SCIENTIFIC REPORT
Ligand fishing with Biacore 3000: selective binding,
recovery and identification by MALDI-MS
Andrei Zhukov, Detlev Suckau2, Jos Buis and Östen Jansson
Biacore AB, Uppsala, Sweden, 2Bruker Daltonics GmbH, Bremen, Germany
THE MICRO-RECOVERY SENSORGRAM
the M13 peptide. During the recovery stage, the
two large dips in the RU reading correspond to
the air bubbles on one line passing through.
Finally, the sensorgram enables the amount of
recovered material to be determined, (for the
brain extract sample this was 1150 RU per flow
cell, corresponding to a total of 275 fmol of CaM
in four flow cells).
CHARACTERIZATION OF THE BINDING OF CALMODULIN USING MALDI-MS
Figure 2
In order to obtain the best possible sensitivity, the
limited amount of material recovered from the
chip will in most cases require sample pre-concentration and desalting to attain highest possible
Sensorgram showing the binding and
micro-recovery of an M13-specific ligand
present in bovine brain extract.
Washing
Binding
Recover
1500
1000
Recovered
Response (RU)
I
n order to demonstrate that Biacore’s SPR technology can be used to isolate and identify target
molecules from a complex biological mixture,
M13, a peptide fragment of myosin light-chain
kinase responsible for the interaction with the signal transduction protein, calmodulin (CaM), was
immobilized on a sensor chip via amine coupling.
Bovine brain extract was then injected over the sensor chip surface at a flow rate of 20 µl/min.
We utilized a micro-recovery function designed
for recovery of material from the sensor chip surface in a very small volume (3-7 µl). In Biacore
3000, used for the SPR section of these experiments, micro-recovery operates by injecting a
series of liquid volumes separated by air bubbles
on one line over the sensor surface (known as
sandwich elution). The air bubbles help to ensure
that the eluted sample is kept within a defined
volume to aid recovery and prevent contamination
or dilution of the recovered material (Figure 1 in
previous article).
The sensorgram in Figure 2 shows the microrecovery of material bound to the M13-modified
chip. As the CaM binds, the response (expressed
in resonance units, RU) increases and then falls
slightly as the wash stage removes any material
adhered to the sensor chip that is not bound to
Biacore Journal – Number 1 2002
PDF name: Biacore Journal 1 02 fix.pdf March 27, 2002 17:48:41
500
0
-500
-1000
-1500
-2000
0
200
400
600
800
1000
1200
1400
Time (s)
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SCIENTIFIC REPORT
phase columns, using pre-structured MALDI targets. This was demonstrated in an experiment in
which a total of 460 fmol CaM was captured on
the chip. This was achieved by injecting brain
extract followed by recovery in 4 µl. Only 0.5 µl
of the eluate was mixed with 0.5 µl dihydroxybenzoic acid matrix solution on an AnchorChip
target (Bruker Daltonics) with 600 µm diameter
sample spots. The molecular ion region of the
resulting spectrum is shown in Figure 5.
However, the presence of contaminating substances arising from the omission of such purification or preparative measures may, in certain
applications, interfere with the capacity of
MALDI to provide reliable molecular weight
data.
Intensity
3.00
0
10 000
20 000
m/z
Figure 3
MALDI MS spectrum for untreated bovine
brain extract.
Figure 4
MALDI MS spectrum of SPR CaM eluate.
Recovery buffer was 0.5% TFA.
IDENTIFYING CALMODULIN FROM ENZYMATIC DIGESTS
sensitivity. This can be carried out using small
reverse phase columns integrated at the outlet of
pipette tips. Untreated bovine brain extract produced a spectrum with a large number of peaks
(Figure 3). However, material recovered from the
chip after the injection of brain extract, followed
by concentration and desalting on a reverse phase
column, produced a clear spectrum containing
only the single and doubly charged CaM molecular ion (Figure 4). This data agrees with the
known covalent structure of CaM, including Nterminal acetylation and K115-trimethylation.
These spectra were obtained on a Bruker
ReflexIII MALDI-TOF mass spectrometer operated in the linear mode using delayed extraction
and deflection of lower mass molecules with a 6
kDa cut-off.
However, the preparation of the eluate can
also be carried out without the use of reverse
MH
16792
[M+2H]
8397
5000
7500
10000
12500
m/z
15000
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PDF name: Biacore Journal 1 02 fix.pdf March 27, 2002 17:48:41
17500
20000
In addition to carrying out MALDI-TOF MS
analysis on CaM captured by and then eluted
from a sensor chip, we have recently identified
the captured protein based on proteolysis of the
analyte followed by mass spectrometric peptide
mapping. The following example further demonstrates the feasibility of identifying a protein that
is captured on a chip after fishing from a complex
mixture, using SPR-MS.
Brain extract in a buffer composed of HEPESbuffered saline and 2 mM CaCl2 was injected
over M13 peptide immobilized on a sensor chip.
M13 selectively captured CaM and binding levels
of 2000 RU per flow cell were obtained. If CaM
alone was captured, this SPR signal corresponds
to roughly 480 fmol of CaM (in total from four
flow cells) available for recovery.
CaM was recovered in 4 µl, using EGTA to
release the CaM and 50 mM NH4HCO3, pH 7.8,
to provide a solution in which trypsin is active.
Trypsin was added to a final concentration of 5
µg/ml and the sample was incubated overnight at
room temperature. After a desalting/concentration step using ZipTip-µC18 reverse phase
columns (Millipore), the masses of tryptic fragments in the sample were analyzed using MALDI
TOF mass spectrometry.
The resulting spectrum was submitted to a
database search that unambiguously identified
bovine CaM. The spectrum and the peptide coverage map (55% sequence coverage) are shown in
Figure 6. The two CaM post-translational modifications previously described were defined in the
BioTools software for this peptide map. M/z
1563.73 includes the acetylated N-terminus and
m/z 2401.18 the trimethyl-K115 residue.
Biacore Journal – Number 1 2002
SCIENTIFIC REPORT
As well as allowing the identification of the
eluted material and to a large extent the characterization of structural details by MS analysis of
the digest, this experiment also just as importantly demonstrates that we could easily perform a
digestion with the quantities eluted from SPR.
Further confirmation of structural details could
then be obtained from MALDI-MS/MS methods
such as PSD, CID or LIFT, a proprietary
TOF/TOF technology (Bruker Daltonics).
CONCLUSIONS
SPR/MS tandem can be used to solve a typical
proteomics problem, providing functional as well
as structural data, including a target sequence
and post-translational modifications. The calmodulin experiments demonstrate that the two technologies can be interlinked even without modification of the existing software and hardware. In
addition, it was shown to be possible to prepare
the sample recovered by SPR detection for MS
analysis without the use of reverse phase
columns.
11000
13000
m/z
15000
17000
Figure 5
Molecular ion region of the spectrum produced for CaM SPR eluate when using
dihydroxybenzoic acid matrix solution on
an AnchorChip target. Recovery buffer was
0.5% TFA.
References
1. T. Natsume, H. Nakayama, Ö. Jansson, T.
Isobe, K. Takio and K. Mikoshiba
Combination of biomolecular interaction analysis and mass spectrometric amino acid sequencing
Anal Chem 72: 4193-8 (2000)
Figure 6
Spectrum and coverage map for tryptic
digests of CaM eluted from a sensor chip.
Recovery buffer was 50 mM NH4HCO3, 1 mM
EGTA.
1754.8
91-
2. D. Nedelkov, R. W. Nelson
Analysis of human urine biomarkers via biomolecular interaction analysis mass spectrometry.
Am J Kidney Diseases 38: 481-7 (2001)
3. J. R. Krone, R. W. Nelson, D. Dogruel, P.
Williams and R. Granzow
BIA/MS: Interfacing biomolecular interaction
analysis with mass spectrometry
Anal Biochem 244: 124-32 (1997)
2401.1
107-
1563.7
11844.9
14-
1265.6
95-
1250
10
ADQLTEEQIA
1500
20
EFKEAFSLFD
2490.0
127-
1750
2500
2000
2250
2750
m/z
30
20
50
60
KDGDGTITTK ELGTVMRSLG QNPTEAELQD MINEVDADGN
70
90
80
GTIDFPEFLT MMARKMKDTD SEEEIREAFR
100
110
120
VFDKDGNGYI SAAELRHVMT NLGEKLTDEE
130
150
140
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Biacore Journal – Number 1 2002
PDF name: Biacore Journal 1 02 fix.pdf March 27, 2002 17:48:41
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