Download - Flagellar glycosylation in Clostridium botulinum

Flagellar glycosylation in Clostridium botulinumSusan M. Twine1, Catherine J. Paul1,2, Evgeny Vinogradov1, David J. McNally1, Jean-RobertBrisson1, James A. Mullen1, David R. McMullin1, Harold C. Jarrell1, John W. Austin2, John F. Kelly1

and Susan M. Logan1

1 NRC-Institute for Biological Sciences, Ottawa, Canada

2 Bureau of Microbial Hazards, HFPB, Health Canada, Sir Frederick G. Banting Research Centre, Ottawa, Canada

Clostridium botulinum is a Gram-positive spore-form-

ing anaerobic bacterium that produces the potent

botulinum neurotoxin (BoNT). Botulism is a descend-

ing symmetrical paralysis caused by BoNT. Foodborne

botulism occurs after ingestion of food contaminated

with the neurotoxin, and is associated with a case

fatality rate in the USA of 4% [1]. Infant botulism, an

intestinal toxaemia, typically occurs in infants under

the age of 12 months, and is caused by spores that

colonize and produce toxin in the intestine. Infant bot-

ulism is now the most common form of botulism in

the USA, and adult intestinal colonization botulism is

extremely rare, with less than 15 cases reported [1a].

Wound botulism occurs when C. botulinum infects

wounds and produces toxin. It is largely associated

with injection drug use and is now the most common

form of botulism in the UK [2]. The process by which

the organism is able to colonize the gastrointestinal

tract or wound tissue is currently unknown, but

bacterial cell surface-associated factors are envisaged

as playing an important role in this process.

Glycosylation of proteins is known to impart novel

physical properties and biological roles to proteins

from both eukaryotes and prokaryotes. Glycoproteins

Keywords

Clostridium botulinum; flagellin; legionaminic

acid; protein glycosylation

Correspondence

S. M. Logan, Institute for Biological

Sciences, National Research Council, Room

3037, 100 Sussex Drive, Ottawa, ON,

K1A 0R6, Canada

Fax: +1 613 952 9092

Tel: +1 613 990 0839

E-mail: [email protected]

(Received 6 June 2008, revised 3 July 2008,

accepted 7 July 2008)

doi:10.1111/j.1742-4658.2008.06589.x

Flagellins from Clostridium botulinum were shown to be post-translationally

modified with novel glycan moieties by top-down MS analysis of purified

flagellin protein from strains of various toxin serotypes. Detailed analyses

of flagellin from two strains of C. botulinum demonstrated that the protein

is modified by a novel glycan moiety of mass 417 Da in O-linkage. Bio-

informatic analysis of available C. botulinum genomes identified a flagellar

glycosylation island containing homologs of genes recently identified in

Campylobacter coli that have been shown to be responsible for the

biosynthesis of legionaminic acid derivatives. Structural characterization of

the carbohydrate moiety was completed utilizing both MS and NMR

spectroscopy, and it was shown to be a novel legionaminic acid derivative,

7-acetamido-5-(N-methyl-glutam-4-yl)-amino-3,5,7,9-tetradeoxy-d-glycero-

a-d-galacto-nonulosonic acid, (aLeg5GluNMe7Ac). Electron transfer disso-

ciation MS with and without collision-activated dissociation was utilized to

map seven sites of O-linked glycosylation, eliminating the need for chemical

derivatization of tryptic peptides prior to analysis. Marker ions for novel

glycans, as well as a unique C-terminal flagellin peptide marker ion, were

identified in a top-down analysis of the intact protein. These ions have the

potential for use in for rapid detection and discrimination of C. botulinum

cells, indicating botulinum neurotoxin contamination. This is the first

report of glycosylation of Gram-positive flagellar proteins by the ‘sialic

acid-like’ nonulosonate sugar, legionaminic acid.

Abbreviations

BoNT, botulinum neurotoxin; CAD, collision activated dissociation; ETD, electron transfer dissociation; FGI, flagellar glycosylation island; Leg,

5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-nonulosonic acid; aLeg5GluNme7Ac, 7-acetamido-5(N-methyl-glutam-4-yl)-amino-

3,5,7,9-tetradeoxy-D-glycero-a-D-galacto-nonulosonic acid; VR, variable region.

4428 FEBS Journal 275 (2008) 4428–4444 ª 2008 National Research Council – Institute for Biological Sciences. Journal compilation ª 2008 FEBS

of bacteria have received considerable attention

recently, most notably those identified in pathogenic

species and localized on the bacterial cell surface where

they may be involved in interactions with the host. In

Gram-negative bacteria, examples of surface-associated

glycoproteins are the pilins of Pseudomonas aeruginosa

[3–5] and Neisseria spp.[6], the adhesins TibA and

AIDA-1 of Escherichia coli [7,8] and HMW1 of

Haemophilus influenzae [9], and the flagellins of

P. aeruginosa[10], Helicobacter pylori [11,12] and

Campylobacter jejuni ⁄ coli [13]. Although the full signif-

icance of glycosylation of these proteins has yet to be

defined, there are a number of reports describing the

contribution of these modifications to virulence and

colonization [9,14,15].

For Gram-positive bacteria, extensive characteriza-

tion of S-layer proteins from a number of organisms

has revealed glycosylation as a major structural modi-

fication [16,17]. The platelet aggregation-associated

protein of Streptococcus sanguis was one of the first

prokaryotic virulence factors shown to be modified by

an N-linked rhamnose polymer [18]. More recently, a

unique family of high-molecular-mass serine-rich pro-

teins found in staphyococcal and streptococcal species

that play a major role in bacterial interactions with

host components has been characterized. The Fap1

fimbriae of Streptococcus parasanguis and the Fap1-like

protein adhesins GspB of Streptococcus gordonii [19]

and SrpA of S. sanguis [20] are all glycosylated in a

novel fashion through O-linkage. The glycan moieties

of the Fap1 fimbriae were recently shown to play a

role in biofilm development [21]. The flagellins of

Listeria monocytogenes are glycosylated with b-GlcNAc

O-linked at up to six sites per flagellin monomer,

although the biological significance of this modification

is not yet understood [22].

Glycosylation of proteins within the genus

Clostridium has been reported. S-layer proteins from

Clostridium difficile have been identified as glycosylated

[23] and the S-layer protein S102-70 of Clostrid-

ium thermohydrosulfuricum is modified with a

tyrosine-linked hexasaccharide [24]. The Clostrid-

ium thermocellum S-layer protein p130 is glycosylated,

as are several of the component proteins of its

cellulosome [25,26]. The Clostridium cellulolyticum

cellulosome contains up to four glycoproteins, and

these are thought to be N-linked [27]. Flagellins of

Clostridium tyrobutyricum, Clostridium acetobutylicum

and C. difficile have been examined, and indirect

staining and aberrant molecular mass indicated that

these proteins are also glycosylated [28–31].

Many of the standard methods for identification

of strains of C. botulinum involve determining the

BoNT serotype or gene sequence. These methods

have limited ability to unequivocally identify a strain

due to a high degree of structural and DNA

sequence conservation across the various BoNT types

[32]. The designation of BoNT as a bio-warfare and

terrorism agent has led to renewed interest in the

development of techniques capable of (a) detecting

C. botulinum ⁄BoNT contamination, (b) identifying

and characterizing the BoNT type and ⁄or subtype,

and (c) forensic identification of individual strains

[33,34]. Previously, Paul et al. [35,36] demonstrated

that variations in the flaA1 and flaA2 gene sequences

could be used to distinguish strains independently of

the BoNT type; however, this method has only lim-

ited ability to distinguish between strains, as several

unrelated C. botulinum strains possessed the same fla

variable region (VR) type and BoNT serotype. In

addition, it has been shown that the flagellins of

C. botulinum are post-translationally modified: flagel-

lin proteins isolated from strains with the same

flaVR type and BoNT serotype migrated differently

on SDS–PAGE gels.

The present study examines in detail the structural

basis of this mass difference, and demonstrates that

flagellins of C. botulinum are glycosylated with novel

O-linked glycan moieties. MS analysis of the flagellin

protein provides an opportunity to explore strain

diversity at the flagellin post-translational level, and to

utilize flagellin-specific marker ions for identification

and characterization of C. botulinum. The identifica-

tion of flagellin in culture supernatants containing

BoNT demonstrates that flagellin detection may be

utilized as a surrogate biomarker for BoNT detection.

Results

Intact mass analysis of C. botulinum flagellins

In a previous study of C. botulinum flagellin diversity,

it was shown that sheared flagella preparations from a

number of group I strains of C. botulinum contained

one to three species of flagellin monomers with molec-

ular masses ranging from 29 to 32 kDa as observed by

SDS–PAGE analysis [36]. Peptide MS ⁄MS analysis

confirmed that these flagellin monomers were the prod-

ucts of either the flaA1 or flaA2 genes of each strain

[35]. In the present study, we have determined the

mass of flagellin proteins from a number of strains

belonging to distinct toxin types and of both the prote-

olytic group (I) and non-proteolytic group (II). The

mass of each flagellin protein was obtained following

infusion into a QTOF2 mass spectrometer (Table 1).

In each case, the MS spectrum showed an envelope of

S. M. Twine et al. Flagellar glycosylation in Clostridium botulinum

FEBS Journal 275 (2008) 4428–4444 ª 2008 National Research Council – Institute for Biological Sciences. Journal compilation ª 2008 FEBS 4429

multiply charged protein ions, from which the

reconstructed molecular mass profile was calculated.

Flagellin from strains belonging to groups I and II

and toxin types A, B, A ⁄B and F all produced flagel-

lins with a molecular mass approximately 7–10% lar-

ger than that predicted from the translated sequence of

respective flagellin structural genes (Table 1). In con-

trast, the group II type E strains examined (Bennett

and Gordon) produced a larger flagellin monomer

protein, the product of the flaB structural gene

(52 155 Da). The intact mass of these flagellin mono-

mers matched precisely the mass predicted from the

translated protein sequence, indicating that these fla-

gellins were not post-translationally modified. A repre-

sentative spectrum of flagellin from C. botulinum strain

FE9909ACS Alberta is shown in Fig. 1A, and the

reconstructed molecular mass profile shown in Fig. 1B.

Three major peaks at 31 462, 31 879 and 32 297 Da

were observed in the reconstructed molecular mass

profile (Fig. 1A). Peaks of much lower intensity were

observed at 31 045, 30 627 and 21 531 Da (data not

shown). Each of the major intact mass peaks were

separated by a mass of 417 Da.

Top-down analysis of C. botulinum flagellins

Examination of intact proteins by top-down MS has

been shown to readily identify the labile glycan oxoni-

um ions found on flagellin glycoproteins [37]. Analysis

of the group I C. botulinum flagellins using this method

revealed at least two distinct marker ions for each

flagellin examined (Table 1 and Fig. 1A,C–H). The

first of these was common to all C. botulinum flagellins

(m ⁄ z 512.2), bute the second ion identified was unique

to particular strains of C. botulinum (Table 1) (m ⁄ z418, Fig. 1A,C; m ⁄ z 259, Fig. 1D,E; m ⁄ z 301, Fig. 1D;

m ⁄ z 299, 317, Fig. 1H). The inset in Fig. 1A is an

expanded view of the lower mass region of the

FE9909ACS Alberta FlaA spectrum, showing the

presence of two marker ions of high intensity at m ⁄ z

Table 1. Diversity of flagellin proteins determined by mass spectrometry. Flagellin from a diverse array of strains was purified from cultured

cells by mechanical shearing, and the accurate mass of the protein was determined by infusion into QTOF2 mass spectrometer (Waters).

Marker ions are labile protein ions observed during tandem mass spectrometry experiments using intact protein. The 512.32+ ion was

observed as a labile marker ion in all Clostridium botulinum flagellins studied. ND, not determined.

Strain FlaVR Region Source MWa Exact protein massb (kDa) Marker ionsc

Group I

Type A

FE9909ACS Alberta 3 Alberta, Canada Feces 29.4 32.30, 31.89, 31.40 512.32+, 418.1+

FE303A1YO 3 Ontario, Canada Feces 29.4 32.30, 31.88, 31.46 512.32+, 418.1+

MUL0109ASA 1 Gulf of Kuwait, Kuwait Mullet fish 29.80 32.63, 32.60, 32.27, 32.24, 31.88 512.32+, 418.1+

FE0205A1AK 1 Alberta, Canada Feces 29.59 32.32, 32.35, 32.28 512.32+, 301.1+, 259.1+

17A 5 – – 30.0 32.88, 32.45, 32.24 512.32+

Type B

PA9508B 2 Quebec, Canada Pate 29.6 32.92, 32.88, 32.49 512.32+, 259.1+

FE9904BMT 4 Ontario, Canada Feces 29.4 32.96, 32.80, 32.47 512.32+, 299.1+ (317.1+)

Type AB

FE9504ACG 1 Quebec, Canada Feces 29.7 32.36 512.32+, 259.1+

Type F

Langeland 3 Denmark Liver paste 29.4 32.32, 31.91, 31.49 512.32+, 418.1+

H461297F 1 Wisconsin, USA Honey 29.8 32.27, 32.31, 32.35 512.32+, 301.1+, 259.1+

Group II

Type B

17B 9 Pacific, USA Sediment 29.1 32.18d 512.32+, 697.2+

Kap-B3 California, USA Kapchunka (fish) 29.1 32.89d 512.32+

Type E

Bennett 8 Newfoundland, Canada Gastric fluid 52.1 52.15 512.32+

Gordon 10 Quebec, Canada Clinical ND 52.15 512.32+

Type F

610F 9 Oregon, USA Salmon 29.3 32.18, 32.20, 32.22* 512.32+, 697.2+

a Predicted molecular mass from DNA sequence. b Protein molecular mass of intact Clostridium flagellins, determined by mass spectrometry.

Intact proteins were infused into the QTOF2 mass spectrometer and the ion profile was recorded. The exact mass was determined using a

maximum-entropy algorithm. c Ions observed during tandem mass spectrometry of multiple charged intact protein ions. Examples are shown

in Fig. 1. d Peaks of much lower intensity, that were not well resolved above the baseline noise, were observed at approximately 38 and

43 kDa. These correspond to protein bands previously visualized by SDS–PAGE of flagellin preparations [36].

Flagellar glycosylation in Clostridium botulinum S. M. Twine et al.


418.2 and 512.1. Increasing the RF lens 1 voltage pro-

moted the formation of fragment ions in the ori-

fice ⁄ skimmer region of the mass spectrometer, allowing

MS ⁄MS spectra of the marker ions to be recorded.

Second-generation ion spectra of m ⁄ z 512.1 gave a

typical peptide MS ⁄MS spectrum, with a clear series of

type y and b ions (Fig. 2A), the sequence of which

(PQGVLQLLR) corresponded to the C-terminal

amino acid sequence of FlaA1 and FlaA2 proteins.

This peptide is found in flagellins from all C. botulinum

isolates for which flagellin gene sequence data have

been obtained, including the strain Hall A for which

the genome has been sequenced. Second-generation

ion spectra of other flagellin marker ions showed no

peaks characteristic of peptide type y or b ions. The

MS ⁄MS spectrum of m ⁄ z 259.1 showed fragments ions

at m ⁄ z 200.1, 182.1, 158.1, 154.1, 126.1 and 112.1,

characteristic of a di-N-acetylhexuronic acid, previ-

ously observed as part of a trisaccharide modification

on Methanococcus voltae flagellin [38] (Fig. S1A ). The

MS ⁄MS spectra of m ⁄ z 301.1 revealed fragment ions

identical to those observed for fragmentation of the

m ⁄ z 259.1 di-N-acetylhexuronic acid moiety, with the

additional mass of 42 Da. It appears that this m ⁄ z301.1 marker ion corresponds to a tri-N-acetylhexu-

ronic acid moiety (Fig. S1B). In contrast, the MS ⁄MS

spectrum of the ion observed at m ⁄ z 299.1 resulted

from neutral loss of water from m ⁄ z 317.1. The

MS ⁄MS of the latter oxonium ion showed distinct

fragment ions at m ⁄ z 299.1, 261.1, 239.1, 222.1, 221.1,

180.1, 162.1 and 135.1 (Fig. 2C and Fig. S1C), charac-

teristic of the fragmentation pattern of nonulosonic

acids such as pseudaminic or legionaminic acid. These

derivatives have been found previously on Campylo-

bacter and Helicobacter flagellins [12,39].

Second-generation ion spectra of the m ⁄ z 418 ion

gave an MS ⁄MS spectrum with predominant peaks at

274, 240 and 181 Da (Fig. 2C). The fragmentation

Fig. 1. Electrospray mass spectrometry and

tandem mass spectrometry analyses of

intact flagellin protein from strains of

C. botulinum. (A) Electrospray mass spec-

trometry of intact flagellin from C. botulinum

strain FE9909ACS Alberta. The inset shows

the predominant ions observed in the lower

mass region of the mass spectrum at m ⁄ z512.32+ and 418.2+. (B) The reconstructed

molecular mass profile of C. botulinum

strain FE9909ACS Alberta, showing three

major peaks at 33 297, 31 879 and

31 462 Da. (C–H) Tandem mass spectro-

metry analyses of multiply charged protein

ions: (C) m ⁄ z 1089.530+ from C. botulinum

Alberta, (D) m ⁄ z 1155.428+ from

C. botulinum FE0205A1AK, (E) m ⁄ z940.535+ from C. botulinum PA9508B, (F)

m ⁄ z 1094.448+ from C. botulinum Bennett,

(G) m ⁄ z 1039.031+ from C. botulinum 17B,

(H) m ⁄ z 1144.229+ from C. botulinum

FE9904BMT. Spectra were acquired at a

collision energy of 15–25 V using argon as

the collision gas. Ions at m ⁄ z 512.3 were

observed in the tandem mass spectrometry

analysis of all purified flagellin proteins stud-

ied. The corresponding singly charged ion at

m ⁄ z 1023.6+ was observed in some but not

all spectra. Other intense ions were

observed in all spectra recorded, except

those of strain Bennett and Gordon, at (C)

m ⁄ z 418.2+, (D) m ⁄ z 301.1+, 259.1+, (E)

m ⁄ z 259.1+, (G) m ⁄ z 647.2+ and (H) m ⁄ z299.1+, 317.1+.



pattern of this novel glycan moiety closely resembles

that previously observed for pseudaminic acid (shown

in Fig. 2C) and legionaminic acid, with an additional

unknown mass of 143 Da. The corresponding frag-

ment ion was observed at m ⁄ z 144 and is indicated in

Fig. 2B. The m ⁄ z 418 oxonium ion has been observed

as a flagellin modification on multiple strains of

C. botulinum, including BoNT serotype strains A and

F (Table 1). Based upon the fragmentation pattern of

the glycan oxonium ion, it appears that this is a novel

structure.

As for the group I flagellins, top-down analysis of

all group II strain flagellins also identified the m ⁄ z512.3 marker ion, which corresponds to the C-terminal

peptide. In contrast to the group II type E strains,

which produced a higher-molecular-mass flagellin that

corresponded precisely with the predicted mass of the

FlaB gene product, the flagellins from group II type B

and F strains appeared to be glycosylated. In addition

to the C-terminal marker ion (m ⁄ z 512.3), they pro-

duced a second unique marker ion, m ⁄ z 697.2, in

top-down analysis (Fig. 1G). The structural nature of

this ion remains to be determined, although MS ⁄MS

fragmentation patterns indicate that it is probably gly-

can in nature. A number of fragment ions in common

with other nonulosonate sugars were observed in the

fragmentation pattern (data not shown).

Bioinformatic analysis of the C. botulinum type F

Langeland genome

While many of the oxonium ion fragmentation pat-

terns observed by top-down analysis of FlaA could be

matched to profiles of known carbohydrates, the m ⁄ zion at 418 observed from FlaA of FE9909ACS

Alberta, FE0303AYO and Langeland appeared to be a

novel derivative of a nonulosonate sugar. The com-

plete genome sequence of the C. botulinum BoNT ⁄Fstrain Langeland has recently become available (Gen-

bank accession number NC_009700), and the genome

was therefore searched by blastp for enzymes known

to be involved in the production of legionaminic and

pseudaminic acid. The genes involved in the biosynthe-

sis of these two nonulosonate sugars in Ca. jejuni and

H. pylori have been characterized in detail [40–42]. In

addition, the recently completed genome sequence of

strain Hall A ATCC3502 revealed a large cluster of

genes between flgB and fliD, many of which appeared

to be to be involved in carbohydrate biosynthesis [43].

Our analysis identified a flagellar glycosylation island

(FGI) in the Langeland genome that was located in a

similar genomic context to that observed in the

ATCC3502 genome between the flgB and fliD homo-

A

B

C

Fig. 2. Second-generation product ion spectra of ions obtained

from in-source dissociation of multiply charged flagellin ions.

MS ⁄ MS spectra were acquired by increasing the RF lens 1 voltage

from 40 to 90 V, forming fragment ions in the orifice ⁄ skimmer

region of the mass spectrometer and promoting the formation of

labile ions from the intact flagellin. (A) MS ⁄ MS spectrum of m ⁄ z512.3, yielding a clear series of peptide type y and b ions, giving

the sequence QGVLQLLR. This peptide sequence corresponded to

the conserved C-terminal peptide of C. botulinum flagellin [36].

(B) MS ⁄ MS spectrum of m ⁄ z 418.2+, yielding a series of predomi-

nant fragment ions, which did not correspond to peptide type y and

b ions. (C) MS ⁄ MS fragmentation pattern of pseudaminic acid (data

originally published in [46]). Common fragment ions at m ⁄ z 181.1,

221.1, 257.1, 275.1 are found in the spectra in (B) and (C). The

MS ⁄ MS spectrum of the 418.2+ ion has an additional fragment ion

at m ⁄ z 144.1 (as indicated by an asterisk), corresponding to loss of

a mass of 143.1 Da from the 418.2 oxonium ion.



logs (Fig. 3). The order of the first 29 ORFs of the

FGI (FGI-I) was completely conserved between Lange-

land and ATCC3502, with the homology of each pre-

dicted protein product never below 80% amino acid

identity. A number of genes in this conserved region

appear to be involved in carbohydrate biosynthesis

and show homology to capsular biosynthetic proteins

of group B Streptococcus agalactia, in particular the

predicted gene products of CLI_2752–2755, which

show homology to CpsE, CpsD, CpsB and CpsC of

S. agalactia. Homologs of the sialic acid biosynthetic

genes neuB (CLI_2745) and neuA (CLI_2746) were

also present [44]. In contrast, the region immediately

downstream of the conserved region and extending to

the flaA structural genes (CBO2730 and CBO2731

ATCC3502; CLI_2781 in Langeland) differed signifi-

cantly between Langeland and ATCC3502 in terms of

their genetic content. This second section of the FGI

(FGI-II) contains a 22 kb deletion in Langeland, and

the organization of the remaining ORFs differs sub-

stantially. The ATCC3502 genome contained tandem

copies of the flagellin structural genes (flaA1 and

flaA2), but only a single flaA gene was found at the

analogous position in the Langeland FGI. Both

Langeland and ATCC3502 genomes contained a num-

ber of carbohydrate biosynthetic genes in this region,

including homologs of a second set of the sialic acid

biosynthetic genes, neuA and neuB. However, only the

Langeland locus contained predicted gene products

with sequence similarity to proteins recently shown to

be involved in legionaminic acid biosynthesis in

Ca. coli [45]. These genes and the Campylobacter

homologs are listed in Table 2.

Nano-LC-MS ⁄ MS analysis of a C. botulinum

FE9909ACS Alberta flagellin peptide digest

To precisely assign the location of the post-translational

modifications, flagellar tryptic peptides of C. botulinum

strain FE9909ACS Alberta were analysed by nano-LC-

Table 2. Identification of legionaminic acid biosynthetic gene homologs in C. botulinum type F Langeland. The E values were obtained by

BLASTP of the Campylobacter jejuni 11168 protein sequence against the C. botulinum type F Langeland genome, and results are for the

Langeland ORF that gave the highest E value.

C. jejuni gene

number Gene name

Langeland

homologous ORF E value Function in Campylobacter jejuni Reference

Cj1319 Unknown CLI_2770 3e-98 Unknown [52]

Cj1320 Unknown CLI_2769 4e-58 Unknown [52]

Cj1325 ptmH CLI_2776 3e-06 Methyl transferase [52]

Cj1327 ptmC CLI_2775 1e-58 Legionaminic acid synthase [62]

Cj1328 ptmD CLI_2777 1e-68 Legionaminic acid biosynthetic gene [62]

Cj1329 ptmE CLI_2778 1e-67 Legionaminic acid biosynthetic gene [62]

Cj1331 ptmB CLI_2773 4e-23 CMP-legionaminic acid synthase [13,52]

Fig. 3. Flagellar glycosylation island locus of C. botulinum strain Langeland.



MS ⁄MS. Many of the MS ⁄MS spectra could be readily

assigned, but a number of peptide spectra were observed

that did not match any expected tryptic peptides. In

each case, an intense ion at m ⁄ z 418 was observed for

each of these unassignable peptides. This ion did not

correspond to any peptide fragmentation and also

accounted for the mass difference between predicted

and observed peptide ion masses, confirming that the

putative oxonium ion observed in top-down studies of

the intact protein corresponds to the flagellin glycan

modification. The MS ⁄MS spectrum of tryptic peptide

169-178 (VGSINIGSGK) is shown in Fig. 4A, with the

type y and b ions series indicated. Nano LC-MS ⁄MS

analyses were performed upon tryptic and endo protei-

nase GluC (Staphylococcus areus protease V8; New Eng-

land Biolabs, Ipswich, MA, USA) digests of the protein,

and resulted in 98% sequence coverage. This process

identified both modified and unmodified peptides

(Fig. 4C).

Determination of glycan attachment site by

electron transfer dissociation

Examination of the modified peptide sequences showed

no evidence of the classic eukaryotic N-linked or the

more recently defined prokaryotic N-linked consensus

sequons [46]. The glycan linkage was therefore sus-

pected to be O-linked. Although the modified glyco-

peptides were readily detected, based upon mass

differences from the predicted sequence, the precise

sites of glycosylation were not. The labile nature of the

glycosidic bond between the hydroxyl amino acid of

serine or threonine and the carbohydrate residue made

it impossible to detect fragment ions corresponding to

the intact modification on the native glycopeptide. To

date, mapping of O-linked glycans has required chemi-

cal derivatization via alkaline hydrolysis of each glyco-

peptide prior to collision-activated dissociation (CAD)

to map the precise site of glycosylation. Electron trans-

fer dissociation (ETD) differs from traditional CAD in

that it preserves the post-translational modifications of

peptides, thereby allowing direct determination of the

sites of post-translational modification without prior

chemical treatment [47–50]. HPLC fractions containing

glycopeptides from FE9909ACS Alberta FlaA were

fragmented using ETD, and seven sites of modification

were identified at Ser126, Ser139, Ser142, Ser165,

Ser171, Ser176 and Ser182 (Fig. 4C). The ETD spec-

trum of peptide T is shown in Fig. 4B, and the corre-

sponding c and z ions are highlighted. The mass

increase imparted by these modifications of the intact

protein was consistent with the measured masses indi-

cating occupancy of 3–7 sites, with a glycan of mass

417 Da on each FlaA monomer. Some microhetero-

geneity was observed at certain serine residues, for

example peptide T was found in two forms, with either

one or both serine residues modified. Accurate mass

measurements were performed on the glycan oxonium

ions in the MS ⁄MS spectra to determine a plausible

A

B

C

Fig. 4. MS ⁄ MS and electron transfer dissociation mass spectrum

of the VGS*INIGS*GK tryptic peptide (asterisks indicate modified

amino acids). (A) MS ⁄ MS spectrum of peptide T of FlaA. Peptide

type y and b fragmentation ions are indicated, and an intense ion at

m ⁄ z 418 was observed, corresponding to the putative glycan oxoni-

um ion. (B) ETD mass spectrum obtained on the [M + 3H]3+ ion at

m ⁄ z 589.4, corresponding to the same peptide T as FlaA. Observed

fragment ions of type c and z are indicated. This peptide was found

to harbor two glycan modifications of 417 Da, on serines 171 and

186 respectively. (C) Peptide sequence coverage obtained from

MS ⁄ MS sequencing of FlaA digested with trypsin or GluC.

Sequenced peptides are indicated in bold, and glycosylation sites

are underlined.



empirical formula for the glycan moiety. The known

masses of peptide fragment ions were used as internal

standards as previously described [39]. The accurate

mass of the glycan oxonium ion was determined to be

418.1822 Da (± 0.0015 Da). Thus the top-ranked

plausible elemental composition for the glycan residue

was C17H28N3O9.

NMR structural analysis of the flagellin

glycopeptide

Structural elucidation of the glycan was accomplished

using a purified tryptic glycopeptide (glycopeptide 1),

and glycopeptide 2, which was produced from exten-

sive proteolytic digestion of intact flagellin using pro-

teinase K (Figs 5 and 6). MS analysis showed that

glycopeptide 2 had the sequence GSAK. Homonuclear

and heteronuclear NMR experiments on these samples

allowed complete assignment of the 1H and 13C reso-

nance shifts (Table 3) and determination of the glycan

structure, which was identical for both peptides.

For the tryptic glycopeptide 1, due to the small

amount material that was available, the signal-to-noise

ratio was maximized by concentrating the sample from

150 to 50 lL by the use of two immiscible liquid plugs.

Diffusion-weighted NMR spectroscopy was used to

differentiate resonances of the higher-molecular-mass

peptidyl glycan from those of contaminants. Unusual

resonances were observed that did not correspond to

known structures, especially the presence of a singlet

at 2.74 p.p.m. (Fig. 5). This experiment effectively

attenuated signals from the lower-molecular-mass

contaminants (higher diffusion rates), leaving reso-

nances from the larger peptide as well as the glycan as

the dominant peaks in the NMR spectrum (Fig. S2).

Selective TOCSY experiments on the putative glycan

resonances were then used to assign the proton reso-

nances and measure coupling constants (Fig. 5). The

Fig. 5. Structure and proton NMR analyses

of the O-linked glycan found on the glyco-

peptides isolated from the flagellin of

C. botulinum. (A) Stucture and atom num-

bering for aLeg5GluNMe7Ac. (B) 1H-NMR

spectrum (16 scans) of protein-

ase K-digested glycopeptide 2 (GS*AK).

(C) 1H-NMR spectrum (256 scans) of the

tryptic glycopeptide (VGS*INIGS*GK). (E–G)

Selective TOCSY experiments for the tryptic

glycopeptide using a mixing time of 80 ms

for the Leg-H3ax (D), Leg-H9 (E) and

glutamate H4 (F) resonances.



chemical shifts and proton coupling constants, as well

as the strong H4–H6 and H3ax–H5 NOEs (Fig. S3),

were characteristic of those found for legionaminic acid

[51–53]. Legionaminic acid was determined to have the

a anomeric configuration based on the low field shift

of H3eq (2.7 p.p.m.), which indicates an axial carboxyl

group. Following assignment of the 13C chemical shifts

from the 1H–13C HSQC spectrum (Fig. 6), comparison

of the proton and carbon chemical shifts with those

reported previously for authentic standards [51,52]

indicated that legionaminic acid had the d-glycero-

d-galacto configuration. This conclusion was also

supported by a strong H9–H7 NOE [45] (Fig. S2).

In addition to the signals for legionaminic acid and

known amino acids from the peptides (Table S1), the1H-NMR spectra of the glycopeptides contained a spin

system for glutamic acid and a methyl group. An ace-

tyl resonance was also observed, which, for the tryptic

peptide, appeared as a doublet due to the two sites of

glycosylation (Fig. 5). Proton and carbon resonances

were assigned (Table 3) from TOCSY (Fig. 5) and

HSQC experiments (Fig. 6). The location of these

groups was determined from the HMBC experiment

on glycopeptide 2 (Fig. 6). The HMBC correlation

(NMe:E2) between the CH3 proton resonance and the

C2 resonance of Glu and the NOE between the methyl

resonance and H2 resonance of Glu (data not shown)

indicated N-methylation of its amino group. The

chemical shift of the glutamate C2 at dC 64.4 p.p.m.

(approximately 9 p.p.m. from its non-methylated posi-

tion) also indicated N-methylation. The signal of the

C5 (carbonyl group) of glutamate was identified from

the HMBC correlation E4:5, between H4 and C5

(Fig. 6). The HMBC correlations, X5:E5, from H5 of

5,7-diacetamido-3,5,7,9-tetradeoxy-d-glycero-d-galacto-

nonulosonic acid (Leg) to C5 of Glu, and the X7:Ac1

correlation from H7 of Leg to C1 (C¼O) of acetate,

identified the respective acylation positions as

Leg5GluNMe7Ac. The HMBC correlation, S3:X2,

between H3 of Ser and C2 of Leg, also confirmed

Fig. 6. Heteronuclear NMR experiments for

the O-linked glycan aLeg5GluNMe7Ac for

glycopeptide 2 (GS*AK). In the overlap of

the 1H-13C HSQC and HMBC spectra, the

single-bond H–C correlations are indicated

by a residue code and atom number using

the single letter code for the amino acids, X

for Leg, Ac for the acetyl group, and NMe

for the N-methyl group. Multiple bond corre-

lations are indicated as H:C.



O-linked glycosylation to Ser. Based on these NMR

results, the glycan O-linked to Ser on the flagellin gly-

copeptides was determined to be 5,7,9-tetradeoxy-d-

glycero-a-d-galacto-nonulosonic acid, N-acylated with

N-methylated glutamate at C5 and acetate at C7

(aLeg5GluNMe7Ac).

Detection of flagellin signature ions in

C. botulinum culture supernatants

We next examined a C. botulinum culture supernatant,

containing BoNT, to determine whether unique flagel-

lin fragment ions can be used as surrogate markers for

detection of BoNT contamination. A culture superna-

tant from C. botulinum strain FE9909ACS Alberta was

prepared, which contained 20 000 mouse lethal doses ⁄mL by the mouse bioassay. This preparation was then

heat-inactivated. We examined this fraction by MS-

based methods, and were indeed able to detect flagellin

protein as well as the C. botulinum flagellin-specific

marker ions at m ⁄ z 418 and m ⁄ z 512. (Fig. 7A,B). The

deconvoluted mass profile is presented in the inset to

Fig. 7A, and the protein mass is in close agreement

with that obtained for purified flagellin from this strain

(Fig. 1B). Multiply charged protein ions at m ⁄ z 1197

and 1346 were selected for tandem mass spectrometry.

Spectra were acquired at 15–25 V collision energy using

argon as the collision gas, with marker ions characteris-

tic C. botulinum flagellin observed at m ⁄ z 418.2, 512.3

and 1023.6. Increasing the RF lens 1 voltage from 40

to 90 V resulted in non-specific fragmentation in the

orifice ⁄ skimmer region of the mass spectrometer,

increasing the intensity of flagellin marker ions at m ⁄ z418.2 and 512, and allowing MS ⁄MS spectra to be

obtained for each ion. The MS ⁄MS spectrum of m ⁄ z512.3 yielded a clear series of peptide type y and b ions,

giving the sequence PQGVLQLLR. MS ⁄MS spectra

were obtained for m ⁄ z 418.2, yielding a series of frag-

ment ions characteristic of the glycan modification of

C. botulinum strain FE9909ACS Alberta flagellin,

thereby confirming that the labile flagellin marker ions

can be detected in a complex protein mixture that con-

tains BoNT.

Discussion

In this study, we have demonstrated that the flagellins

of C. botulinum are glycosylated in O-linkage with

novel glycans at up to seven sites per monomer. Top-

down MS analysis of flagellin proteins prepared from

Table 3. NMR data for the O-linked nonulosonic acid derivative,

aLeg5GluNMe7Ac, found on glycopeptides from flagella of

C. botulinum. Carbon and proton chemical shifts were referenced

to an internal acetone standard (dH 2.225 p.p.m. and dC

31.5 p.p.m.). The error for dH is ± 0.02 p.p.m., that for dC is

± 0.4 p.p.m., and that for JH,H is ± 0.2 Hz. J, coupling constant;

d, chemical shift. AC, acetyl; GluNMe, N-methyl-glutamyl.

Residue

Proton

(1H)

dH

(p.p.m.)

Carbon

(13C)

dC

(p.p.m.) JH,H J (Hz)

Leg C1 174.6

C2 101.8 J3ax,3eq 13.2

H3ax 1.68 C3 40.7 J3ax,4 11.0

H3eq 2.71 J3eq,4 3.9

H4 3.59 C4 69.9 J4,5 10.0

H5 3.69 C5 53.3 J5,6 10.4

H6 3.92 C6 73.0 J6,7 3.0

H7 3.85 C7 55.2 J7,8 3.0

H8 3.95 C8 68.4 J8,9 6.3

H9 1.16 C9 19.4

GluNMe C1 174.6

H2 3.61 C2 64.4

H3 2.12 C3 26.1

H4 2.38 C4 32.9

C5 176.0

Me 2.73 Me 33.1

Ac C=O 175.2

CH3 2.02 CH3 23.3

A

B

Fig. 7. Detection of flagellin in culture supernatants from C. botu-

linum strain FE9909ACS Alberta. (A) Electrospray mass spectrum

of crude BoNT preparation. The inset shows the deconvoluted

mass profile, indicating an intact protein mass of 32 300 Da. The

arrows indicate protein ions that were selected for tandem mass

spectrometry. (B) Tandem mass spectrometry analyses of a multi-

ply charged protein ion at m ⁄ z 1197.027+. Ions at m ⁄ z 418.2+,

512.32+ and 1023.6+ were observed in the tandem mass spectrum.



strains belonging to a number of unique toxin types

revealed diversity in glycan structure amongst isolates.

Glycan mass and MS ⁄MS fragmentation patterns of

each of the glycan oxonium ions showed that group I

C. botulinum strains are able to glycosylate flagellin

monomers with derivatives of either a nonulosonate

sugar, a di-acetamido-substituted hexuronic acid sugar

or a tri-acetamido-substituted hexuronic acid sugar. In

contrast, preliminary studies of group II C. botulinum

strains revealed that, while type B and type F strains

produce flagellins of increased mass that were glycosy-

lated with a novel glycan of mass 696 Da, the FlaB

flagellin from group II type E strains is distinctive in

that it is a higher-molecular-mass flagellin protein

that is not glycosylated. It has been demonstrated

indirectly that numerous clostridial species appear to

glycosylate their flagellin proteins, but this is the first

study to provide specific structural details of clostridial

flagellar glycan modifications. Previous work demon-

strating the sensitivity of the glycan from flagella of

C. acetobutylicum to neuraminidase treatment provided

the first evidence that glycosylation may involve

a sialic acid-related sugar [28]. More recent comparative

genomic analysis revealed the presence of homologs of

sialic acid biosynthetic genes in the completed genomes

of C. acetobutylicum and C. tetani [43,54].

In the present study, NMR and MS were used for

structural assignment of a novel 417 Da glycan found

on flagellins of the group I type A strain FE9909ACS

Alberta and the type F strain Langeland. Using a com-

bination of MS and NMR techniques, we have demon-

strated that these strains of C. botulinum glycosylate

flagellin not with sialic acid but with a novel derivative

of the sialic acid-like nonulosonate sugar legionaminic

acid, Leg5GluNMe7Ac. Legionaminic acid belongs to

the class of nonulosonic acids that consists of 5,7-diami-

no-3,5,7,9-tetradeoxy nonulosonate derivatives uniquely

found in microorganisms. The sugars of this class have

been reported as components of a variety of capsular

polysaccharides and lipopolysaccharides from a number

of Gram-negative bacteria [51]. More recently, these

nonulosonate derivatives have been shown to be com-

ponents of the pilin glycoprotein from P. aeruginosa [3]

as well as of the flagellar glycoproteins of Ca. jejuni ⁄ coli,H. pylori and Aeromonas caviae [12,12,39].

This is the first report of the biosynthesis of a nonu-

losonate sugar in a Gram-positive, anaerobic bacteria.

It remains to be established whether the presence of

these nonulosonate sugars facilitiates a host ⁄pathogeninteraction or imparts a novel biological function to

C. botulinum cells. However, the increasing number of

examples of pathogenic species that incorporate these

sugars on prominent cell-surface glycoconjugate struc-

tures suggests a potential role in host ⁄pathogeninteraction. It is noteworthy that three of the four

strains associated with infant botulism (FE9909ACS

Alberta, FE0303AYO and FE9904BMT) produced

glycan fragmentation patterns characteristic of legio-

naminic acid derivatives, while the flagellins from three

of four strains not associated with C. botulinum

infections (PA9508, 17A and H461297F) were modified

with a di-N-acetylhexuronic acid derivative. It has been

suggested that the similarity of the bacterial nonulo-

sonic acids to sialic acids may provide an immune

‘cloak’, whereby the host response to the invading

bacterium is less intense [51].

Future work will be directed towards examination

of flagellin glycans from distinct epidemiological clus-

ters of C. botulinum to determine whether there is

indeed a correlation between glycan structure and

pathogenic potential. Until recently, the genetic manip-

ulation of Clostridium spp. has been difficult. However,

with the recent report of a mutagenesis system based

on the mobile GpII intron for Clostridium spp., it will

now be possible to perform functional studies on puta-

tive flagellar glycosylation genes [55]. This system will

provide the means to determine the role of these dis-

tinct glycan moieties in both flagellar assembly and

other biological interactions that may be important for

C. botulinum pathogenesis.

Many of the tools for the identification and genetic

discrimination of group I C. botulinum strains are

based on a very limited number of structurally and

genetically similar toxin types. The recent work of

Paul et al. [35,36] using flagellin gene sequencing has

provided an additional means to more accurately dis-

tinguish C. botulinum isolates. We demonstrate here

that top-down MS analysis of the flagellin protein

can be utilized to rapidly detect C. botulinum through

identification of a common C-terminal peptide marker

ion (m ⁄ z 512). In addition, this top-down analysis has

revealed the presence of unique flagellar glycan mar-

ker ions that are found exclusively on individual

strains and could also be utilized for strain typing.

Genomic comparisons of the FGI from the two

C. botulinum strains for which the genome sequence is

available (ATCC3502 and Langeland) indicated that

diversity in the gene content at this locus may also

provide additional discriminatory power for typing of

isolates.

In a similar manner, extensive genomic analysis of

multiple Campylobacter strains revealed that the FGI

was a hypervariable region of the chromosome. Diver-

gence in genetic content at the Campylobacter flagellar

glycosylation locus led to identification of a cluster of

genes within this locus that were shown to be a genetic



marker for a livestock-associated clade [56]. Two of

the genes identified from this cluster – ptmG (Cj1324)

and ptmH (Cj1325 ⁄ 6) – have been shown to be

involved in the biosynthesis of the flagellar glycans

Leg5Am7Ac (ptmG) and Leg5AmNMe7Ac (ptmH)

[45]. In the present study, bioinformatic analysis identi-

fied a homolog of ptmH in the C. botulinum Langeland

genome (CLI_2776), together with homologs of other

legionaminic acid biosynthetic genes (see Table 2). It

remains to be established whether diversity in flagellar

glycan biosynthetic capacity in C. botulinum is related

to host specificity and the colonization ability of

isolates.

Detailed characterization of prokaryotic glycopro-

teins still presents technical challenges. MS and

MS ⁄MS techniques provide substantial information on

the nature of a particular glycan, the type of linkage

and the site of attachment, although the lability of par-

ticular glycan modifications may still be problematic.

In this study we utilized the electron transfer dissocia-

tion soft fragmentation technique to map all glycan

attachment sites without the need for chemical deriva-

tization. In this manner, the amino acid sequence and

glycan attachment sites for all glycopeptides, regardless

of size or charge state, were successfully identified. A

larger glycopeptide ion failed to yield informative frag-

ment ions by ETD alone, instead mainly generating

charge-reduced precursor ions. In this instance, frag-

ment ions were generated by performing gentle colli-

sion-activated dissociation with a low Q value on the

charge-reduced precursor ions [57]. This process

favored the production of c and z fragment ions over

production of b and y ions, allowing mapping of the

glycan attachment site.

Detailed structural elucidation of novel glycan moie-

ties or glycopeptides by NMR requires substantially

higher amounts of material (lg) of considerably

greater purity than is required for MS analysis (ng).

These requirements can provide a considerable techni-

cal challenge. This is especially true for glycoproteins

from fastidious microorganisms or organisms that

require bio-containment. In this study, we have dem-

onstrated that, even for the very ‘sample limited’ and

partially purified peptidyl glycan samples that are

obtained from C. botulinum flagellin, diffusion-

weighted experiments and sample concentration with

immiscible plugs enable these challenges to be over-

come, facilitating rigorous definition of structures by

NMR techniques.

Preparation of glycopeptide by trypsin digest pro-

vided sufficient quantity and quality of sample to eluci-

date the basic glycan structure by NMR, as done

previously for determination of pseudaminic acid on

Ca. coli flagellin [39]. Due to the unusual nature of the

N-methyl glutamate modification, larger amounts were

required in order to detect the weak HMBC correla-

tions required to determine unambiguously the loca-

tion of acetyl and N-methyl-glutamyl groups on

legionaminic acid. In this case, extensive proteinase K

hydrolysis of the flagellin protein produced glycan

attached to only three or four amino acids, which

could be more readily fractionated from non-glycosy-

lated peptides by gel chromatographic separation in a

manner similar to that described by Voisin et al. [38].

Different glycopeptide preparations also helped to dis-

tinguish the signals from the amino acid linked to Leg

from those in the peptide. Both purification methods

are complementary, and their use depends on the nat-

ure of the glycan being isolated and characterized.

The ability to use mass spectrometry to rapidly iden-

tify the flagellar modifications of C. botulinum opens

the possibility for screening large numbers of strains

for unique sugars to potentially establish biological

correlations for strains of C. botulinum. This could

include geographic distributions of strains, coloniza-

tion potential and type of disease caused (i.e. coloniza-

tion versus wound botulism). Flagellar modification

with nonulosonate sugars may be correlated with the

ability of organisms such as C. botulinum to establish

infections in the gastrointestinal tract, and will be the

subject of future studies.

Experimental procedures

Bacterial strains

C. botulinum strains were stored and archived at the

Botulism Reference Service for Canada at )86 �C on

Microbank� beads (Pro-Lab Diagnostics, Richmond Hill,

Canada), and their sources have been detailed elsewhere [35].

All C. botulinum cultures were routinely grown in SPGY

broth containing 5% w ⁄ v special peptone (Oxoid Inc.,

Basingstoke, UK), 0.5% w ⁄ v peptone (Difco, Tucker, GA,

USA), 2% w ⁄ v yeast extract (Difco), 0.4% w ⁄ v glucose

(Difco) and 0.1% w ⁄ v sodium thioglycolate (Sigma-Aldrich,

St Louis, MO, USA), adjusted to a pH of 7.2 using HCl.

Cells were grown for 24–48 h in an atmosphere of 10% H2,

10% CO2 and 80% N2 at either 35 �C (group I strains) or

room temperature (group II strains).

Purification of flagella

Flagella proteins were isolated as described previously [36].

In brief, cultures were grown overnight in 100 mL SPGY

broth. Cells were harvested and flagella were sheared from

the cell surface using a 50 mL tissue homogenizer. Follow-



ing removal of whole cells by low-speed centrifugation,

flagellar filaments were collected by centrifugation at

130 000 g for 1 h, pellets were washed in ultrapure water,

re-centrifuged, and resuspended in ultrapure water. Before

any downstream analysis, all flagellin protein preparations

were heat-treated for 25 min at 75 �C to denature any

BoNT present in the sample. The purity of flagellar prepa-

rations was determined by SDS–PAGE.

Mass spectrometry of isolated flagellins

Purified flagellin was dialysed in aqueous 0.2% v ⁄ v formic

acid using a Centricon YM-30 membrane filter (Millipore,

Billerica, MA, USA). The resulting solution was infused in

a QTOF2 hybrid mass spectrometer (Waters, Milford, MA,

USA) at a flow rate of 0.5 lLÆmin)1. Top-down experi-

ments were performed as described by Schirm et al. [37]

using argon collision gas with collision energies ranging

from 20–30 V. The RF lens 1 voltage was increased from

30 to 125 V in order to obtain second-generation fragment

ion spectra.

Solution enzymatic digests

To identify the type and location of glycosylation sites,

flagellin (50–200 lg) was digested with trypsin (Promega,

Madison, WI, USA) at a protein:enzyme ratio of 30 : 1

(v ⁄ v) in 50 mm ammonium bicarbonate at 37 �C overnight

or with endoproteinase GluC (Sigma) at a protein:enzyme

ratio of 25 : 1 (v ⁄ v) in 100 mm phosphate buffer, pH 7.8,

at 37 �C overnight. Protein digests were analysed by nano-

LC-MS ⁄MS using a QTOF2 or QTOF Ultima hybrid

quadrupole time-of-flight mass spectrometer coupled to a

CapLC capillary HPLC system (Waters). MS ⁄MS spectra

were acquired automatically on doubly, triply and quadru-

ply charged ions. Peak lists were automatically generated

by proteinlynx software (Waters) with the following

parameters: smoothing – four channels, two smooths,

Savitzky–Golay mode; centroid – minimum peak width at

half height of four channels, centroid top 80%. Tryptic

peptides were analysed by nano-LC-MS ⁄MS, and spectra

were searched against the National Centre for Biotechno-

logy nonredundant database and an in-house database of

sequenced C. botulinum flagellin proteins using mascot

2.0.1 (Matrix Science, London, UK), as described previ-

ously [35]. MS ⁄MS spectra that did not correspond to

predicted tryptic peptides were examined manually.

HPLC purification of tryptic peptides

Tryptic digests of flagellin were fractionated using an

Agilent 1100 series HPLC with a diode array detector

(Agilent Technologies, Palo Alto, CA, USA). Each tryptic

digest (100 lL) was separated using a 4.6 · 250 mm Jupiter

C18 reverse-phase column with a Phenomenex pre-column

(SecurityGuard, Torrance, CA, USA). Peptides were sepa-

rated using a linear gradient of 5–60% acetonitrile, 0.5%

formic acid over 40 min at a flow rate of 1 mLÆmin)1. A

post-column splitter was used to divert approximately

60 lLÆmin)1 of column eluate to the electrospray interface

of the QTOF2 to allow real-time monitoring of ion elution

profiles, allowing specific peptides to be isolated as they

eluted from the column. A 7 lL aliquot of each fraction

was retained, and the remainder immediately evaporated to

dryness and stored at )20 �C. Aliquots of each fraction

were screened by nano-LC-MS ⁄MS using the QTOF2 to

confirm the peptide contents of each HPLC fraction. Pure

tryptic glycopeptide (glycopeptide 1) was thus obtained.

Accurate mass measurement of C. botulinum

glycan

A glycopeptide-containing HPLC fraction was infused in

the QTOF Ultima at 1 lLÆmin)1 and the MS ⁄MS spectrum

was recorded over a period of 30 min. Accurate mass deter-

mination of the glycan oxonium ion was achieved using a

number of neighboring peptide fragment ions as internal

mass standards.

Determination of glycan linkage sites by electron

transfer dissociation

Electron transfer dissociation preserves delicate modifica-

tions during the fragmentation process and is ideal for

identifying the linkage sites of O-glycans [47,49,58]. Glyco-

peptide-containing HPLC fractions were infused at 1 lLÆmin)1 into the electrospray ionization source of an LTQ XL

linear ion trap (Thermo Fisher Scientific, Nepean, Canada)

capable of performing ETD. Initially, CAD MS ⁄MS analysis

was performed on the glycopeptide ions to confirm their

identity. ETD was performed using fluoranthene as the anio-

nic reagent and with supplementary activation enabled. The

ETD reaction time was adjusted for optimal fragmentation

of each glycopeptide (typically 35 ms). One glycopeptide ion

failed to yield informative fragment ions by ETD alone,

instead mainly generating charged-reduced precursor ions. In

this instance, c and z fragment ions were generated by per-

forming gentle CAD (10 V) on the individual charge-reduced

precursor ions generated by ETD. Production of c and z frag-

ment ions was favored over b and y ions by reducing the

Q value from its default of 0.25 to 0.15 [57].

Preparation of glycopeptide using proteinase K

digestion

A preparation of flagellin (8 mg) in water was adjusted to

pH 8.5 by addition of Na2HPO4. Proteinase K (10 mg) was



added, and the solution was kept at 50 �C for 1 week. The

resulting digest was filtered through a SepPak C18 column

(Waters) (pre-washed with methanol and then water) and

desalted on a Sephadex G15 column (1.6 · 80 cm; Sigma-

Aldrich) in pyridine (0.4%) ⁄ acetic acid (1%) in H2O, moni-

tored by a refractive index detector. All fractions (5 mL

each) eluted before the salt peak were dried and analyzed by1H-NMR. A relatively clean fraction containing a glycopep-

tide (glycopeptide 2) as well as mixtures of this and other

glycopeptides were collected and analyzed by NMR and MS.

NMR analysis of purified glycopeptide

The HPLC-purified glycopeptide 1 sample [VGSINIGSGK

(169–178)] was lyophilized and resuspended in 99% D2O

(Cambridge Isotope Laboratories Inc., Andover, MD,

USA) and analyzed using NMR spectroscopy. To increase

the level of signal to noise by approximately threefold over

a conventional aqueous NMR sample, the sample was

resuspended in a minimum amount of D2O (50 lL) and

centered between two insoluble liquid plugs of FC43

(Sigma-Aldrich) in a 3 mm NMR tube. The aqueous sam-

ple was centered in the RF detection coil of the NMR

probe. The fraction containing glycopeptide 2 was lyophi-

lized and resuspended in 150 uL D2O in a 3 mm NMR

tube. For both samples, standard homo- and heteronuclear-

correlated two-dimensional 1H-NMR, 13C-HSQC, HMBC,

DQCOSY, NOESY and ROESY pulse sequences from

Varian (Palo Alto, CA, USA) were used for general assign-

ments. Selective one-dimensional TOCSY experiments with

a Z-filter were used for complete residue assignments and

measurement of proton coupling constants [59]. NMR spec-

tra were acquired on a Varian INOVA 500 MHz (1H) spec-

trometer with a Varian Z-gradient 3 mm triple resonance

probe (1H, 13C and 31P) or a Varian 600 MHz (1H) spec-

trometer equipped with a Varian 5 mm Z-gradient triple

resonance (1H, 13C and 15N) cryogenically cooled probe

(cold probe) for optimized sensitivity. NMR experiments

were acquired at 25 �C with suppression of the HOD reso-

nance at 4.78 p.p.m. by solvent presaturation. For proton

and carbon experiments, the methyl resonance of acetone

was used as an internal reference (dH 2.225 p.p.m. and dC31.5 p.p.m.). Diffusion-weighted NMR spectroscopy at

500 MHz was performed using a bipolar pulse pair-longitu-

dinal encode ⁄ decode (BPP-LED) to attenuate signals aris-

ing from low-molecular-mass contaminants and enhance

signals originating from larger molecules, thereby simplify-

ing the NMR spectrum [60].

Bioinformatic analysis of flagellar glycosylation

loci

The FGI in the genome sequence of C. botulinum strain

Langeland (Genbank accession number NC_009699) was

identified by first locating the ORFs corresponding to

homologs of the FGI flanking proteins FlgB and FliD by

blastp (http://www.ncbi.nlm.nih.gov/BLAST/). Open read-

ing frames between FlgB and FliD were then compared to

either the Hall A genome sequence (Genbank accession

number NC_009495), the Ca. jejuni NCTC11168 genome

sequence (Genbank accession number NC_002163) or the

complete NCBI protein sequence database to assign puta-

tive identities. The flagellar glycosylation locus map was

compiled using enhance map draw version 2.0 (Sci-Ed

Software, Carey, NC, USA).

Preparation of C. botulinum culture supernatants

Culture supernatants of C. botulinum were prepared using

the standard method employed by the Botulism Reference

Service of Canada with minor modifications. Briefly,

C. botulinum strain FE9909ACS was grown in 100 mL

SPGY broth for 5 days. Bacteria were removed by centrifu-

gation at 10 000 g for 10 min at 4 �C and the supernatant

divided. One half was filter-sterilized using a 0.22 lm filter

for toxin quantification by mouse bioassay (see below). The

remaining supernatant was heated for 25 min at 75 �C to

inactivate BoNT, but was not filter-sterilized. Heated super-

natant was dialyzed into Milli-Q water (Millipore) and con-

centrated tenfold using an Amicon spin filter (molecular

mass cut-off 10 000 Da) and diluted to the desired concen-

trations for MS detection of FlaA.

MS detection of flagellar marker ions in

C. botulinum culture supernatants

Concentrated culture supernatant (50 lL) was injected onto

a protein microtrap (Chromatographic Specialties, Brock-

ville, Canada) connected to the Agilent HPLC and washed

offline with 500 lL of Milli-Q water. An HPLC gradient of

5–60% acetonitrile, 0.2% formic acid (1 mLÆmin)1) over

60 min was used to resolve the protein mixture. A pre-col-

umn splitter was used to direct approximately 60 lLÆmin)1

of the HPLC mobile phase through the trap and into the

electrospray interface of the QTOF2 to allow real-time

monitoring of ion elution profiles. The MS ⁄MS spectra of

multiply charged ion peaks were recorded as described

above. When low-intensity ions at m ⁄ z 418 and 512 were

observed, the RF lens voltage was increased until the inten-

sity of these ions was sufficient to collect MS ⁄MS data.

MS ⁄MS data were then collected for each ion to confirm

that these were the labile flagellin-associated ions.

Mouse bioassay for BoNT

The mouse bioassay was conducted as previously described

[61]. Briefly, samples of flagellin or filter-sterilized culture

supernatant were diluted in gelatin phosphate buffer up to

1 ⁄ 1 000 000. Samples were tested by intraperitoneal injection



into a minimum of two female CFI white mice. Mice were

observed for 72 h for symptoms of BoNT intoxication. The

observational endpoint used was pinched waist with labored

breathing, after which animals were killed. These experiments

were carried out in accordance with the guidelines laid down

by the National Institutes of Health in the USA regarding

the care and use of animals for experimental procedures.

Acknowledgements

This work was supported by project CRTI-02-0091TA

of the Defense Research and Development Canada

Chemical, Biological, Radiological and Nuclear Res-

earch and Technology Initiative (J.W.A. and S.M.L.).

We thank Luc Tessier for technical assistance with mass

spectrometers and Greg Sanders and Jeff Bussey for

conducting mouse bioassays for BoNT activity.

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Supporting information

The following supporting information is available:

Fig. S1. Second-generation product ion spectra of

multiply charged flagellin ions.

Fig. S2. BPP-LED NMR analysis of the tryptic glyco-

peptide.

Fig. S3. NOESY NMR 1D traces of tryptic glycopep-

tide.

Table S1. Tryptic glycopeptide chemical shifts.

This supporting information can be found in the

online version of this article.

Please note: Blackwell Publishing are not responsible

for the content or functionality of any supporting

information supplied by the authors. Any queries

(other than missing material) should be directed to the

corresponding author for the article.



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