Chemistry Reference
In-Depth Information
plate and sealed with adhesive aluminium foil and then removed from the oxygen-free
environment. Analysis was performed by ESI-MS on a Q-TOF mass spectrometer (Q-
TOFmicro, Micromass, Altrincham, UK) interfaced with a NanoMate chip-based nanoESI
source (Advion Biosciences, Ithaca, NY, USA). Time courses were started when the Nano-
Mate tip pierced the aluminium seal covering the 96-well microtitre plate and introduced O
2
into the system to initiate thiol-disulfide exchange. Samples were infused into the Q-TOF
mass spectrometer through the ESI chip (estimated flow rate approximately 0.1 Lmin
−
1
).
Calibration and sample acquisitions were performed in the positive ion mode in the range
m
/
z
500-5000. The results from this ESI-MS screen confirmed the preference of native
BcII for certain disulfide heterodimers from all possible DCL constituents. A sample mass
spectrum from this study is presented in Figure 7.14. Hit candidates (
H-5
,
H-8/9/10
and
(25
239
±
1 Da)
BcII(Zn
II
)
2
-
2,3,4
(25
085
±
2 Da)
BcII(Zn
II
)
2
(25
272
±
1 Da)
BcII(Zn
II
)
2
-
dithiol
100
%
1 min
0
100
CO
2
H
SH
%
1 min
HS
H
0
(25
391
±
2 Da)
CO
2
H
100
BcII(Zn
II
)
2
S
NH
2
H-8/9/10
HS
S
CO
2
H
SH
%
(25
424
±
2 Da)
20 h
CO
2
H
HS
H
BcII(Zn
II
)
2
HS
S
CO
2
H
S
H-5
0
Figure 7.14
Deconvoluted ESI mass spectra from an equimolar mixture of 19 thiols (10
μ
M
each of
4
-
22
) and BcII (15
μ
M): (a) after 1 min; (b) plus anchor fragment dithiol
H
(30
μ
M)
after 1 min of aerial exposure; (c) plus anchor fragment dithiol
H
(30
μ
M) after 20 h of aerial
exposure. Reproduced with permission from Lienard, B.M.R., Selevsek, N., Oldham, N.J. and
Schofield, C.J., Combined mass spectrometry and dynamic chemistry approach to identify
metalloenzyme inhibitors.
ChemMedChem
2007,
2
, 175-179. Copyright Wiley-VCH Verlag
GmbH.
