Chapter 18
Integration of SPR Biosensors with Mass Spectrometry
(SPR-MS)
Dobrin Nedelkov
Abstract
The combination of surface plasmon resonance (SPR) and mass spectrometry (MS) creates a comprehensive protein investigation approach wherein SPR is employed for protein quantification and MS is
utilized to structurally characterize the proteins. In such, MS utterly complements the SPR detection
and reveals intrinsic protein structural modifications that go unregistered via the SPR detection. Protein
complexes and non-specific binding can also be delineated via the SPR-MS approach. Described here are
the protocols and know-how for successful and reproducible integration of SPR and MS. The individual
steps of the entire SPR-MS process are illustrated via an example showing analysis of myoglobin from
human plasma.
Key words: SPR, mass spectrometry, MALDI-TOF, SPR-MS, protein interactions.
1. Introduction
The major technological advances that ushered the field of proteomics have mainly been driven by developments in the mass
spectrometric methods of detection. Mass spectrometry, in the
form of matrix-assisted laser desorption ionization time-of-flight
(MALDI-TOF) and electrospray ionization (ESI), has taken center stage in the investigation of the human proteome. Coupled
with powerful single (2D-gels) or multidimensional (liquid chromatography, LC/LC) separation approaches, mass spectrometry
offers a unique way of detecting proteins and their smaller peptide
fragments. MS interrogation can also be performed on proteins
analyzed by surface plasmon resonance (SPR) biosensors, another
N.J. de Mol, M.J.E. Fischer (eds.), Surface Plasmon Resonance, Methods in Molecular Biology 627,
DOI 10.1007/978-1-60761-670-2_18, © Springer Science+Business Media, LLC 2010
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relatively new technology that has come to dominate the field of
protein interactions since its commercialization in the early 1990s.
The combination of SPR biosensors with mass spectrometry has
become known as SPR-MS or BIA/MS (biomolecular interaction analysis MS) and offers a unique way of analyzing proteins:
SPR is employed for protein quantification, and MS is utilized to
delineate the structural features of the analyzed proteins. Proteins
are initially affinity-captured from solution via ligands covalently
attached on the SPR sensor surface. These ligands are most commonly antibodies, although other types of interacting molecules
can also be utilized. Because the SPR detection is non-destructive,
proteins retrieved on the SPR sensing surface can be further analyzed via mass spectrometry, either directly from the sensor/chip
surface or separately, following an elution and microrecovery. The
MS analysis offers a unique opportunity to delineate non-specific
binding (NSB) of other biomolecules to the surface-immobilized
biomolecules (or to the underivatized sensor surface itself), binding of protein fragments, protein variants (existing due to posttranslational modifications and point mutations), and complexed
(with other molecules) proteins. The SPR sensing alone cannot
detect any of the above events due to its non-discriminatory character (i.e., uniform detection of all binding events via mass change
on the sensor surface).
The SPR-MS approach was first demonstrated in 1996 (1)
and was subsequently described in several proof-of-principle articles (2–3). Almost all of the early work and technology development was performed in our laboratory (4–11). Other investigators have also utilized the approach and advanced it into new
applications (12–22). Described here are the protocols, knowhow, nuances, and the “little tricks” we do in our laboratory for
successful and reproducible integration of SPR and MS, using the
“from-the-chip” MS analysis approach.
2. Materials
Any SPR biosensor that utilizes a planar SPR chip can be utilized
for the SPR analysis and protein retrieval (the dominant player in
the SPR instruments field is Biacore, Biacore Life Sciences, Piscataway, NJ; another manufacturer of SPR biosensors is GWC Technologies, Inc., Madison, WI). In the case of the Biacore instruments, ligands (e.g., antibodies) are most commonly immobilized
on the carboxymethyldextran surface of a CM5 Research Grade
Sensor Chip (Biacore, Cat. No. BR. 1000-14) using amine coupling kit chemicals (Biacore, Cat. No. BR-1000-50).
Integration of SPR Biosensors with Mass Spectrometry (SPR-MS)
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The interface between the SPR biosensor and the mass spectrometer, as practiced in our laboratory, consists of a chip cutter
and matrix applicator. The chip cutter is used for excising the
chip from its plastic support so that it can be placed in an appropriately milled MALDI target. The chip cutter contains a circular
heated cutter-head that, when pressed against the plastic mount,
excises a chip/plastic mount of a defined circular shape that fits
into our MALDI mass spectrometer target (23). The MALDI
matrix applicator is an aerosol-spraying device also developed in
our laboratory (23). The device consists of an aspirating/sheath
gas needle (∼30 μm orifice) backed by ∼30 psi of compressed air.
The air/matrix solution ratio can be adjusted to produce a fine
mist of matrix solution that is aimed at the entire surface of the
cutout chip. The matrix of choice is α-cyano-4-hydroxycinnamic
acid (ACCA, Aldrich, Milwaukee, WI, USA, Cat. No. 47,687-0),
which is further processed by powder-flash re-crystallization from
a low-heat saturated acetone solution of the original stock.
MALDI-TOF mass spectrometry analysis from the chip surface can be performed on any commercially available mass spectrometer. In our laboratory, a custom-made MALDI-TOF mass
spectrometer (Intrinsic Bioprobes, Inc.) is used for the SPR-MS
analysis. The instrument consists of a linear translation stage/ion
source capable of precise targeting of each of the flow cells under
a focused laser spot. Ions generated during a 4 ns laser pulse
(357 nm, nitrogen) are accelerated to a potential of 30 kV over a
single-stage ion extraction source distance of ∼2 cm, before entering a 1.5 m field-free drift region. The ion signals are detected
using a two-stage hybrid (channel plate/discrete dynode) electron multiplier. Time-of-flight spectra are produced by signal
averaging of individual spectra from 50 to 100 laser pulses (using
a 500 MHz; 500 Ms/s digital transient recorder). Custom software is used in acquisition and analysis of the mass spectra. All
protein spectra are obtained in the positive ion mode.
3. Methods
The individual steps of the entire SPR-MS process will be
illustrated via an example showing analysis of myoglobin from
human plasma. Two antibodies to myoglobin were utilized:
goat anti-human (Fitzgerald, Concord, MA, Cat. No. 70-MG60,
5.2 mg/mL) was immobilized in flow cell 1 (FC1) on a surface of a CM5 Research Grade Sensor Chip (see Note 1), using
standard EDC/NHS coupling protocol and reagents supplied
by Biacore. Rabbit anti-human myoglobin antibody (Fitzgerald,
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Cat. No. 70-MR13, 3.9 mg/mL) was immobilized in a similar
manner in flow cell 2 (FC2) on the same chip. Both antibodies were diluted 100-fold into 10 mM acetate buffer, pH 5.0.
The SPR signals observed at the end of the immobilization process indicated successful immobilization of ∼100 fmoles antibody
in each flow cell (see Note 2). HBS-EP (0.01 M HEPES, pH
7.4, 0.15 M NaCl, 0.005% (v/v) polysorbate 20, and 3 mM
EDTA) at a flow rate of 5 μL/min (see Note 3) was used as a
running buffer in the SPR experiments. Following buffer equilibration in both flow cells, a single 50-μL aliquot of twofold
diluted human plasma (ProMedDX, Norton, MA) was injected
over both flow cells simultaneously. The resulting sensorgram
(Fig. 18.1)indicates binding responses of 324 RUs (response
units) in FC1 and 1,031 RUs in FC2. Immediately following the
end of the injection, the buffer flow was stopped (see Note 4),
the chip removed from the biosensor, washed with three 200-μL
aliquots of ultra pure sterile water, cut from its plastic housing
(see Note 5), and prepared for MALDI-TOF mass spectrometry by application of MALDI matrix (see Note 6) with the help
of the matrix applicator device (see Note 7). Figure 18.2 shows
the mass spectra obtained from the surfaces of the two flow cells.
Both mass spectra contain multiply charged myoglobin signals,
at expected m/z values (MWmyoglobin =17,052.6 Da). The signals and the overall quality of the mass spectrum obtained from
the surface of flow cell 2 are little better than those obtained
from FC1. This is somewhat expected, as there was more material bound in FC2, judging from the SPR response in Fig. 18.1.
Hence, from the SPR-MS experiment it was concluded that the
rabbit anti-human myoglobin antibody exhibited better affinity
for human myoglobin and was chosen for further experiments.
Fig. 18.1. SPR sensorgrams showing the injection of a 50-μL aliquot of twofold diluted
human plasma over FC1 and FC2, derivatized with goat and rabbit anti-human myoglobin antibodies, respectively.
Integration of SPR Biosensors with Mass Spectrometry (SPR-MS)
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Fig. 18.2. Mass spectra taken from the surfaces of FC1 and FC2, showing the presence
of multiply charged myoglobin ions (MWmyoglobin =17,052.6 Da).
4. Notes
1. Prior to insertion into the instrument, the sensor chip should
be removed from the plastic housing cassette and washed
with several aliquots of ultra pure water, to remove any residual stabilizers that might interfere with the post-SPR mass
spectrometry analysis.
2. It is highly recommended that the ligand is immobilized in
a high density on the flow cell surface. High ligand densities can be somewhat detrimental to the generation of good
kinetics data (i.e., re-binding of the analyte might complicate the kinetics analysis), but are very beneficial for the subsequent MS analysis because the amount of protein analyte
captured from solution is increased.
3. Another way of increasing the amount of captured analyte
is to decrease the flow rate of the sample over the ligandderivatized flow cell surface. Decreased rates promote mass
transport effects, which in turn increase the amount of protein analyte captured on the chip. On the other hand, at
high flow rates typically used for SPR kinetics experiments
(60–100 μL/min), low-concentration analytes will not be
captured in amounts permissible to downstream MS analysis.
4. To minimize the loss of protein during the post-capture
steps, it is of great importance to minimize the time between
the end of the SPR analysis and the subsequent preparation
steps for MS. Following the end of the sample injection step,
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the SPR response should be promptly recorded, the flow
of the running buffer stopped, and the chip removed from
the biosensor (the fastest way to undocking is via the root
control software of the biosensor). The chip itself should be
removed from the cassette and washed with several aliquots
of ultra pure water to remove the residual buffer which
contains detergent molecules that cause interferences in the
mass spectra.
5. We have used, in the past, and with great success, a chip
cutter with circular heated cutter-head (Intrinsic Bioprobes
Inc., Tempe, AZ) for excising a chip/plastic mount of a
defined circular shape that fits into an appropriately configured MALDI mass spectrometer target (12). However,
most commercially available mass spectrometers can accommodate the large footprint of the entire chip-housing, as
long as the MALDI target is appropriately machined/milled
to accommodate the entire chip.
6. In most MS experiments, higher quality mass spectra are
obtained with α-cyano-4-hydroxycinnamic acid (ACCA) for
proteins smaller than ∼25 kDa, whereas sinapic acid (SA)
yields better results for higher molecular mass (>25 kDa)
proteins. For the MS analysis from the chips, however,
ACCA is superior in that it is a better energy-absorbing
matrix and, consequently, requires less laser power. Lower
laser power means that more spectra can be obtained from a
single spot and the fast burning through the matrix/sample
layer is avoided. (It should be noted that the analyte is captured in a very thin layer on the surface of the chip, and
aggressive interrogation with the laser (at high frequency)
can rapidly deplete the sample spot.) Hence, for big proteins, the appearance and intensity of multi-charged ion analyte signals obtained with ACCA are better indicators of
the presence and the mass of the on-chip retained proteins.
Re-application of more matrix (following initial application
and MS analysis) does not yield better signals and generally
results in decreased S/N ratio.
7. The application of MALDI matrix is the most critical step
in the entire SPR-MS process. The matrix mist generated
by the matrix applicator should moisten, but not completely
wet the chip surface (i.e., the tiny matrix droplets should
stay as individual drops on the surface and not be connected
into one large liquid drop). The matrix droplets will desorb
the proteins from their respective capturing affinity ligand
and, upon rapid drying, the matrix/protein mixture will be
re-deposited on the same area from where the proteins were
originally captured in the SPR analysis.
Integration of SPR Biosensors with Mass Spectrometry (SPR-MS)
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Acknowledgments
This publication was supported in part by grant number 5 R44
CA82079-04 from the National Institutes of Health. Its contents
are solely the responsibility of the author and do not necessarily
represent the official views of the National Institutes of Health.
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