Profiling of: Haemophilus influenzae strain R2866 with carbohydrate-based covalent probes

Article abstract of DOI:10.1039/d0ob01971b

We demonstrate the application of four covalent probes based on anomerically pure d-galactosamine and d-glucosamine scaffolds for the profiling of Haemophilus influenzae strain R2866. The probes have been used successfully for the labelling of target prot

Full text of DOI:10.1039/d0ob01971b

Organic &
Biomolecular Chemistry
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Profiling of Haemophilus influenzae strain R2866
with carbohydrate-based covalent probes†
Cite this: Org. Biomol. Chem., 2021,
19, 476
Camille Metier,a Jennifer Dow,b Hayley Wootton,a Steven Lynham,c Brendan Wrenb
d
and Gerd K. Wagner
*
We demonstrate the application of four covalent probes based on anomerically pure D-galactosamine
and D-glucosamine scaffolds for the profiling of Haemophilus influenzae strain R2866. The probes have
been used successfully for the labelling of target proteins not only in cell lysates, but also in intact cells.
Differences in the labelling patterns between lysates and intact cells indicate that the probes can pene-
trate into the periplasm, but not the cytoplasm of H. influenzae. Analysis of selected target proteins by
LC-MS/MS suggests predominant labelling of nucleotide-binding proteins, including several known anti-
bacterial drug targets. Our protocols will aid the identification of molecular determinants of bacterial
pathogenicity in Haemophilus influenzae and other bacterial pathogens.
Received 25th September 2020,
Accepted 12th October 2020
DOI: 10.1039/d0ob01971b
rsc.li/obc
readily water soluble, which is a considerable advantage for
applications in biological media. Sugars such as D-glucose and
Introduction
The profiling of pathogenic bacteria with chemical probes is a α-lactose are preferred bacterial carbon sources10 and, together
powerful strategy to uncover molecular determinants of bac- with other sugars, serve as building blocks for complex glycans
terial pathogenicity, virulence, and antimicrobial resistance.1–3 of the bacterial cell wall (e.g., lipopolysaccharide, peptidogly-
Typically, such probes contain a reactive moiety for the for- can).11 Carbohydrates and carbohydrate-recognising proteins
mation of a covalent linkage, enabling the irreversible label- are often directly involved in multiple aspects of bacterial
ling of target proteins. Both targeted4 and target-agnostic5 pathogenicity,12 including adhesion,12 immune evasion,12
approaches have been explored for such applications, with virulence,13,14 and biofilm formation.15 Carbohydrate-based
natural products being a particularly common scaffold for covalent probes are therefore excellent tools for the profiling of
probe development.6–8 For target deconvolution in complex bacterial cells and the labelling of proteins involved in bac-
systems, such covalent probes can be conveniently interfaced terial pathogenicity.
with bioanalytical methods such as mass spectrometry and gel
electrophoresis.9
Several examples for the labelling of bacterial proteins with
covalent probes based on a carbohydrate, or carbohydrate-like,
Carbohydrates are an attractive scaffold for the develop- scaffold have previously been reported. Usually, these probes
ment of covalent probes for the profiling of bacterial patho- are designed to specifically recognise a single target protein.
gens, both from a chemistry and microbiology perspective. Thus, a glucosamine-based probe has been used successfully
Even a simple monosaccharide allows the facile installation of to capture the β-N-acetylhexosaminidase NagZ, which is
both an electrophilic warhead for covalent labelling, and a involved in the induction of resistance to β-lactams, from non-
reporter group for detection (Fig. 1). Moreover, most sugars are purified lysates of P. aeruginosa.16 More recently, Titz et al.
have described a galactose-derived probe for the labelling and
in vitro imaging of the lectin LecA, a virulence factor and
aKing’s College London, Department of Chemistry, Britannia House, 7 Trinity Street,
biofilm building block from P. aeruginosa.17 A probe based on
London, SE1 1DB, UK
bLondon School of Hygiene and Tropical Medicine, Department of Infection Biology,
Keppel Street, London, WC1E, 7HT, UK
cProteomics Facility, Centre of Excellence for Mass Spectrometry, King’s College
London, The James Black Centre, 125 Coldharbour Lane, London, SE5 9NU, UK
dQueen’s University Belfast, School of Pharmacy, Medical Biology Centre, 97 Lisburn
Road, Belfast, BT9 7BL, UK. E-mail: G.Wagner@qub.ac.uk
†Electronic supplementary information (ESI) available: Synthetic protocols for
fluorophore controls 9 and 10; additional Fig. S1–S6; LC-MS/MS analysis of SDS-
page gel bands; spectroscopic data for compounds 2–7, 9 and 10. See DOI:
10.1039/d0ob01971b
Fig. 1 General design of carbohydrate-based covalent probes.
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the cyclitol mimic cyclophellitol aziridine has been used for acetamido) derivative 5. Finally, the reporter group was
the labelling of the retaining GH29 α-L-fucosidase from attached at the anomeric position via a short linker. 5 was
Bacteroides thetaiotaomicron, both in recombinant form and in reacted with propynol ethoxylate under Fischer glycosylation
lysates from an E. coli culture overexpressing the target conditions to give glycoside 6 as a mixture of both anomers,
enzyme.18
which were separated by normal phase chromatography. Each
We have recently used a covalent probe based on a anomer was reacted separately in a Cu-catalysed 1,3-dipolar
D-glucosamine scaffold for the profiling of K. pneumoniae cycloaddition with azide 9,28 introducing a dansyl fluorophore,
lysates.19 In the present study, we have extended our general to give the D-galactosamine-based target molecules 7-α and 7-
approach to a different scaffold based on D-galactosamine, and β. The successful cycloaddition was evidenced by 1H NMR
another bacterial pathogen, Haemophilus influenzae. (CDCl3), which showed the diagnostic triazole signal at 7.61
H. influenzae is a Gram-negative coccobacillus responsible for and 7.67 ppm for 7-α and 7-β respectively. The corresponding
life-threatening invasive diseases such as pneumonia and D-glucosamine derivatives 8-α and 8-β as well as their deacety-
meningitis.20
A
cytoplasmic glycosylation system of lated parent sugars 11-α and 11-β (Scheme 1B) were prepared
H. influenzae has been reported, and many carbohydrate as previously reported.19
binding proteins21 involved in the pathogenicity of the bacter-
ium, including lectins22 and glycosyltranferases,23 have been
Assignment of anomers
identified. H. influenzae strain R2866 is an unusually virulent
The identity of the individual anomers was unambiguously
non-typeable strain first isolated from the blood of children
with meningitis infection.24,25 Although lacking a capsular
polysaccharide structure, H. influenzae R2866 displays elevated
serum resistance and a virulence level approaching that of
encapsulated type b H. influenzae,24 and these traits have been
closely related to the presence of a terminal galactoside
epitope of the outer-membrane lipooligosaccharide (LOS)
structure.24,26
In this study, we sought to assess the capacity of different
carbohydrate-based probes to simultaneously label multiple
target proteins in H. influenzae R2866. We reasoned that such
a protocol may aid not only the profiling of this strain, but
also the identification of antibacterial targets. We were particu-
larly interested in the application of our probes not only with
cell lysates, but also, for the first time, with intact cells. Our
results show that probes derived from D-glucosamine and
assigned for both series by 1D and 2D NMR analysis. The weak
orbital overlap between H1 and H2 protons in the α-anomer is
associated with a small coupling constant 3J1,2, as observed for
3
7-α (3J1,2 3.5 Hz). A large coupling constant J1,2 on the other
hand, is characteristic of the β-anomer (7-β: 3J1,2 8.5 Hz). These
assignments are further supported by ROESY spectra of each
anomer. The 1,3-diaxial orientation of the H1, H3, and H5
protons in the β-anomer leads to diagnostic H1–H3 and H1–H5
crosspeaks, as observed in the ROESY spectrum of 7-β (ESI
Fig. S1†). In contrast, these crosspeaks are absent from the
ROESY spectrum of 7-α (ESI Fig. S2†) due to the equatorial
orientation of H1 in the α-anomer. Assignments for 8-α and 8-
β were made as previously reported.19
Labelling of a bacterial glycosyltransferase in cell lysates
D-galactosamine are indeed suitable for such applications. In order to assess the capability of our probes to label a carbo-
Moreover, notable differences between the profiles obtained hydrate-recognising protein, we carried out labelling experi-
from lysates and intact cells suggest, that our protocols can ments with the retaining α-1,4-galactosyltransferase LgtC from
also provide additional information about the localisation of Neisseria meningitidis, which catalyses the transfer of
target proteins within the bacterial cell.
D-galactose from a UDP α-D-galactose donor to a lactose accep-
tor.29 LgtC was overexpressed in BL21* E. coli cells. Cells were
lysed, and cell lysates were incubated for 30 min at 30 °C with
the D-glucosamine derivatives 8-α and 8-β, or the fluorophore
control 10. To assess the potential effect of the acetate protect-
ing groups on labelling, we also included the deacetylated
Results and discussion
Chemical synthesis of carbohydrate-based probes
The two anomers 7-α and 7-β of D-galactosamine-based target sugars 11-α and 11-β (Scheme 1B) in these experiments.
molecule 7 were synthesised in six steps from D-galactosamine Samples were separated on a 12% SDS-page gel under denatur-
1 (Scheme 1A). We reasoned that O-acetylated probes were ing conditions and analysed by in-gel fluorescence emission
more likely than free sugars to penetrate the bacterial mem- and Coomassie staining (Fig. 2).
brane and were therefore more suited for labelling experi-
ments with intact bacterial cells.
In the case of the acetylated probes, both anomers 8-α and
8-β produced a fluorescently labelled band of LgtC, although
First, O-selective acetylation of 1 was achieved in three steps this band was weaker for the β-anomer. Interestingly, in the
via the strategy of Wulffen et al.27 1 was converted into the case of the deacetylated probes, strong fluorescent labelling
corresponding imine 2 by reaction with p-anisaldehyde, fol- was only observed with the α-anomer 11-α. As expected, no lab-
lowed by quantitative O-acetylation of 2. Imine hydrolysis of 3 elling was observed with the fluorophore control 10. These
under acidic conditions gave O-acetylated D-galactosamine 4. results demonstrate that 8-α and 8-β are capable of labelling
Next, the electrophilic warhead was installed in position 2 by target proteins in complex biological media. They also suggest
reaction of 4 with chloroacetyl chloride, to give the 2-(2-chloro- that the presence of the acetate protecting groups, in conjunc-
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Scheme 1 (A) Synthesis of D-galactosamine-based probes 7-α and 7-β. Reagents and conditions: (i) p-Anisaldehyde (1 equiv.), NaOH (1 M), 0 °C; (ii)
acetic anhydride (8 equiv.), pyridine (excess), rt, 16 hours; (iii) acetone, HCl (4 M, 1.2 equiv.), 56 °C, 5 min; (iv) chloroacetyl chloride (3 equiv.) TEA (2
equiv.), DCM, rt, 2 hours; (v) propynol ethoxylate (3 equiv.), BF3 × Et2O (4 equiv.), DCM, 40 °C, 8 hours; (vi) dansyl azide 9 (1 equiv.), CuSO4–5H2O (1
equiv.), sodium ascorbate (1.5 equiv.), DIPEA (3 equiv.), THF : water (10 : 1), rt, 3 hours. (B) D-Glucosamine-based probes 8-α, 8-β, 11-α and 11-β.19 (C)
Control compounds 9 and 10 (see ESI† for synthesis).
tion with the anomeric configuration, may affect labelling trols. Controls included a DMSO-only sample, as well as the
efficacy and/or target recognition.
dansyl fluorophore 10 (0.5 mM), which is lacking both the
sugar scaffold and the electrophilic warhead (Scheme 1C).
Lysate fractions treated with either probe or control were separ-
Profiling of H. influenzae R2866 lysates
Next, we set out to assess the suitability of the pure anomers 7- ated on a 4–12% SDS-page gel, and analysed by in-gel fluo-
α, 7-β, 8-α and 8-β for the profiling of H. influenzae R2866. rescence emission and Coomassie staining (Fig. 3).
First, we investigated all four probes for the labelling of target
For all four probes, a defined labelling pattern of proteins
proteins in cell lysates. To identify optimum growth conditions over a wide MW range (∼20–100 kDa) was observed (Fig. 3).
for labelling experiments, growth kinetics of R2866 were estab- This pattern was absent from the control lanes, with the excep-
lished over 24 hours. We observed a doubling time of 60 min, tion of a strong band of an autofluorescent protein at
with the stationary phase reached at 8–9 hours, and a ∼28 kDa. There was no significant difference in the labelling
maximum OD590 of 1.9 (ESI Fig. S3†). For labelling experi- profile between the four different probes. This suggests that
ments, cell cultures of H. influenzae R2866 were grown in Brain the configuration at position 4 (D-gluco vs. D-gala series) or
Heart infusion (BHI) broth until mid-logarithmic phase was position 1 (α vs. β anomer) of the sugar scaffold has only a
reached (OD590 0.9; 5 hours). The cells were then harvested by minor influence on the target profile. The similar labelling
centrifugation and pellets equivalent to 1 mL of cell culture intensity observed for both anomers, both for 7 and 8, is remi-
were lysed with BugBuster protein extraction reagent. Cell niscent of the observation in the labelling experiments with
debris was removed by centrifugation, and the supernatant LgtC, and suggest that the acetate groups remain intact under
was incubated with probes 7-α, 7-β, 8-α or 8-β (0.5 mM) or con- these conditions.
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Fig. 4 Effect of probe concentration on the labelling of H. influenzae
R2866 lysates. General conditions as in Fig. 2. Following cell lysis and
centrifugation, the supernatant was divided into fractions (9 μL) and
incubated with stock solutions (1 μL, 5–0.25 mM stock in DMSO) of 7-α
or 10 (control). Protein bands were detected by fluorescence scanning
(left) and Coomassie staining (right). The asterisk denotes the low-abun-
dance protein at 50 kDa labelled by all four probes (see text).
Fig. 2 Labelling of recombinant LgtC with 8-α, 8-β, 11-α and 11-β in
E. coli lysates. General conditions: E. coli cells overexpressing LgtC were
lysed (8 h after IPTC induction) by mixing with BugBuster (100 μL for
100 mg of cell pellet, prepared from 10× stock in HEPES buffer) for
30 min at rt. Following centrifugation, the supernatant was divided into
fractions (9 μL) and incubated with stock solutions (1 μL, 5 mM stock in
DMSO) of 8-α, 8-β, 11-α, 11-β or 10 (fluorophore control) for 1 hour at
30 °C. Loading buffer (2.5 μL) was added to each fraction (10 μL).
Samples were incubated at 50 °C for 10 min and loaded onto a 12%
SDS-page gel. The gel was run with MES running buffer at 160 V for
70 min. Protein bands were detected by fluorescence scanning (left) and
Coomassie staining (right).
weakly, labelled by the fluorescent probes. Taken together, this
suggests that protein abundance is not the determining factor
for labelling by these probes.
Under these experimental conditions, labelling efficiency
was dependent on probe concentration, as expected (Fig. 4).
The resolution of the labelling pattern is significantly reduced
at probe concentrations lower than 250 μM, although individ-
ual bands with strong fluorescent labelling, such as the low-
abundance protein at 50 kDa, can still be detected at probe
concentrations as low as 50 μM (Fig. 4).
Protein labelling in intact H. influenzae R2866 cells
Following the successful profiling of H. influenzae R2866
lysates, we next tested the suitability of probes 7-α, 7-β, 8-α
and 8-β for the labelling of proteins in intact cells. First, we
carried out bacterial growth assays to ensure that the probes
did not compromise cell viability. No effect on bacterial
growth was observed, suggesting that the probes were suited
for profiling experiments with intact cells (ESI Fig. S4†). Next,
we assessed the capacity of the probes to penetrate the bac-
terial membrane. Cells were grown as described and harvested
by centrifugation. Pellets equivalent to 4 mL of cell cultures
were incubated for 2 hours with 7-α, 7-β, 8-α, 8-β, 10 (fluoro-
phore control, Scheme 1), or DMSO (control). After incubation,
unbound probe was removed by consecutive washes (5%
DMSO in PBS) and centrifugation.
After four washes, no fluorescence was detected in the
supernatant (Fig. 5A). Cell pellets that had been incubated
with probes or fluorophore control remained fluorescent,
while cells incubated with DMSO control showed no fluo-
rescence (Fig. 5B). Cell pellets were collected, lysed, and centri-
fuged. The fluorescence of samples that had been incubated
Fig. 3 Protein labelling with 7-α, 7-β, 8-α and 8-β in H. influenzae
R2866 cell lysates. General conditions: Cells were grown to OD590 0.9
and lysed by mixing with BugBuster (100 μL, ×1 in PBS, for pellet of 4 mL
of cell culture) for 30 min at rt. Following centrifugation, the supernatant
was divided into fractions (9 μL) and incubated with stock solutions
(1 μL, 5 mM stock in DMSO) of 7-α, 7-β, 8-α, 8-β or 10 (fluorophore
control), or DMSO (1 μL, control) for 1 hour at 30 °C. Loading buffer
(2.5 μL) was added to each fraction (10 μL) and loaded onto a 4–12%
SDS-page gel. The gel was run with MES running buffer at 150 V for
70 min. Protein bands were detected by fluorescence scanning (left) and
Coomassie staining (right). The asterisk denotes the low-abundance
protein at 50 kDa labelled by all four probes (see text).
Particularly strong fluorescence was observed for a band at with probes or fluorophore control persisted in the soluble
∼50 kDa (Fig. 3, asterisk). The corresponding band in the fraction after cell lysis (Fig. 5C). These results provided a first
Coomassie stain was relatively weak, indicating that this is a indication that all four probes as well as the fluorophore
low-abundance protein. In contrast, several high-abundance control can penetrate into the periplasm and/or cytoplasm of
proteins that give strong Coomassie bands were not, or only H. influenzae R2866, and hence that the carbohydrate scaffold
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escent protein at ∼28 kDa, a number of additional bands were
observed for control 9. Most likely, these are due to protein lab-
elling by the reactive azide group in 9 (Scheme 1C).
While the labelling pattern of intact cells was very similar
for all four probes, there were notable differences between this
profile (Fig. 6), and the profile obtained from labelling of cell
lysates (Fig. 3). Most significantly, the strongly fluorescent
band at ∼50 kDa that had been observed with the cell lysate
labelling protocol was absent from the intact cell experiment.
To further investigate these intriguing differences, we decided
to compare both labelling protocols side by side in a single
experiment.
Fig. 5 Protein labelling in intact H. influenzae R2866 cells. Cell pellets
were incubated for 2 hours with 7-α, 7-β, 8-α, 8-β or 10 (fluorophore
control) at 1 mM, or DMSO (control). (A) Supernatant after repeated
washes (5% DMSO in PBS) of pellets that had been incubated with probe
(results shown for 7-α) or DMSO (control); (B) cell pellets that had been
incubated with probe, DMSO (control) or 10 (fluorophore control) after
four washes (5% DMSO in PBS); (C) soluble fraction of the pellets from
(B) after lysis and centrifugation. Fluorescence emission at 365 nm.
Direct comparison of H. influenzae R2866 profiling in intact
cells and cell lysates
A bacterial culture was grown as described, half of which was
used for protein labelling through cells (1 mM of 7-α/β and 8-
α/β, 2 hours) while the other half was lysed and subsequently
incubated with the glucosamine-based probe (0.5 mM,
1 hour). The lysate samples from both experiments were
loaded on a single 4–12% SDS-page gel to enable the direct
comparison of the labelling profiles (Fig. 7).
The labelling profiles obtained with the two different proto-
cols showed a number of differences, confirming our obser-
vations in the previous, separate experiments. Several targets
were labelled exclusively with one protocol, but not the other
(Fig. 7). The most notable difference was the presence of a
strongly fluorescent band at ∼50 kDa, which is detected only
by labelling of cell lysates, but not of intact cells.
in 7 and 8 does not impede penetration of the bacterial
membrane.
To further confirm these results, we next investigated the
labelling of intracellular proteins by our probes. Cell lysates
were separated on a 4–12% SDS-page gel and analysed by fluo-
rescence emission and Coomassie staining (Fig. 6). As for the
application of 7 and 8 with cell lysates, a defined labelling
pattern of proteins over a wide MW range (∼13–100 kDa) was
observed for all four probes under these conditions (Fig. 6).
Labelling efficiency was again concentration-dependent (ESI,
Fig. S5†). While the only strong fluorescent band in the control
lanes for DMSO and 10 resulted from the unknown autofluor-
A possible explanation for these differences is, that the
probes penetrate the outer bacterial membrane and reach the
periplasm of H. influenzae R2866, but cannot cross the inner
membrane to reach the cytoplasmic space. The probes would
therefore be able to only capture periplasmic proteins in the
labelling experiment with intact cells, but both periplasmic
and cytoplasmic proteins in the protocol with cell lysates. We
therefore hypothesised that the proteins labelled exclusively in
Fig. 6 Protein labelling with 7-α, 7-β, 8-α and 8-β in intact
H. influenzae R2866 cells. General conditions: Cells were grown to
OD590 0.9. Cell pellets were suspended in PBS (900 μL) and incubated
with stock solutions (100 μL, 10 mM in DMSO) of probes 7-α, 7-β, 8-α,
8-β or fluorophore controls 9 or 10, or with DMSO (control), for two
hours at 30 °C. Cells were washed (4×, 5% DMSO in PBS), lysed with
BugBuster (100 μL, ×1 in PBS), and shaken for 30 min at rt, and centri-
fuged. Loading buffer (2.5 μL) was added to the clear supernatant (10 μL)
and loaded onto a 4–12% SDS-page gel. Gels were run with MES
running buffer at 150 V for 70 min. Protein bands were detected by
fluorescence scanning (left) and Coomassie staining (right).
Fig. 7 Direct comparison of protein labelling with 7-α, 7-β, 8-α and 8-β
in intact cells and cell lysates from H. influenzae R2866. See Fig. 3 and 5
for general conditions. The asterisk denotes the low-abundance protein
at 50 kDa which is detected, by all four probes, in cell lysates only.
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the bacterial cell lysate experiment may be located in the bac- between profiles from the lysate and intact cell experiments.
terial cytoplasm.
The exclusive labelling of a cytoplasmic protein in lysate
samples, but not intact cells, suggests that these probes can
indeed penetrate the outer, if probably not the inner, bacterial
membrane.
Analysis of selected target proteins by LC-MS/MS
To further assess this hypothesis, we sought to establish the
identity of selected target proteins. Selected bands were
excised from the requisite gel and analysed by LC-MS/MS fol-
lowing in-gel reduction, alkylation and digestion with trypsin.
The resulting peptide fragments were matched against the
current H. influenzae taxonomy in the Uniprot database (ESI†).
This analysis suggests two probable assignments for the
band at ∼50 kDa that is detected selectively by labelling of cell
lysates: FtsZ (45 kDa), a cytoplasmic GTPase that is central for
the bacterial cell division machinery;30 and Elongation Factor
Tu (43 kDa), another cytoplasmic GTPase involved in polypep-
tide chain elongation in protein biosynthesis.31 Interestingly,
both proteins have been identified as attractive targets for
small-molecule antibacterial drug discovery.30,31 These results
are in keeping with our hypothesis that the protein labelled
exclusively with the cell lysate protocol is located in the
cytoplasm.
The cell envelope of Gram-negative bacteria represents a
formidable permeability barrier, and considerable efforts are
being made to identify small molecules that can cross this
barrier.34–36 The ability of these probes to penetrate the bac-
terial membrane, as reported in this study, is therefore of con-
siderable interest. Initial target identification experiments
suggest that these probes preferentially label nucleotide-
binding proteins. This is consistent with the labelling of LgtC,
where labelling may occur at the sugar-nucleotide donor
binding site. Interestingly, potential target proteins include
several known antibacterial drug targets. These probes there-
fore represent exciting new tool compounds to study the role
of such nucleotide-binding proteins for bacterial pathogenicity
of Haemophilus and, potentially, other Gram-negative
pathogens.
High-probability assignments for other target proteins (ESI,
Fig. S6†) include many nucleotide-binding proteins, including
the chaperone protein DnaK (68 kDa), periplasmic NAD
nucleotidase (66 kDa), and DNA-directed RNA polymerase
subunit beta, a nucleotidyl-transferase (157 kD). DnaK is an
outer membrane protein known to mediate sulfatide reco-
gnition32 and a biofilm protein in non-typeable Haemophilus
Influenzae.33 These results suggest that probes 7 and 8 may act
as nucleotide mimics, possibly because of the combination of
a simple sugar with the dansyl fluorophore, which may be
reminiscent of a nucleobase.
Experimental
Chemistry
General. All chemical reagents were obtained commercially
and used as received, with the exception of propynol ethoxylate
which was distilled prior to use. Compounds 8-α, 8-β, 11-α
and 11-β were prepared as previously reported.19 Normal
phase chromatography was performed on silica gel (particle
size 40–63 μm). Thin layer chromatography (TLC) was per-
formed on precoated aluminium plates (Silica Gel 60 F254,
Merck). Compounds were visualised by staining with p-anisal-
dehyde and/or exposure to UV light (365 nm). All compounds
were characterized by 1H-NMR, 13C-NMR, and MS. 1H-NMR
spectra were recorded at 298 K on a Bruker Ascend™ 400
Conclusion
We have synthesised both anomers of a D-galactosamine-based spectrometer at 400 MHz. 13C-NMR spectra were recorded at
fluorescent probe in six steps from D-galactosamine with an 298 K on either a Bruker Ascend™ 400 spectrometer at
overall yield of 22%. These probes, alongside their 100 MHz, or a Bruker Ascend™ 600 spectrometer at 150 MHz.
D-glucosamine congeners, were used successfully for the label- Chemical shifts (δ) are reported in ppm and referenced to
ling of multiple target proteins in both cell lysates and intact residual solvent peaks. Peak assignments were made with the
cells of Haemophilus influenzae R2866. In both the aid of 2D NMR spectra (COSY, HSQC, HMBC and DEPT).
D-galactosamine and D-glucosamine series, the labelling pro- HRMS spectra were recorded at the EPSRC National Mass
files and intensities for the fully acetylated probes were practi- Spectrometry Service Centre, Swansea. LC-MS analysis was per-
cally identical for both anomers. This is consistent with obser- formed on an Agilent Technologies 1260 Infinity II LC system,
vations for the labelling of the recombinant galactosyltransfer- equipped with a ZORBAX Eclipse XDB-C8 column (4.6 ×
ase LgtC, where labelling was comparable between both 150 mm, 5 μm), and coupled to an Advion expressionL CMS
anomers of the fully acetylated D-glucosamine-based probes 8- mass spectrometer. Mobile phase: 0.1% formic acid in water/
α and 8-β, but significantly different between both anomers of methanol; flow rate: 1 mL min−1; detection wavelength: 210/
the corresponding free sugars 11-α and 11-β. This suggests 254 nm. All solvents used for reverse phase chromatography
that the fully acetylated probes are also the active species in were HPLC grade.
the labelling experiments with Haemophilus lysates and intact
cells, and that the acetate groups remain intact under these was dissolved in aq. NaOH (1 M, 9.3 mL) at
conditions. p-Anisaldehyde (1 equiv.) was added dropwise, and the
Compound 2. Galactosamine hydrochloride 1 (9.25 mmol)
0
°C.
While the labelling profile observed with all four probes mixture was shaken vigorously. Upon formation of a white
was practically identical, notable differences were observed solid, the reaction was placed in the freezer (−20 °C) for 1 h to
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complete precipitation. The solid was collected by filtration, NaHCO3 solution (5 mL). DCM (10 mL) was added and the two
washed dropwise with ice-cold H2O (10 mL) and ice-cold layers were separated. The aqueous phase was extracted with
EtOH : Et2O (1 : 1, 10 mL), and dried under vacuum to give 2 as DCM (3 × 10 mL). The organic layers were combined and dried
1
a white powder in 56% yield. H NMR (400 MHz, DMSO-d6) δ over MgSO4. After filtration the mixture was concentrated
3
3
3
3.09 (dd, J 9.5 Hz, J 7.7 Hz, 1H, H-2 sugar), 3.46 (t, J 6.2 Hz, under vacuum. The residue was purified by normal phase
1H, H-5 sugar), 3.50–3.62 (m, 3H, H-3, H-6 sugar), 3.67 (t, 3J column chromatography (EtOAc/hexane) to afford the title
3
1
3.5 Hz, 1H, H-4 sugar), 3.80 (s, 3H, H-methoxy), 4.39 (d, J 4.4 compound as an off-white crystalline product in 85% yield. H
Hz, 1H, H-hydroxyl), 4.50 (d, 3J 7.0 Hz, 1H, H-hydroxyl), NMR (400 MHz, CDCl3) δ 2.02, 2.05, 2.13, 2.17 (4s, 12H,
4.58–4.64 (m, 2H, H-1 anomeric, H-hydroxyl), 6.42 (d, 3J 6.3 CH3CO), 4.00 (s, 2H, H-7), 4.06 (td, 3J 6.5 Hz, 3J 1.0 Hz, 1H,
3
3
3
3
Hz, 1H, H-hydroxyl), 6.98 (d, J 8.8 Hz, 2H), 7.67 (d, J 8.8 Hz, H-5), 4.09–4.20 (m, 2H, H-6), 4.38 (dt, J2–3 11.3 Hz, J2-NH 9.0
2H), 8.13 (s, 1H, H-imine). 13C NMR (100 MHz, DMSO-d6) δ Hz, 1H, H-2), 5.24 (dd, J3–2 11.3 Hz, J3.4 3.3 Hz, 1H, H-3),
3
3
3
55.3 (C-methoxy), 60.7 (C-6 sugar), 67.2 (C-4 sugar), 71.6 (C-3 5.38–5.42 (m, 1H, H-4), 5.82 (d, J1–2 8.8 Hz, 1H, H-1), 6.48 (d,
sugar), 74.5 (C-2 sugar), 75.1 (C-5 sugar), 96.1 (C-1 sugar), 3J 9.2 Hz, 1H, NH). 13C NMR (100 MHz, CDCl3): δ 20.7, 20.7,
113.9 (×2), 129.2, 129.5 (×2), 161.0, 161.2 (C-imine).
20.8, 21.0 (4C, CH3CO), 42.5 (C-7), 50.5 (C-2), 61.5 (C-6), 66.6
Compound 3. 2 (3.20 mmol) was dissolved in pyridine (C-4), 69.9 (C-3), 71.9 (C-5), 92.5 (C-1), 166.9 (C-8), 169.5, 170.2,
(10 mL) at 0 °C. Acetic anhydride (8 equiv.) was added drop- 170.6, 170.6 (CH3CO). LC-MS (m/z) 446.3 [M + Na+] (calculated
wise, the reaction was warmed to room temperature and 446.2).
stirred overnight (16 h). The reaction mixture was poured into
ice (50 mL) under stirring. The precipitate was collected by fil- DCM (3 mL), and the solution was cooled to
Compounds 6-α and 6-β. 5 (1.06 mmol) was dissolved in
°C.
0
tration, washed with ice-cold H2O (200 mL) and ice-cold EtOH Borontrifluoride diethyletherate (4 equiv.) and propynol ethox-
(2 mL), and dried under vacuum, to give 3 as a white powder ylate (3 equiv.) were added dropwise, and the reaction was
1
in 99% yield. H NMR (400 MHz, DMSO-d6) δ 1.82, 1.97, 2.00, stirred at 40 °C for 8 h. The reaction mixture was diluted with
3
3
2.12 (4s, 12H, CH3CO), 3.52 (dd, JH2–H3 10.3 Hz, JH2–H1 8.2 DCM (7 mL) and washed with saturated aq. K2CO3 (2 × 10 mL)
Hz, 1H, H-2 sugar), 3.79 (s, 3H, H-methoxy), 4.04–4.09 (m, 2H, and brine (2 × 10 mL). The organic layer was separated, dried
3
H-6 sugar), 4.46 (t, JH5–H6 6.5 Hz, 1H, H-5 sugar), 5.27 (d, over MgSO4, and concentrated under vacuum. The crude
3
3JH4–H3 3.3 Hz, 1H, H-4 sugar), 5.35 (dd, JH3–H2 10.4 Hz, residue was purified by normal phase column chromatography
3
3JH3–H4 3.4 Hz, 1H, H-3 sugar), 5.98 (d, JH1–H2 8.2 Hz, 1H, H-1 (0–20% acetone in toluene) to give two products. Each product
3
3
anomeric), 6.99 (d, J 8.8 Hz, 2H), 7.67 (d, J 8.8 Hz, 2H), 8.31 was further purified by reverse phase column chromatography
(s, 1H, H-imine). 13C NMR (100 MHz, DMSO-d6) δ 20.3, 20.4, (10–30% EtOAc in hexane) to give the title compounds as pale
20.5, 20.5, (4C, CH3CO), 55.4 (C-methoxy), 61.4 (C-6 sugar), yellow oils in 34% (6-α) and 36% (6-β) yield. Cmpd 6-α: 1H
66.0 (C-4 sugar), 68.4 (C-2 sugar), 70.9 (C-5 sugar), 70.9 (C-3 NMR (400 MHz, CDCl3) δ 1.99, 2.05, 2.16 (3s, 9H, CH3CO),
sugar), 92.8 (C-1 sugar), 114.2, 128.3, 129.9, 161.8, 164.7 2.47 (t, 3J 2.4 Hz, 1H, H-13), 3.67–3.74 (m, 3H, H-9, H-10),
(C-imine), 168.6, 169.2, 169.9, 170.0 (4C, CH3CO).
3.83–3.90 (m, 1H, H-9), 4.01 (d, 3J 3.2 Hz, 2H, H-7), 4.11 (m,
3
3
Compound 4. 3 (2.70 mmol) was dissolved in acetone 2H, H-6), 4.19 (d, J 2.4 Hz, 1H, H-11), 4.28 (t, JH5–H6 6.6 Hz,
(11 mL) and the solution was heated to reflux. Aq. HCl (4 M, 1H, H-5), 4.57 (ddd, 3JH2–H3 11.2 Hz, 3JH2–NH 9.7 Hz, 3JH2–H1 3.7
1.2 equiv.) was added dropwise under stirring. After about Hz, 1H, H-2), 4.95 (d, 3JH1–H2 3.7 Hz, 1H, H-1), 5.24 (dd, 3JH3–H2
3
3
5 min, a white precipitate formed. Heating was continued for a 11.3 Hz, JH3–H4 3.3 Hz, 1H, H-3), 5.41 (dd, JH4–H3 3.2 Hz,
further 2 min. The precipitate was collected by filtration and 3JH4–H5 1.2 Hz, 1H, H-4), 6.71 (d, JNH–H2 9.7 Hz, 1H, NH). 13C
3
washed with ice-cold acetone (50 mL), to give 4 as a white NMR (100 MHz, CDCl3) δ 20.9 (3 × CH3CO), 42.6 (C-7), 48.3
powder in 88% yield. 1H NMR (400 MHz, DMSO-d6) δ 1.99, (C-2), 58.5 (C-11), 62.0 (C-6), 67.0 (C-5), 67.5 (C-4), 67.7 (C-9),
3
2.00, 2.12, 2.16 (4s, 12H, CH3CO), 3.39 (dd, JH2–H1 8.2 Hz, 68.6 (C-3), 68.6 (C-10), 75.1 (C-13), 79.5 (C-12), 97.9 (C-1), 166.4
3JH2–H3 6.2 Hz, 1H, H-2 sugar), 4.98–4.09 (m, 2H, H-6 sugar), (C-8), 170.4, 170.6, 170.8 (3C, CH3CO). LC-MS (m/z) 486.3 [M +
3
4.29 (t, J 6.3 Hz, 1H, H-5 sugar), 5.24–5.32 (m, 2H, H-3, H-4 Na+] (calculated 486.1). Cmpd 6-β: 1H NMR (400 MHz, CDCl3) δ
3
3
sugar), 5.89 (d, JH1–H2 8.7 Hz, 1H, H-1 anomeric), 8.71 (s, 3H, 2.01, 2.05, 2.15 (3s, 9H, CH3CO), 2.45 (t, J 2.3 Hz, 1H, H-13),
NH). 13C NMR (100 MHz, DMSO-d6) δ 20.3, 20.5, 20.7, 20.8 3.65–3.73 (m, 2H, H-10), 3.76–3.84 (m, 1H, H-9), 3.93 (t, J 6.7
3
(4C, CH3CO), 49.4 (C-2 sugar), 61.2 (C-6 sugar), 68.8, 65.8 (C-3, Hz, 1H, H-5), 3.97–4.08 (m, 4H, H-2, H-9, H-7), 4.12–4.23 (m,
3
C-4 sugar), 71.1 (C-5 sugar), 90.3 (C-1 sugar), 168.6, 169.3, 4H, H-6, H-11), 4.84 (d, JH1–H2 8.4 Hz, 1H, H-1), 5.30 (dd,
3
3
169.9, 169.9 (4C, CH3CO).
3JH3–H2 11.3 Hz, JH3–H4 3.4 Hz, 1H, H-3), 5.38 (d, JH4–H3 3.4
Compound 5. To a suspension of 4 (0.4 mmol) in DCM Hz, 1H, H-4), 6.49 (d, JNH–H2 8.6 Hz, 1H, NH). 13C NMR
(2 mL), triethylamine (2 equiv.) was added. The reaction (100 MHz, CDCl3) δ 20.8, 20.8, 20.8 (3C, CH3CO), 42.7 (C-7),
mixture was stirred at room temperature until all solid was dis- 52.1 (C-2), 58.6 (C-11), 61.6 (C-6), 66.8 (C-4), 68.9 (C-9), 69.3
solved (approx. 2 min). The solution was cooled to 0 °C, and (C-10), 69.8 (C-3), 71.0 (C-5), 74.9 (C-13), 79.6 (C-12), 101.2
chloroacetyl chloride (3 equiv.) was added dropwise. The reac- (C-1), 166.6 (C-8), 170.3, 170.5, 170.6 (3C, CH3CO). LC-MS (m/z)
tion mixture was stirred at 0 °C for 10 min, and at room temp- 486.3 [M + Na+] (calculated 486.1).
3
erature until TLC (8 : 1 EtOAc : hexane) showed full conversion
Compounds 7-α and 7-β. Under stirring, 6-α or 6-β
(1 h). The reaction was quenched by addition of saturated aq. (0.16 mmol) and 10 (1 equiv.) were dissolved in THF (1 mL). A
482 | Org. Biomol. Chem., 2021, 19, 476–485
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3
3
3
solution of sodium ascorbate (2 equiv.) and copper sulfate Hz, 1H), 7.53 (dd, J 8.5 Hz, J 7.4 Hz, 1H), 7.60 (dd, J 8.6 Hz,
pentahydrate (1.2 equiv.) in H2O (110 μL) was added, followed 3J 7.7 Hz, 1H), 8.24–8.26 (m, 2H), 8.26–8.28 (m, 2H), 8.57 (d, 3J
by DIPEA (5 equiv.). The reaction was covered with aluminium 8.5 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 42.5, 45.6, 45.6,
foil and stirred at room temperature for 3 h. The reaction was 51.1, 115.5, 118.6, 123.3, 128.8, 129.6, 129.8, 130.1, 131.0,
extracted with EtOAc (15 mL). The organic extract was washed 134.6, 152.3. HR-MS (m/z) 320.1182 [M + H]+ (calculated
with H2O (5 mL) and brine (5 mL), and concentrated under 320.1181).
vacuum. The crude residue was purified by normal phase
Compound 10. The title compound was obtained as pre-
column chromatography twice (first solvent system: 50–100% viously described28 from 9 and 1-pentyne, under the cyclo-
1
EtOAc in hexane. Second solvent system: 0–70% acetone in addition reaction conditions described for 7, in 87% yield. H
3
toluene) to give the title compounds as powders in 74% (7-α) NMR (400 MHz, CDCl3) 0.95 (t, J 7.4 Hz, 3H), 1.58–1.68 (m,
and 82% (7-β) yield. Cmpd 7-α: 1H NMR (400 MHz, CDCl3) δ 2H), 2.58–2.63 (m, 2H), 2.86 (s, 6H), 3.44 (dd, J 11.2 Hz, J 6.1
3
3
1.98, 2.00, 2.11 (3s, 9H, CH3CO), 2.89 (s, 6H), 3.37–3.43 (m, Hz, 2H), 4.30–4.36 (m, 2H), 4.41–4.48 (m, 1H, NH), 7.11 (s, 1H,
3
2H), 3.70–3.76 (m, 3H), 3.80–3.88 (m, 1H), 3.98 (s, 2H, H-7), H-triazole), 7.19 (d, J 7.1 Hz, 1H), 7.51–7.57 (m, 2H), 8.18 (dt,
3
3
4.03–4.15 (m, 2H, H-6 sugar), 4.29 (td, JH5–H6 6.4 Hz, JH5–H4 3J 8.7 Hz, 3J 1 Hz, 1H) 8.25 (dd, 3J 7.3 Hz, 3J 1.2 Hz, 1H),
1.4 Hz 1H, H-5 sugar), 4.39–4.52 (m, 2H), 4.56–4.70 (m, 3H, 8.54–8.58 (m, 1H). 13C NMR (100 MHz, CDCl3) δ 14.0, 22.8,
H-2 sugar, H-aliphatic), 5.01 (d, 3JH1–H2 3.6 Hz, 1H, H-1 anome- 27.7, 42.9, 45.6 (×2), 50.0, 115.5, 118.6, 122.1 (C-triazole),
3
3
ric), 5.20 (dd, JH3–H2 11.3 Hz, JH3–H4 3.3 Hz, 1H, H-3 sugar), 123.3, 128.9, 129.6, 129.7, 130.1, 131.0, 134.5, 148.3, 152.2.
5.43 (dd, 3JH4–H3 3.3 Hz, 3JH4–H5 1.4 Hz, 1H, H-4 sugar), 6.14 (t,
3
Biology
3JNH–H15 5.6 Hz, 1H, NH-sulfonamide), 7.14 (d, JNH–H2 9.6 Hz,
3
1H, NH-amide), 7.18 (d, J 7.4 Hz, 1H), 7.50–7.57 (m, 2H), 7.61
Expression of LgtC. Recombinant LgtC was expressed as pre-
3
(s, 1H, H-triazole), 8.19–8.26 (m, 2H), 8.56 (d, J 8.5 Hz, 1H). viously described37 from E. coli clone NMC-41, transformed
13C NMR (100 MHz, CDCl3) δ 20.8, 20.9, 21 (3C, CH3CO), 42.7 with plasmid containing sequences encoding His-tagged LgtC
(C-7), 42.8, 45.6 (×2), 48.3 (C-2 sugar), 50.4, 62.0 (C-6 sugar), and ampicillin resistance.38
64.4, 66.9 (C-5 sugar), 67.6, 67.6 (C-4 sugar), 68.7 (C-3 sugar),
Bacterial culture. H. influenzae R2866 was streaked onto
69.3, 98.0 (C-1 sugar), 115.5, 118.9, 123.3, 124.2 (C-triazole), bacitracin chocolate agar plates (Oxoid) from glycerol stock
128.6, 129.6, 129.6, 130.1, 130.8, 134.7, 144.8 (C-triazole), and incubated at 37 °C. A single colony from the resultant
152.2, 166.9 (C-amide), 170.6, 170.7, 171.3 (3C, CH3CO). plate was used to inoculate 10 mL sBHI broth (Oxoid Brain
LC-MS (m/z) 784.3 [M + H+] (calculated 784.2). Cmpd 7-β: 1H Heart Infusion broth supplemented with 10 µm mL−1 NAD,
NMR (400 MHz, CDCl3) δ 1.97, 1.99, 2.14 (3s, 9H, CH3CO), and 10 µL mL−1 Hemin), followed by incubation at 37 °C, 180
2.89 (s, 6H), 3.33–3.50 (m, 2H), 3.67–3.81 (m, 2H), 3.84–3.99 rpm overnight. For downstream experiments, fresh sBHI was
(m, 3H, H-5 sugar, H-aliphatic), 4.02 (d, 3J 6.9 Hz, 2H, H-7), then inoculated 1 : 33 to an OD590 of 0.1 and incubated aerobi-
4.05–4.20 (m, 3H, H-2, H-6 sugar), 4.40–4.49 (m, 1H), 4.50–4.59 cally at 37 °C and 180 rpm. For growth curves, OD590 was
3
(m, 1H), 4.63 (s, 2H), 4.89 (d, JH1–H2 8.5 Hz, 1H, H-1 anome- recorded at various time points in triplicate cultures.
3
3
ric), 5.21 (dd, JH3–H2 11.2 Hz, JH3–H4 3.4 Hz, 1H, H-3 sugar),
Cell viability. To assess the effect of probes and DMSO
3
3
5.36 (d, J 2.9 Hz, 1H, H-4 sugar), 6.07 (t, JNH–H15 5.8 Hz, 1H, solvent on cell viability, bacterial growth assays were per-
NH-sulfonamide), 7.09 (d, 3JNH–H2 8.8 Hz, 1H, NH-amide), 7.19 formed by observation of growth on bacitracin chocolate agar
3
(d, J 7.6 Hz, 1H), 7.61–7.50 (m, 2H), 7.67 (s, 1H, H-triazole), plates. After incubation of cultures with the respective probe
8.19 (d, 3J 8.7 Hz, 1H), 8.24 (dd, J 7.3 Hz, 1.1 Hz, 1H), 8.56 (d, for 2 h in sBHI at 30 °C, 50 μL of culture were plated on baci-
3
3J 8.5 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 20.8, 20.8, 20.9 tracin chocolate agar plates and incubated at 37 °C for
(3C, CH3CO), 42.8 (C-7), 45.6 (×2), 50.4, 51.8 (C-2 sugar), 61.6 48 hours. Viability was assessed by comparative plate count
(C-6 sugar), 64.5, 67.0 (C-4 sugar), 68.6, 70.4 (C-3 sugar), 70.7, between cultures incubated with and without probe in DMSO.
70.8 (C-5 sugar), 101.3 (C-1 sugar), 115.6, 118.8, 123.6, 124.6
(C-triazole), 128.8, 129.6, 129.7, 130.2, 131.0, 134.3, 144.8
Labelling experiments
Materials. NuPAGE™ 4–12% bis–tris protein gels, 1.0 mm,
(C-triazole), 152.2, 167.3 (C-amide), 170.5, 170.6, 170.8 (3C, 12-well. PageRuler™ prestained protein ladder 15–190 kDa.
CH3CO). LC-MS (m/z) 784.3 [M + H+] (calculated 784.2).
NuPAGE™ MES SDS running buffer (20×).
Compound 9. A solution of dansyl chloride 11 (5.00 mmol),
Protein labelling in intact cells. Cell cultures of H. influenzae
2-bromoethylamine hydrobromide (1 equiv.) and triethylamine R2866 were grown as described. Upon reaching mid log phase
(2 equiv.) in DCM (25 mL) was stirred at room temperature for (OD590 0.9), 4 mL cell cultures were centrifuged at 13 000g for
4 h. The solvent was removed under vacuum. The resulting 1 minute. The supernatant was discarded. Cell pellets were
residue was dissolved in MeCN (50 mL). NaN3 (2.5 equiv.) was resuspended in PBS (900 μL), and stock solutions of probe or
added, and the reaction was stirred at reflux overnight. The fluorophore control (100 μL, 10× desired concentration in
solvent was removed under vacuum, and the crude product DMSO), or DMSO (100 μL, control) were added. Samples were
was purified by flash column chromatography (solvent system: incubated for 2 hours at 30 °C with regular mixing, and centri-
1
0–50% EtOAc in Hexane) to give 9 as an oil in 87% yield. H fuged (2 min, 13 000g). Cell pellets were separated from the
NMR (400 MHz, CDCl3) δ 2.89 (s, 6H), 3.03–3.08 (m, 2H), supernatant, washed with a solution of 5% DMSO in PBS, and
3.29–3.33 (m, 2H), 4.99 (t, 3J 5.9 Hz, 1H, NH), 7.21 (d, 3J 7.6 centrifuged. This procedure was repeated four times to remove
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unbound fluorophore. After each cycle, the fluorescence of Monoisotopic precursor ions were filtered using charge state
pellets and washes was examined at 365 nm (Fig. 4). (+2 to +7) with an intensity threshold set between 5.0 × 103 to
BugBuster protein extraction reagent (Millipore, 1× in PBB, 1.0 × 1020 and a dynamic exclusion window of 35 s 10 ppm.
100 μL) was added to the cell pellets. Samples were incubated MS2 precursor ions were isolated in the quadrupole set to a
for 30 min at rt with shaking, and centrifuged (10 min, mass width filter of 1.6 m/z. Ion trap fragmentation spectra
13 000g). Supernatant and cell debris were separated, and the (ITMS2) were collected with an AGC target setting of 1.0 × 104
fluorescence of both was examined at 365 nm. Supernatant with a maximum injection time of 35 ms with CID collision
(10 μL) from each sample was incubated with loading buffer energy set at 35%. This method takes advantage of multiple
(no dye, 2.5 μL) for 10 min at 50 °C. Samples (10 μL) were analyzers in the Orbitrap Fusion Lumos and drives the system
loaded on precast SDS-page gels (NuPAGE™ 4 to 12%, bis–tris, to use all available parallelizable time, resulting in decreasing
1.0 mm, Novex). Gels were run in 1× MES running buffer at dependence on method parameters.
150 V for 70 min and analysed by fluorescence emission and
Coomassie staining.
Database searching. Raw mass spectrometry data were pro-
cessed into peak list files using Proteome Discoverer
Protein labelling in cell lysates. Cell cultures of H. influenzae (ThermoScientific; v2.2). The raw data file was processed and
R2866 were grown as described above. Cell pellets were iso- searched using the Sequest search algorithm39 against the
lated by centrifugation and lysed with BugBuster protein current Haemophilus influenzae database from Uniprot (HI;
extraction reagent as described. Following centrifugation, the 4957 reviewed entries).
supernatant from each sample was kept on ice, and the cell
debris was discarded. The supernatants (9 μL) were incubated
with stock solutions of probe or fluorophore control (1 μL, 10× Conflicts of interest
desired concentration in DMSO), or DMSO (1 μL, control) for
1 hour at 30 °C with regular mixing. Samples (10 μL) were incu-
There are no conflicts to declare.
bated with loading buffer (2.5 μL) for 10 min at 50 °C and ana-
lysed by SDS-page as described.
Acknowledgements
Protein mass spectrometry
We thank King’s College London for a studentship (to CM),
the EPSRC National Mass Spectrometry Facility, Swansea, for
Enzymatic digestion. In-gel reduction, alkylation and diges-
tion with trypsin was performed on selected gel bands prior to
the recording of mass spectra, and Dr Jon Cuccui (LSHTM) for
subsequent analysis by mass spectrometry. Cysteine residues
helpful discussions. The LgtC-expressing clone NMC-41 was a
were reduced with dithiothreitol and derivatised by treatment
gift from Prof Warren Wakarchuk (University of Alberta).
Funding from the Biotechnology
Research Council (grant BB/N001591/1, to BW) is gratefully
acknowledged.
with iodoacetamide to form stable carbamidomethyl deriva-
tives. Trypsin digestion was carried out overnight at room
temperature after initial incubation at 37 °C for 2 hours.
& Biological Sciences
LC-MS/MS. Peptides were extracted from the gel pieces by a
series of acetonitrile and aqueous washes. The extract was
pooled with the initial supernatant and lyophilised. The
sample was then resuspended in 40 μl of resuspension buffer
(2% ACN in 0.05% FA) and analysed by LC-MS/MS (10 μl).
Chromatographic separation was performed using a U3000
UHPLC NanoLC system (ThermoFisherScientific, UK).
Peptides were resolved by reversed phase chromatography on a
75 μm C18 Pepmap column (50 cm length) using a three-step
linear gradient of 80% acetonitrile in 0.1% formic acid. The
gradient was delivered to elute the peptides at a flow rate of
250 nl min−1 over 60 min starting at 5% B (0–5 minutes) and
increasing solvent to 40% B (5–40 minutes) prior to a wash
step at 99% B (40–45 minutes) followed by an equilibration
step at 5% B (45–60 minutes). The eluate was ionised by elec-
trospray ionisation using an Orbitrap Fusion Lumos
(ThermoFisherScientific, UK) operating under Xcalibur v4.1.5.
The instrument was first programmed to acquire using an
Orbitrap-Ion Trap method by defining a 3 s cycle time between
a full MS scan and MS/MS fragmentation. Orbitrap spectra
(FTMS1) were collected at a resolution of 120 000 over a scan
range of m/z 375–1500 with an automatic gain control (AGC)
setting of 4.0 × 105 with a maximum injection time of 35 ms.
Notes and references
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