omenn G & Haab B (2009)

Mucin glycosylation is altered by pro-inflammatory

signaling in pancreatic-cancer cells. J Proteome Res 8,

1876–1886.

45 Lee A, Kolarich D, Haynes PA, Jensen PH, Baker MS

& Packer NH (2009) Rat liver membrane

glycoproteome: enrichment by phase partitioning and

glycoprotein capture. J Proteome Res 8, 770–781.

46 Bourne Y, Astoul CH, Zamboni V, Peumans WJ,

Menu-Bouaouiche L, Van Damme EJ, Barre A &

Rouge P (2002) Structural basis for the unusual

carbohydrate-binding specificity of jacalin towards

galactose and mannose. Biochem J 364, 173–180.

47 Takahashi K, Hiki Y, Odani H, Shimozato S, Iwase

H, Sugiyama S & Usuda N (2006) Structural analyses

of O-glycan sugar chains on IgA1 hinge region using

SELDI-TOFMS with various lectins. Biochem Biophys

Res Commun 350, 580–587.

48 Gimenez E, Benavente F, Barbosa J & Sanz-Nebot V

(2007) Towards a reliable molecular mass

determination of intact glycoproteins by matrix-

assisted laser desorption ⁄ ionization time-of-flight mass

spectrometry. Rapid Commun Mass Spectrom 21,

2555–2563.

49 Loureiro RF, de Oliveira JE, Torjesen PA, Bartolini P

& Ribela MTCP (2006) Analysis of intact human

follicle-stimulating hormone preparations by reversed-

phase high-performance liquid chromatography.

J Chromatogr A 1136, 10–18.

50 Neususs C & Pelzing M (2009) Capillary zone

electrophoresis–mass spectrometry for the characteriza-

tion of isoforms of intact glycoproteins. In Methods in

molecular biology, vol. 492 (Lipton M & Pasa-Tolic L,

eds), pp. 201–213. Humana Press, Totowa, NJ.

51 Peter-Katalinic J (2005) O-glycosylation of proteins. In

Methods in Enzymology, vol. 405 (Burlingame AL,

ed.), pp. 139–171. Academic Press, San Diego, CA.

52 Lee YC & Rice KG (1993) Fractionation of glycopep-

tides and oligosaccharides from glycoproteins by

HPLC. In Glycobiology: A Practical AQpproach

(Fukuda M & Kobata A eds), pp. 127–163. Oxford

University Press, New York, NY.

53 Bigge JC, Patel TP, Bruce JA, Goulding PN, Charles

SM & Parekh RB (1995) Nonselective and efficient

fluorescent labeling of glycans using 2-amino

benzamide and anthranilic acid. Anal Biochem 230,

229–238.

54 Xia B, Royall JA, Damera G, Sachdev GP &

Cummings RD (2005) Altered O-glycosylation and

sulfation of airway mucins associated with cystic

fibrosis. Glycobiology 15, 747–775.

55 Anumula KR (1994) Quantitative determination of

monosaccharides in glycoproteins by high-performance

liquid chromatography with highly sensitive fluores-

cence detection. Anal Biochem 220, 275–283.

56 Wuhrer M, Dennis RD, Doenhoff MJ, Bickle Q,

Lochnit G & Geyer R (1999) Immunochemical

characterisation of Schistosoma mansoni glycolipid

antigens. Mol Biochem Parasitol 103, 155–169.

57 Iwase H, Ishii-Karakasa I, Fujii E, Hotta K, Hiki Y

& Kobayashi Y (1992) Analysis of glycoform of

O-glycan from human myeloma immunoglobulin a1

by gas-phase hydrazinolysis following pyridyl-

amination of oligosaccharides. Anal Biochem 206,

202–205.

58 Kuraya N & Hase S (1992) Release of O-linked sugar

chains from glycoproteins with anhydrous hydrazine

and pyridylamination of the sugar chains with

improved reaction conditions. J Biochem 112,

122–126.

59 Patel T, Bruce J, Merry A, Bigge C, Wormald M,

Parekh R & Jaques A (1993) Use of hydrazine to

release in intact and unreduced form both N- and

O-linked oligosaccharides from glycoproteins.

Biochemistry 32, 679–693.

60 Huang Y, Mechref Y & Novotny MV (2001)

Microscale nonreductive release of O-linked glycans

for subsequent analysis through MALDI mass

spectrometry and capillary electrophoresis. Anal Chem

73, 6063–6069.

61 Xie Y, Liu J, Zhang J, Hedrick JL & Lebrilla CB

(2004) Method for the comparative glycomic analyses

of O-linked, mucin-type oligosaccharides. Anal Chem

76, 5186–5197.

62 Hanisch F-G, Jovanovic M & Peter-Katalinic J (2001)

Glycoprotein identification and localization of

O-glycosylation sites by mass spectrometric analysis of

deglycosylated ⁄ alkylaminylated peptide fragments.

Anal Biochem 290, 47–59.

63 Koizumi K, Tanimoto T, Okada Y & Matsuo M

(1990) Isolation and characterization of three

positional isomers of diglucosylcyclomaltoheptaose.

Carbohydr Res 201, 125–134.

64 Davies M, Smith KD, Harbin A-M & Hounsell EF

(1992) High-performance liquid chromatography of

oligosaccharide alditols and glycopeptides on a

graphitized carbon column. J Chromatogr A 609,

125–131.

65 Coppin A, Florea D, Maes E, Cogalniceanu D &

Strecker G (2003) Comparative study of carbohydrate

chains released from the oviducal mucins of the two

very closely related amphibian species Bombina

bombina and Bombina variegata. Biochimie 85, 53–64.

66 Guerardel Y, Kol O, Maes E, Lefebvre T, Boilly B,

Davril M & Strecker G (2000) O-glycan variability of

egg-jelly mucins from Xenopus laevis: characterization

of four phenotypes that differ by the terminal

glycosylation of their mucins. Biochem J 352, 449–463.

67 Jang-Lee J, Curwen RS, Ashton PD, Tissot B,

Mathieson W, Panico M, Dell A, Wilson RA &



Haslam SM (2007) Glycomics analysis of Schistosoma

mansoni egg and cercarial secretions. Mol Cell

Proteomics 6, 1485–1499.

68 Mourad R, Morelle W, Neveu A & Strecker G (2001)

Diversity of O-linked glycosylation patterns between

species. Characterization of 25 carbohydrate chains

from oviducal mucins of Rana ridibunda. Eur J

Biochem 268, 1990–2003.

69 Storr SJ, Royle L, Chapman CJ, Hamid UMA,

Robertson JF, Murray A, Dwek RA & Rudd PM

(2008) The O-linked glycosylation of secretory ⁄ shedMUC1 from an advanced breast cancer patient’s

serum. Glycobiology 18, 456–462.

70 Parry S, Wong N-K, Easton RL, Panico M, Haslam

SM, Morris HR, Anderson P, Klotz KL, Herr JC,

Diekman AB et al. (2007) The sperm agglutination

antigen-1 (SAGA-1) glycoforms of CD52 are

O-glycosylated. Glycobiology 17, 1120–1126.

71 Kaneko MK, Kato Y, Kameyama A, Ito H, Kuno

A, Hirabayashi J, Kubota T, Amano K, Chiba Y,

Hasegawa Y et al. (2007) Functional glycosylation of

human podoplanin: glycan structure of platelet

aggregation-inducing factor. FEBS Lett 581, 331–

336.

72 Robbe C, Michalski JC & Capon C (2006) Structural

determination of O-glycans by tandem mass

spectrometry. In Methods in molecular biology:

glycobiology protocols, vol. 347 (Brockhausen I, ed.),

pp. 109–123. Humana Press, Totawa, NJ.

73 Backstrom M, Link T, Olson FJ, Karlsson H,

Graham R, Picco G, Burchell J, Taylor-Papadimitriou

J, Noll T & Hansson GC (2003) Recombinant MUC1

mucin with a breast cancer-like O-glycosylation

produced in large amounts in Chinese-hamster ovary

cells. Biochem J 376, 677–686.

74 Schulz BL, Packer NH & Karlsson NG (2002)

Small-scale analysis of O-linked oligosaccharides from

glycoproteins and mucins separated by gel electro-

phoresis. Anal Chem 74, 6088–6097.

75 Wilson NL, Robinson LJ, Donnet A, Bovetto L,

Packer NH & Karlsson NG (2008) Glycoproteomics

of milk: differences in sugar epitopes on human and

bovine milk fat globule membranes. J Proteome Res 7,

3687–3696.

76 Karlsson H, Larsson JMH, Thomsson KA, Hard I,

Backstrom M & Hansson GC (2009) High-throughput

and high-sensitivity nano-LC ⁄MS and MS ⁄MS for

O-glycan profiling. In Methods in molecular biology:

Glycomics, vol. 534 (Packer NH & Karlsson NG, eds),

pp. 117–132. Humana Press, Totawa, NJ.

77 Royle L, Matthews E, Corfield A, Berry M, Rudd

PM, Dwek RA & Carrington SD (2008) Glycan

structures of ocular surface mucins in man, rabbit

and dog display species differences. Glycoconj J 25,

763–773.

78 Costello CE, Contado-Miller JM & Cipollo JF (2007)

A glycomics platform for the analysis of permethylated

oligosaccharide alditols. J Am Soc Mass Spectrom 18,

1799–1812.

79 Hanisch F-G & Muller S (2009) Analysis of

methylated O-glycan alditols by reversed-phase

nanoLC coupled CAD-ESI mass spectrometry. In

Methods in molecular biology: glycomics, vol. 534

(Packer NH & Karlsson NG, eds), pp. 107–116.

Humana Press, Totawa, NJ.

80 Degroote S, Maes E, Humbert P, Delmotte P,

Lamblin G & Roussel P (2003) Sulfated oligosaccha-

rides isolated from the respiratory mucins of a secretor

patient suffering from chronic bronchitis. Biochimie

85, 369–379.

81 Pons A, Richet C, Robbe C, Herrmann A,

Timmerman P, Huet G, Leroy Y, Carlstedt I, Capon

C & Zanetta J-P (2003) Sequential GC ⁄MS analysis of

sialic acids, monosaccharides, and amino acids of gly-

coproteins on a single sample as heptafluorobutyrate

derivatives. Biochemistry 42, 8342–8353.

82 Shen X & Perreault H (1998) Characterization of

carbohydrates using a combination of derivatization,

high-performance liquid chromatography and mass

spectrometry. J Chromatogr A 811, 47–59.

83 Geyer R & Geyer H (1994) Saccharide linkage analysis

using methylation and other techniques. In Methods in

enzymology, vol. 230 (Lennarz WJ & Hart GW, eds),

pp. 86–108. Academic Press, San Diego, CA.

84 Larsen MR, Hojrup P & Roepstorff P (2005)

Characterization of gel-separated glycoproteins using

two-step proteolytic digestion combined with

sequential microcolumns and mass spectrometry. Mol

Cell Proteomics 4, 107–119.

85 An HJ, Peavy TR, Hedrick JL & Lebrilla CB (2003)

Determination of N-glycosylation sites and site

heterogeneity in glycoproteins. Anal Chem 75,

5628–5637.

86 Mirgorodskaya E, Hassan H, Wandall HH, Clausen H

& Roepstorff P (1999) Partial vapor-phase hydrolysis

of peptide bonds: a method for mass spectrometric

determination of O-glycosylated sites in glycopeptides.

Anal Biochem 269, 54–65.

87 Czeszak X, Ricart G, Tetaert D, Michalski JC & Lem-

oine J (2002) Identification of substituted sites on

MUC5AC mucin motif peptides after enzymatic O-gly-

cosylation combining beta-elimination and

fixed-charge derivatization. Rapid Commun Mass Spec-

trom 16, 27–34.

88 Mirgorodskaya E, Krogh T & Roepstorff P (2000)

Characterisation of protein glycosylation by

MALDI-TOF MS. In Methods in molecular biology:

mass spectrometry of proteins and peptides, vol. 146

(Chapman J, ed.), pp. 273-292. Humana Press, Totawa,

NJ.



89 Tajiri M, Yoshida S & Wada Y (2005) Differential

analysis of site-specific glycans on plasma and cellular

fibronectins: application of a hydrophilic affinity

method for glycopeptide enrichment. Glycobiology 15,

1332–1340.

90 Rawn JD & Lienhard GE (1974) Binding of boronic

acids to chymotrypsin. Biochemistry 13, 3124–3130.

91 Sparbier K, Koch S, Kessler I, Wenzel T & Kostrzewa

M (2005) Selective isolation of glycoproteins and

glycopeptides for MALDI-TOF MS detection

supported by magnetic particles. J Biomol Tech 16,

407–413.

92 Xu Y, Wu Z, Zhang L, Lu H, Yang P, Webley PA &

Zhao D (2009) Highly specific enrichment of glycopep-

tides using boronic acid-functionalized mesoporous sil-

ica. Anal Chem 81, 503–508.

93 Hagglund P, Bunkenborg J, Elortza F, Jensen ON &

Roepstorff P (2004) A new strategy for identification

of N-glycosylated proteins and unambiguous assign-

ment of their glycosylation sites using HILIC

enrichment and partial deglycosylation. J Proteome

Res 3, 556–566.

94 Kieliszewski MJ, O’Neill M, Leykam J & Orlando R

(1995) Tandem mass spectrometry and structural eluci-

dation of glycopeptides from a hydroxyproline-rich

plant cell wall glycoprotein indicate that contiguous

hydroxyproline residues are the major sites of

hydroxyproline O-arabinosylation. J Biol Chem 270,

2541–2549.

95 Hagglund P, Matthiesen R, Elortza F, Hojrup P,

Roepstorff P, Jensen ON & Bunkenborg J (2007) An

enzymatic deglycosylation scheme enabling identifica-

tion of core fucosylated N-glycans and O-glycosylation

site mapping of human plasma proteins. J Proteome

Res 6, 3021–3031.

96 Larsen MR, Jensen SS, Jakobsen LA & Heegaard NH

(2007) Exploring the sialiome using titanium dioxide

chromatography and mass spectrometry. Mol Cell Pro-

teomics 6, 1778–1787.

97 Zhang H, Li XJ, Martin DB & Aebersold R (2003)

Identification and quantification of N-linked glycopro-

teins using hydrazide chemistry, stable isotope labeling

and mass spectrometry. Nat Biotechnol 21, 660–666.

98 Tian Y, Zhou Y, Elliott S, Aebersold R & Zhang H

(2007) Solid-phase extraction of N-linked

glycopeptides. Nat Protoc 2, 334–339.

99 Schwientek T, Mandel U, Roth U, Muller S &

Hanisch FG (2007) A serial lectin approach to the

mucin-type O-glycoproteome of Drosophila

melanogaster S2 cells. Proteomics 7, 3264–3277.

100 Gerken TA, Gupta R & Jentoft N (1992) A novel

approach for chemically deglycosylating O-linked

glycoproteins. The deglycosylation of

submaxillary and respiratory mucins. Biochemistry 31,

639–648.

101 Gooley AA, Classon BJ, Marschalek R & Williams

KL (1991) Glycosylation sites identified by detection

of glycosylated amino acids released from Edman

degradation: the identification of Xaa-Pro-Xaa-Xaa as

a motif for Thr-O-glycosylation. Biochem Biophys Res

Commun 178, 1194–1201.

102 Carr SA, Huddleston J & Bean MF (1993) Selective

identification and differentiation of N- and O-linked

oligosaccharides in glycoproteins by liquid chromatog-

raphy–mass spectrometry. Protein Sci 2, 183–196.

103 Demelbauer UM, Plematl A, Allmaier G & Rizzi A

(2004) Determination of glycopeptide structures by

multistage mass spectrometry with low-energy colli-

sion-induced dissociation: comparison of electrospray

ionization quadrupole ion trap and matrix-assisted

laser desorption ⁄ ionization quadrupole ion trap

reflection time-of-flight approaches. Rapid Commun

Mass Spectrom 18, 1575–1582.

104 Henning S, Peter-Katalinic J & Pohlentz G (2007)

Structure elucidation of glycoproteins by direct nanoE-

SI MS and MS ⁄MS analysis of proteolytic glycopep-

tides. J Mass Spectrom 42, 1415–1421.

105 Mirgorodskaya E, Roepstorff P & Zubarev RA (1999)

Localization of O-glycosylation sites in peptides by

electron capture dissociation in a Fourier transform

mass spectrometer. Anal Chem 71, 4431–4436.

106 Deguchi K, Ito H, Baba T, Hirabayashi A, Nakagawa

H, Fumoto M, Hinou H & Nishimura S (2007)

Structural analysis of O-glycopeptides employing

negative- and positive-ion multi-stage mass spectra

obtained by collision-induced and electron-capture

dissociations in linear ion trap time-of-flight mass

spectrometry. Rapid Commun Mass Spectrom 21,

691–698.

107 Zubarev RA, Kelleher NL & McLafferty FW (1998)

Electron capture dissociation of multiply charged

protein cations. a nonergodic process. J Am Chem Soc

120, 3265–3266.

108 Syka JEP, Coon JJ, Schroeder MJ, Shabanowitz J &

Hunt DF (2004) Peptide and protein sequence analysis

by electron transfer dissociation mass spectrometry.

Proc Natl Acad Sci USA 101, 9528–9533.

109 Wu S-L, Huhmer AFR, Hao Z & Karger BL (2007)

On-line LC-MS approach combining collision-induced

dissociation (CID), electron-transfer dissociation

(ETD), and CID of an isolated charge-reduced species

for the trace-level characterization of proteins with

post-translational modifications. J Proteome Res 6,

4230–4244.

110 Kjeldsen F, Haselmann KF, Budnik BA, Sorensen ES

& Zubarev RA (2003) Complete characterization of

posttranslational modification sites in the bovine milk

protein PP3 by tandem mass spectrometry with

electron capture dissociation as the last stage. Anal

Chem 75, 2355–2361.



111 Sparrow LG, Gorman JJ, Strike PM, Robinson CP,

McKern NM, Epa VC & Ward CW (2007) The

location and characterisation of the O-linked glycans

of the human insulin receptor. Proteins 66, 261–265.

112 Gerken TA, Owens CL & Pasumarthy M (1997)

Determination of the site-specific O-glycosylation

pattern of the porcine submaxillary mucin tandem

repeat glycopeptide. Model proposed for the

polypeptide:GalNAc transferase peptide binding site.

J Biol Chem 272, 9709–9719.

113 Pisano A, Packer NH, Redmond JW, Williams KL &

Gooley AA (1994) Characterization of O-linked

glycosylation motifs in the glycopeptide domain of

bovine j-casein. Glycobiology 4, 837–844.

114 Pisano A, Redmond JW, Williams KL & Gooley AA

(1993) Glycosylation sites identified by solid-phase

Edman degradation: O-linked glycosylation motifs on

human glycophorin A. Glycobiology 3, 429–435.

115 Zachara N, Packer NH, Temple MD, Slade MB,

Jardine DR, Karuso P, Moss CJ, Mabbutt BC, Curmi

PMG, Williams KL et al. (1996) Recombinant

prespore-specific antigen from Dictyostelium discoide-

um is a b-sheet glycoprotein with a spacer peptide

modified by O-linked N-acetylglucosamine. Eur J

Biochem 238, 511–518.

116 Zachara N & Gooley AA (2000) Identification of

glycosylation sites in mucin peptides by Edman

degradation. In Methods in Molecular Biology

(Corfield A ed), pp. 121–128. Humana Press, Totowa,

NJ.

117 Wiesner J, Premsler T & Sickmann A (2008) Applica-

tion of electron transfer dissociation (ETD) for the

analysis of posttranslational modifications. Proteomics

8, 4466–4483.

118 Mikesh LM, Ueberheide B, Chi A, Coon JJ, Syka

JEP, Shabanowitz J & Hunt DF (2006) The utility of

ETD mass spectrometry in proteomic analysis. Bio-

chim Biophys Acta 1764, 1811–1822.

119 Rademaker GJ, Haverkamp J & Thomas-Oates J

(1993) Determination of glycosylation sites in O-linked

glycopeptides: a sensitive mass spectrometric protocol.

Organic Mass Spectrom 28, 1536–1541.

120 Rademaker GJ, Pergantis SA, Blok-Tip L, Langridge

JI, Kleen A & Thomas-Oates JE (1998) Mass spectro-

metric determination of the sites of O-glycan attach-

ment with low picomolar sensitivity. Anal Biochem

257, 149–160.

121 Mirgorodskaya E, Hassan H, Clausen H & Roepstorff

P (2001) Mass spectrometric determination of O-glyco-

sylation sites using b-elimination and partial acid

hydrolysis. Anal Chem 73, 1263–1269.

122 Czeszak X, Morelle W, Ricart G, Tetaert D &

Lemoine J (2004) Localization of the O-glycosylated

sites in peptides by fixed-charge derivatization with a

phosphonium group. Anal Chem 76, 4320–4324.

123 Haselmann KF, Budnik BA, Olsen JV, Nielsen ML,

Reis CA, Clausen H, Johnsen AH & Zubarev RA

(2001) Advantages of external accumulation for elec-

tron capture dissociation in Fourier transform mass

spectrometry. Anal Chem 73, 2998–3005.

124 Renfrow MB, Mackay CL, Chalmers MJ, Julian BA,

Mestecky J, Kilian M, Poulsen K, Emmett MR,

Marshall AG & Novak J (2007) Analysis of O-glycan

heterogeneity in IgA1 myeloma proteins by Fourier

transform ion cyclotron resonance mass spectrometry:

implications for IgA nephropathy. Anal Bioanal Chem

389, 1397–1407.

125 Hakansson K, Cooper HJ, Emmett MR, Costello CE,

Marshall AG & Nilsson CL (2001) Electron capture

dissociation and infrared multiphoton dissociation

MS ⁄MS of an N-glycosylated tryptic peptide to yield

complementary sequence information. Anal Chem 73,

4530–4536.

126 Robinson EW, Leib RD & Williams ER (2006) The

role of conformation on electron capture dissociation

of ubiquitin. J Am Soc Mass Spectrom 17, 1470–1480.

127 Sihlbom C, van Dijk Hard I, Lidell ME, Noll T,

Hansson GC & Backstrom M (2009) Localization of

O-glycans in MUC1 glycoproteins using electron-

capture dissociation fragmentation mass spectrometry.

Glycobiology 19, 375–381.

128 Coon JJ, Ueberheide B, Syka JEP, Dryhurst DD,

Ausio J, Shabanowitz J & Hunt DF (2005) Protein

identification using sequential ion ⁄ ion reactions and

tandem mass spectrometry. Proc Natl Acad Sci USA

102, 9463–9468.

129 Swaney DL, McAlister GC, Wirtala M, Schwartz JC,

Syka JEP & Coon JJ (2007) Supplemental activation

method for high-efficiency electron-transfer dissocia-

tion of doubly protonated peptide precursors. Anal

Chem 79, 477–485.

130 Pitteri SJ, Chrisman PA, Hogan JM & McLuckey SA

(2005) Electron transfer ion ⁄ ion reactions in a three-

dimensional quadrupole ion trap: reactions of doubly

and triply protonated peptides with SO2*-. Anal Chem

77, 1831–1839.

131 Perry RH, Cooks RG & Noll RJ (2008) Orbitrap mass

spectrometry: instrumentation, ion motion and appli-

cations. Mass Spectrom Rev 27, 661–699.

132 McAlister GC, Berggren WT, Griep-Raming J,

Horning S, Makarov A, Phanstiel D, Stafford G,

Swaney DL, Syka JEP, Zabrouskov V et al. (2008) A

proteomics grade electron transfer dissociation-enabled

hybrid linear ion trap–OrbiTrap mass spectrometer.

J Proteome Res 7, 3127–3136.

133 Loignon M, Perret S, Kelly J, Boulais D, Cass B,

Bisson L, Afkhamizarreh F & Durocher Y (2008)

Stable high volumetric production of glycosylated

human recombinant IFNalpha2b in HEK293 cells.

BMC Biotechnol 8, 65, doi:10.1186/1472-6750-8-65.



134 Schroeder MJ, Webb DJ, Shabanowitz J, Horwitz AF

& Hunt DF (2005) Methods for the detection of

paxillin post-translational modifications and

interacting proteins by mass spectrometry. J Proteome

Res 4, 1832–1841.

135 Breloy I, Schwientek T, Gries B, Razawi H, Macht M,

Albers C & Hanisch F-G (2008) Initiation of mamma-

lian O-mannosylation in vivo is independent of a con-

sensus sequence and controlled by peptide regions

within and upstream of the {alpha}-dystroglycan

mucin domain. J Biol Chem 283, 18832–18840.

136 Perdivara I, Petrovich R, Allinquant B, Deterding

LJ, Tomer KB & Przybylski M (2009) Elucidation

of O-glycosylation structures of the b-amyloid pre-

cursor protein by liquid chromatography–mass spec-

trometry using electron transfer dissociation and

collision induced dissociation. J Proteome Res 8,

631–642.

137 Thomsson KA, Schulz BL, Packer NH & Karlsson

NG (2005) MUC5B glycosylation in human saliva

reflects blood group and secretor status. Glycobiology

15, 791–804.



Mucin-type O-glycosylation - putting the pieces together

Documents