Báo cáo khoa học: Mucin-type O-glycosylation – putting the pieces together docx

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 enzym

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: nicki.packer@mq.edu.au

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.

[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-glyN-gly-cosylated 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]

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 glygly-cans 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.

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 addib-elimina-tion, yields side reacb-elimina-tions 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 – 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 massesa[M–H]

Possible composition (reduced glycans, alditol form)

675.2 (Hex) 1 (HexNAc) 1 (NeuAc) 1

733.3 (Hex)1(HexNAc)2(deoxyhexose)1

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.

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-acetylhexosaminineuramini-dase, 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 glycosylafragmenta-tion 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 glycosylafragmenta-tion 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 fragmentaactiva-tion 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⁄ 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 novosite 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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