Detection of in vivo matrix metalloproteinase activity using microdialysis sampling and liquid chromatography/mass spectrometry

Article abstract of DOI:10.1021/ac901703g

Matrix metalloproteinases (MMPs) are a family of endoproteases that break down extracellular matrix and whose upregulation contributes to several diseases. A liquid chromatography/tandem mass spectrometry (LC/ MS/MS) method was developed to quantify MMP-1

Full text of DOI:10.1021/ac901703g

Anal. Chem. 2009, 81, 9961–9971
Detection of in Vivo Matrix Metalloproteinase
Activity Using Microdialysis Sampling and Liquid
Chromatography/Mass Spectrometry
Ying Wang,†,# Dmitri V. Zagorevski,† Michelle R. Lennartz,‡ Daniel J. Loegering,§ and
Julie A. Stenken*,|
Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, 110 Eighth Street,
Troy, New York 12180, Center for Cell Biology and Cancer Research, Center for Cardiovascular Sciences, Albany
Medical College, 43 New Scotland Avenue, Albany, New York 12208, and Department of Chemistry and
Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701
Matrix metalloproteinases (MMPs) are a family of en-
doproteases that break down extracellular matrix and
whose upregulation contributes to several diseases. A
liquid chromatography/tandem mass spectrometry (LC/
MS/MS) method was developed to quantify MMP-1 and
MMP-9 substrates and their N-terminal peptide products
in samples obtained from implanted microdialysis sam-
pling probes. In vitro studies with purified human MMP-1
and MMP-9 were used to optimize the assay and deter-
mine the effectiveness of the local delivery of a broad-
spectrum MMP inhibitor, GM 6001. Localized delivery
of GM 6001 at 10 µM was sufficient to completely inhibit
product formation in vitro. In vivo studies in male
Sprague-Dawley rats were performed with microdialysis
probes implanted into the subcutaneous tissue. Directly
after microdialysis probe implantation, infusions of the
MMP-1 and MMP-9 substrates (50 µM each) resulted in
recovered product concentrations of approximately 2 µM.
During a 50 µM GM 6001 coinfusion with the substrates,
a 30% and 25% reduction in product formation for the
MMP-1 and MMP-9 substrates was obtained, respec-
tively. Blank dialysates were negative for enzymatic activity
that could cleave the MMP substrates. This method
allowed for the activity of different MMPs surrounding the
microdialysis probe to be observed during in vivo sampling.
trations of these modulators and their temporal sequence of
release is of great importance for understanding the multifaceted
series of reactions during the FBR. These modulators work in
concert in an in vivo environment and are not easily mimicked in
vitro,4,5 suggesting that developing in vivo bioanalytical collection
and detection methodologies for these molecules is crucial.
Of all these modulators, MMPs have long been underestimated
since they have been simplistically viewed as proteolytic enzymes
that serve to hydrolyze components of the extracellular matrix
(ECM). It has become increasingly apparent that MMPs also
release many bioactive cytokines and growth factors that are
entrapped in the ECM.6,7 MMPs are a large family of zinc-
dependent endoproteases that can cleave various protein compo-
nents within the ECM or at the cell surface.8,9 In addition to their
role in wound healing, these matrix remodeling enzymes play
critical roles in numerous biological processes from embryonic
development to cancer metastasis.
The presence of MMPs in biological samples is typically
determined by (1) bioassays or activity assays using labeled
substrates, (2) immunoassays and/or immunohistochemical stain-
ing, and (3) zymography.10-13 Bioassays using labeled substrates
can be carried out by using either labeled natural protein
substrates, e.g., radiolabeled proteins, fluorescently labeled pro-
teins, biotinylated gelatin or succinylated gelatin, or initially
quenched fluorogenic peptide substrates.14 Zymography is an
electrophoretic technique to analyze the activities of MMPs. The
Implantation of materials leads to a sequence of events known
as the foreign body reaction (FBR), which includes the release
of synergistic bioactive molecules including cytokines, eicosanoids,
and matrix metalloproteinases (MMPs).1-4 Knowing the concen-
(3) Luttikhuizen, D. T.; Amerongen, M. J.; Feijter, P. C.; Petersen, A. H.;
Harmsen, M. C.; Luyn, M. J. A. Biomaterials 2006, 27, 5763–5770
(4) Vaday, G. G.; Lider, O. J. Leukocyte Biol. 2000, 67, 149–159.
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(5) Sternlicht, M. D.; Werb, Z. Annu. Rev. Cell Dev. Biol. 2001, 17, 463–516
(6) Somerville, R. P. T.; Oblander, S. A.; Apte, S. S. Genome Biol. 2003, 4,
216.1–11
(7) McCawley, L. J.; Matrisian, L. M. Curr. Opin. Cell Biol. 2001, 13, 534–
540
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* To whom correspondence should be addressed. Phone: 479-575-7018. Fax:
479-575-4049. E-mail: jstenken@uark.edu.
.
† Rensselaer Polytechnic Institute.
(8) Matrix Metalloproteinase Protocols; Clark, I. M., Ed.; Methods in Molecular
‡ Center for Cell Biology and Cancer Research, Albany Medical College.
§ Center for Cardiovascular Sciences, Albany Medical College.
| University of Arkansas.
Biology, Vol. 151; Humana Press, Inc.: Totowa, NJ, 2001.
(9) Egeblad, M.; Werb, Z. Nat. Rev. Cancer 2002, 2, 161–174
(10) Catterall, J. B.; Cawston, T. E. Methods Mol. Biol. 2003, 225, 353–364
(11) Iked, K.; Tate, G.; Suzuki, T.; Mitsuya, T. Gynecol. Oncol. 2005, 97, 323–
329
(12) Kleiner, D. E.; Stetler-Stevenson, W. G. Anal. Biochem. 1994, 218, 325–
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(13) Gogly, B.; Groult, N.; Hornebeck, W.; Godeau, G.; Pellat, B. Anal. Biochem.
1998, 255, 211–216
(14) Lombard, C.; Saulnier, J.; Wallach, J. Biochimie 2005, 87, 265–272
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# Present address: Regeneron Pharmaceuticals, Inc., 81 Columbia Turnpike,
Rensselaer, NY 12144.
.
(1) Anderson, J. M.; Rodriguez, A.; Chang, D. T. Semin. Immunol. 2008, 20,
86–100
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(2) Jones, J. A.; McNally, A. K.; Chang, D. T.; Qin, L. A.; Meyerson, H.; Colton,
E.; Kwon, I. L. K.; Matsuda, T.; Anderson, J. M. J. Biomed. Mater. Res.
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10.1021/ac901703g CCC: $40.75  2009 American Chemical Society
Published on Web 11/11/2009
Analytical Chemistry, Vol. 81, No. 24, December 15, 2009 9961

main limitations of zymography are that it is tedious, difficult to
quantitate, and limited to a few MMPs, such as MMP-1, -2, -7, -9,
and -13.
Microdialysis sampling is a well-established diffusion-based
sample collection method that has gained wide acceptance for
collection of solutes in vivo.15,16 During microdialysis sampling,
the exchange of solutes can occur in both directions across the
membrane, both into and out of the probe, depending on the
concentration gradient of the solute. Microdialysis sampling
effectiveness is determined via the bidirectional extraction ef-
ficiency (EE), which can be calculated as shown in eq 1, where
Cinlet, Coutlet, Csample stand for the analyte concentration in
perfusion fluid, dialysate, and external sample medium, respec-
tively.17 This equation allows the calculation of extraction for
a delivered substrate that is locally infused through the dialysis
probe.
Figure 1. Microdialysis sampling schematic for in situ MMPs
detection: MMP-1 substrate and MMP-2/-9 substrate diffuse to the
surrounding medium and react with MMP-1 and MMP-9, forming the
products that diffuse back into the probe.
gained via individual perfusion of each substrate. To test the
presence of different MMPs with each individual substrate
requires switching the perfusion fluid.
Cinlet - Coutlet
Cinlet - Csample
EE )
(1)
Microdialysis sampling combined with liquid chromatography/
electrospray ionization mass spectrometry (LC/ESI-MS) has been
applied to achieve an in situ and multisubstrate detection of
elastase enzymatic activity external to the microdialysis sampling
probes.26 With mass spectrometry, analyte detection is based on
the m/z ratio requiring no modification to the substrate structure
or synthesis of chromophore-containing compounds.27 Thus, the
distinct advantage of the mass spectrometry approach over
monitoring fluorescent products is that the activity of multiple
MMPs could be simultaneously monitored.
This work focuses on the in vitro and in vivo method
development and validation of MMP activity using microdialysis
sampling combined with LC/MS detection. MMP-1, -2/-9 were
chosen as the initial targets since they are known to be involved
in the initiation and progression of FBR associated with biomaterial
implantation.28 The substrate specificities for MMP-2 and -9
overlap, and the substrate used in this study can be cleaved by
both enzymes. These substrates were infused through the mi-
crodialysis sampling probe as denoted in Figure 1. A broad-
spectrum MMP inhibitor (GM 6001)29,30 was included in the
perfusate to determine if MMPs surrounding the dialysis probe
could be inhibited, providing validation that MMPs and not other
proteases were cleaving the infused substrates. Additionally,
zymography was performed to confirm the presence of MMPs
around the implanted microdialysis probes.
Microdialysis sampling can be performed to collect analytes
from the sample medium in the recovery mode, or it can be used
to deliver certain substances with a molecular weight smaller than
the membrane molecular weight cutoff (MWCO) to the fluid
outside the probe in the delivery mode. Coupled with appropriate
analytical methods and the judicious choice of substrates, mi-
crodialysis sampling has been applied toward in situ detection of
enzyme activities.18-20 The in vivo localized delivery of a substrate
followed by collection of an enzymatic product during microdi-
alysis sampling has been demonstrated by several researchers.21,22
Since microdialysis sampling has been so widely used in
biomedicine, it is not surprising that several researchers have
focused their attention on determining MMP activity in various
in vivo settings. MMPs are known to be upregulated in tumors.
These enzymes have been collected into 100 kDa MWCO
microdialysis probes implanted into human breast tumors.23
Additionally, MMPs are believed to play important roles with
respect to atherosclerotic plaque formation and remodeling. An
online fluorescence-based sensor has been described to monitor
the colorimetric products formed from MMP substrates infused
through microdialysis sampling probes.24,25 These substrates all
produce a common fluorescent coumarin. Selectivity can only be
(15) Stenken, J. A. Microdialysis Sampling. In Encyclopedia of Medical Devices
and Instrumentation, 2nd ed.; Webster, J. G., Ed.; John Wiley & Sons, Inc.:
Hoboken, NJ, 2006; Vol. 4, pp 400-420.
(16) Bourne, J. A. Clin. Exp. Pharmacol. Physiol. 2003, 30, 16–24
(17) Bungay, P. M.; Morrison, P. F.; Dedrick, R. L. Life Sci. 1990, 46, 105–
119
(18) Bergstro¨m, S. K.; Goiny, M.; Danielsson, R.; Ungerstedt, U.; Andersson,
M.; Markides, K. E. J. Chromatogr., A 2006, 1120, 21–26
(19) Modi, S. J.; LaCourse, W. R. J. Chromatogr., A 2006, 1118, 125–133
.
EXPERIMENTAL SECTION
Chemicals. MMP-1 (proenzyme (55 kDa), from human
rheumatoid synovial fibroblasts, g15 mU/mg protein), MMP-9
(proenzyme (92 kDa), monomer, from human neutrophils, g1000
mU/mg protein), MMP-1 substrate I, fluorogenic (DNP-Pro-Leu-
.
.
.
(20) Nilsson, C.; Nilsson, F.; Turner, P.; Sixtensson, M.; Nordberg Karlsson,
E.; Holst, O.; Cohen, A.; Gorton, L. Anal. Bioanal. Chem. 2006, 385, 1421–
1429
(21) Scott, D. O.; Lunte, C. E. Pharm. Res. 1993, 10, 335–342
(22) Stenken, J. A.; Holunga, D. M.; Decker, S. A.; Sun, L. Anal. Biochem. 2001,
290, 314–323
(23) Nilsson, U. W.; Dabrosin, C. Cancer Res. 2006, 66, 4789–4794
.
(26) Wang, Y.; Zagorevski, D. V.; Stenken, J. A. Anal. Chem. 2008, 80, 2050–
.
2057
(27) Saghatelian, A.; Jessani, N.; Joseph, A.; Himphrey, M.; Cravatt, B. F. Proc.
Natl. Acad. Sci. U.S.A. 2004, 27, 10000–10005
(28) Xu, P.; Sefton, M. V. J. Biomed. Mater. Res. 2004, 71, 226–232
.
.
.
.
.
(24) Etoh, T.; Joffs, C.; Deschamps, A. M.; Davis, J.; Dowdy, K.; Hendrick, J.;
(29) Galardy, R. E.; Cassabonne, M. E.; Giese, C.; Gilbert, J. H.; Lapierre, F.;
Baicu, S.; Mukherjee, R.; Manhaini, M.; Spinale, F. G. Am. J. Physiol. 2001,
Lopez, H.; Schaefer, M. E.; Stack, R.; Sullivan, M.; Summers, B. Ann. N.Y.
281, H987–H994
(25) Spinale, F. G.; Koval, C. N.; Deschamps, A. M.; Stroud, R. E.; Ikonomidis,
J. S. Circulation 2008, 118, S16–S23
.
Acad. Sci. 1994, 732, 315–323
(30) Galardy, R. E.; Grobelny, D.; Foellmer, H. G.; Fernandez, L. A. Cancer Res.
1994, 54, 4715–4718
.
.
.
9962 Analytical Chemistry, Vol. 81, No. 24, December 15, 2009

Table 1. Structure and Molecular Ion of MMP-1 and MMP-2/-9 Substrate and Productsa
MMP-1
MMP-2/-9
sequence
m/z of [M + H]+
sequence
m/z of [M + H]+
substrate
DNP-Pro-Leu-Ala∼Leu-Trp-Ala-Arg
992.5
466.2
545.3
DNP-Pro-Leu-Gly∼Met-Trp-Ser-Arg
1012.5
452.2
579.3
N-terminal product (NTP) DNP-Pro-Leu-Ala
C-terminal product (CTP) Leu-Trp-Ala-Arg
DNP-Pro-Leu-Gly
Met-Trp-Ser-Arg
a DNP denotes the 2,4-dinitrophenyl derivative to the terminal amine; “∼” denotes the enzyme cleavage site.
Ala-Leu-Trp-Ala-Arg-OH, DNP denotes the 2,4-dinitrophenyl de-
rivative to the terminal amine), MMP-2/MMP-9 substrate I,
fluorogenic (DNP-Pro-Leu-Gly-Met-Trp-Ser-Arg-OH), and MMP
inhibitor GM 6001 (N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-
solvent B (v/v 40%) was used to separate the analytes. Samples
(5 µL) were injected onto the LC column using the system
autosampler.
MS conditions were set as follows: capillary voltage was set to
3.5 kV; -143.5 V on the capillary exit and -40.0 V at the skimmer.
Octopole 1 dc was set to -12.0 V and octopole 2 dc was set to
-2.0 V. Octopole rf amplitude was set to 200 Vpp. Ion lenses 1
and 2 were set to -5.0 and -60.0 V, respectively. Further ion
source parameters were nebulizer gas (nitrogen) 25.0 psi and
drying gas (nitrogen) 10.0 L/min at a drying temperature of
350 °C.
Calibration Curves and Analyte Quantification. For sub-
strates and products, standard solutions of MMP-1 substrate
(0.5-50 µM), MMP-2/-9 substrate (0.5-50 µM), MMP-1 NTP
(0.1-50 µM), and MMP-2/-9 NTP (0.1-50 µM) were prepared
from the stock solutions (1 mM in DMSO) with PBS. Table 1
shows the peptide sequences and the m/z values for the [M +
H]+ ions of the substrates and their N-terminal products.
Standards were analyzed in triplicate by LC/MS/MS in product
ion monitoring mode. The peak area from the extracted ion
chromatograms (EIC) of MS/MS of m/z 992 for the MMP-1
substrate, MS/MS of m/z 1012 for the MMP-2/-9 substrate,
MS/MS of m/z 466 for the MMP-1 NTP, and MS/MS of m/z
452 for the MMP-2/-9 NTP versus concentration were used to
generate calibration curves.
methylpentanoyl]-L-tryptophan methylamide) were purchased
from EMD Chemicals, Inc. (Gibbstown, NJ). Stock solutions of
the MMP-1 substrate, MMP-2/-9 substrate, and GM 6001 were
prepared in DMSO and stored at -80 °C.
The MMP-1 N-terminal peptide product (NTP, DNP-Pro-Leu-
Ala, >95% purity) and MMP-2/-9 NTP (DNP-Pro-Leu-Gly, >95%
purity) were custom-synthesized from Biomatik Corp. (Cambridge,
ON, Canada). The sequence and molecular weight was confirmed
by mass spectrometry. Stock solutions of MMP-1 NTP and MMP-
2/-9 NTP were prepared in DMSO and stored at 4 °C.
Phosphate-buffered saline (PBS, 1×, pH 7.4, sterile, Dulbecco’s
formula, consisting of CaCl2 0.90 mM, MgSO4 0.49 mM, KCl
2.68 mM, KH2PO4 1.47 mM, NaCl 136.89 mM, Na2HPO4 8.10
mM) was purchased from MP Biomedicals (Irvine, CA).
Trypsin (from bovine pancreas, TPCK-treated, essentially salt-
free, lyophilized powder, 13 500 BAEE units/mg solid) and
trypsin inhibitor SBTI (type I-S) were purchased from Sigma
(St. Louis, MO). Stock solutions of trypsin (500 µg/mL) were
prepared in 1 mM HCl and stored at -80 °C until use.31 The
activation buffer for pro-MMP activation was prepared by
dissolving 50 mM Tris-HCl, 0.1 M NaCl, 5 mM CaCl2 in water
with the pH adjusted to 7.5. All other chemicals were reagent
grade or better. HPLC grade water was purchased from Fisher
Scientific.
Novex 10% zymogram (gelatin) gel (1.0 mm, 10 well), 12%
zymogram (casein) gel (1.0 mm, 10 well), zymogram renaturing
buffer (10×), zymogram developing buffer (10×), Tris-glycine
SDS sample buffer (2×), Tris-glycine SDS running buffer (10×),
XCell SureLock Mini-Cell system, SimplyBlue SafeStain, and
DryEase Mini-Gel drying system were all purchased from Invit-
rogen Corp. (Carlsbad, CA). Zymography was performed accord-
ing to manufacturer’s instructions.
Standard solutions of MMP inhibitor GM 6001 (0.1-50 µM)
were prepared by diluting the stock solution (2.5 mM in DMSO)
with PBS. Standards were analyzed by LC/MS in triplicate. Peak
area of EIC of molecular ion (MS of m/z 389) versus concentration
was used to generate the calibration curve.
Dialysates were analyzed in duplicate using LC/MS/MS. MS
of m/z 389 for GM 6001, MS/MS of m/z 992 for MMP-1 substrate,
MS/MS of m/z 1012 for MMP-2/-9 substrate, MS/MS of m/z 466
for MMP-1 NTP, and MS/MS of m/z 452 for MMP-2/-9 NTP were
monitored on each segment. Peak areas from the above EIC were
used for quantitation using the appropriate calibration curves.
Analyte Stability and Intra-/Interday Variation. The stabili-
ties of the MMP substrates, MMP-1 and -9 NTP, and GM 6001
were tested by storing each as a 50 µM solution in PBS in the
dark at 4 °C or at room temperature for 1, 3, and 7 days. The
intraday variances were tested on four runs of 50 µM standards
using the LC/MS/MS method. The interday difference was tested
on 50 µM standards on three different days.
Liquid Chromatography/Electrospray Ionization Mass
Spectrometry System. An Agilent 1100 series high-performance
liquid chromatograph (HPLC) with an MSD 1100 ion trap mass
analyzer (Agilent Technologies, Santa Clara, CA) operating with
electrospray ionization (ESI) in positive ion mode was used for
separation and detection. A binary pump was used to deliver the
solvents. Solvent A contained 0.2% formic acid in water, and solvent
B contained 0.2% formic acid in acetonitrile. An Alltech Alltima 5
µm C18 column (150 mm × 4.6 mm i.d.) with guard column (Grace
Company, Deerfield, IL) was employed for analyte separation. The
mobile phase flow rate was 0.5 mL/min. An isocratic elution with
Pro-MMPs Activation. The MMPs are obtained in their
proenzyme form and require activation using trypsin.32 Upon
activation, human MMP-1 is converted from a 55 kDa proenzyme
to a 45 kDa active enzyme. Similarly, human MMP-9 is converted
(32) Duncan, M. E.; Richardson, J. P.; Murray, G. I.; Melvin, W. T.; Fothergill,
(31) Walsh, K. A. Methods Enzymol. 1970, 19, 41–63
.
J. E. Eur. J. Biochem. 1998, 258, 37–43.
Analytical Chemistry, Vol. 81, No. 24, December 15, 2009 9963

from a 92 kDa proenzyme to an 86 kDa active enzyme. One
volume (5 µL) of pro-MMP-1 or pro-MMP-9 at a concentration of
approximately 100 µg/mL was incubated with two volumes of
trypsin at a concentration of 15 µg/mL in the activation buffer,
giving a final trypsin concentration of 10 µg/mL. After incubation
at 37 °C for 2 h, the reaction was terminated by the addition of
one volume of trypsin inhibitor SBTI at a concentration of 200
µg/mL (diluted form stock with PBS). The two reaction mixtures
were combined together and diluted 12.5 times in PBS to get
enough volume for microdialysis experiments with a final solution
volume of 500 µL. This reaction mixture contained activated
MMPs and was used for further microdialysis experiments.
Microdialysis Sampling in Vitro. Microdialysis sampling was
performed with a 1 mL Hamilton syringe (Hamilton Company,
Reno, NV) with plastic tip and a BAS baby bee syringe pump with
controller (BASi, West Lafayette, IN). CMA 20/04 microdialysis
probes with a 10 mm 20 kDa MWCO poly(arylethersulphone)
(PAES) membrane (CMA/Microdialysis, North Chelmsford, MA)
were used. All in vitro experiments were performed using a 1 µL/
min flow rate in a 37 °C water bath.
EE% of Substrates: Quiescent versus Well-Stirred. Mi-
crodialysis probes were used to locally deliver mixtures of MMP-1
substrate (50 µM) and MMP-2/-9 substrate (50 µM) to 2 mL
solutions of PBS at 37 °C. The PBS sample medium was kept
quiescent in one set of experiments and well-stirred in the other
set of experiments. The dialysates were collected 10 min after
the probe was immersed in the sample medium to allow for
equilibration. Two probes were employed for each set of experi-
ments, and dialysates were collected from both probes.
30 min for 3.5 h. Dialysates were analyzed using LC/MS/MS,
and the analytes were quantified using the calibration curves.
In Vitro Collection of MMPs. Two microdialysis sampling
probes were immersed into the activated MMPs solution with PBS
perfused at 1 µL/min to determine if any of the active MMPs
could pass through the probe membrane. The dialysate collection
started 10 min after the probe was immersed in the sample
medium to allow for equilibration. Dialysates were collected from
both probes and were subsequently spiked with MMP-1 substrate
(50 µM) and MMP-2/-9 substrate (50 µM) to achieve an initial
concentration of 20 µM for each substrate. The reaction mixtures
were kept dark at 25 °C for 12 h and then analyzed using LC/
MS/MS.
In Vivo Microdialysis Sampling with Freely Moving Rats.
Male Sprague-Dawley rats (210-250 g, purchased from Taconic,
NY) were used and had free access to food and water as well as
a 12 h on/off light cycle. All surgical procedures were performed
using aseptic technique. The Albany Medical College IACUC
committee approved the protocols, and these protocols also met
the guidelines set forth by the NIH for the care and use of
laboratory animals.
Before probe implantation, the microdialysis probes were
perfused with 70% ethanol then sterile water in a biosafety cabinet.
Under isoflurane (Abbott Laboratories, North Chicago, IL) anes-
thesia, two identical PAES probes (CMA 20/04, 10 mm, 20 kDa
MWCO membrane) were implanted in the dorsal subcutaneous
space using an incision of less than one inch with one probe
implanted on each side of the dorsal spine. The microdialysis
probe was inserted into the subcutaneous space, and the incision
was closed using surgical staples. The inlet and outlet tubing was
cleaned with 70% ethanol and tunneled out from the neck of the
rat. Immediately after surgery, the rats were connected to a CMA
120 freely moving animals system (CMA Microdialysis, North
Chelmsford, MA). Microdialysis sampling was performed with two
1 mL CMA glass syringes with plastic tip controlled by a CMA
102 microdialysis dual-channel pump (CMA Microdialysis, North
Chelmsford, MA). Food and water were provided to the rats
during sample collection.
EE% of Inhibitor GM 6001. The loss (EE) of inhibitor GM
6001 from the microdialysis probes was measured in vitro.
Solutions of GM 6001 (10, 20, or 50 µM) were prepared in PBS
buffer with or without the mixture of MMP-1 substrate (50 µM)
and MMP-2/-9 substrate (50 µM). These solutions were perfused
(1 µL/min) through microdialysis probes placed in a quiescent
PBS sample medium (2 mL). The dialysate collection started 10
min after the probe was immersed in the sample medium to allow
for equilibration. Three probes were employed and dialysates were
collected for 30 min.
After connecting the fluid lines in the freely moving animals
system, both probes were perfused with sterile PBS buffer with
the following sequence 5 µL/min for 10 min, 1 µL/min for 10
min, and then a blank PBS sample (no substrate included) was
collected at 1 µL/min for 30 min. The 5 µL/min flush for the first
10 min was performed ensure that fluid lines were open after the
implantation of the probe. Additionally, as microdialysis sampling
is an invasive procedure, any solutes released due to the surgery
or probe insertion into the tissue will be flushed out with a higher
flux as mass removal during microdialysis sampling is directly
correlated with perfusion flow rate. The perfusate was then
switched to a substrate mixture (MMP-1 substrate (50 µM) and
MMP-2/-9 substrate (50 µM) in PBS) that was infused in one
probe. In the other probe, the substrate mixture combined with
GM 6001 (50 µM) was perfused. After initiating the switch in
perfusate from collection of blanks to infusion of substrates, the
probes were again perfused at 5 µL/min for 10 min, 1 µL/min for
10 min, and then the dialysates were collected every 30 min for
3.5 h.
Microdialysis Sampling for in Situ MMPs Detection. The
inhibitor was first included into the sample medium containing
activated MMPs to see whether their enzymatic activity could be
effectively inhibited by GM 6001. GM 6001 stock solution was
added to the activated MMPs solution to achieve a final GM 6001
concentration of 10, 20, or 50 µM. This solution was kept at 37 °C
and used as sample medium outside the probe. In separate vials,
the same volume of activated MMPs solution with no inhibitor
added was used as a control. The substrate mixture solution with
MMP-1 substrate (50 µM) and MMP-2/-9 substrate (50 µM) in
PBS was perfused through microdialysis sampling probes im-
mersed in either the sample medium containing GM 6001 or the
control. Dialysates were collected every 30 min for 3.5 h.
In a separate set of experiments, the GM 6001 inhibitor was
included in the perfusate at concentrations of 10, 20, or 50 µM to
determine if a local inhibitor infusion could be used to completely
inhibit the MMPs. The same enzyme and substrate concentrations
were used as described above. Dialysates were collected every
9964 Analytical Chemistry, Vol. 81, No. 24, December 15, 2009

After the perfusion of the substrates through the probes was
complete, the tubing lines were placed in a surgical pocket that
was created at the nape of the neck of the rat. This pocket was
closed using surgical staples. The microdialysis probes were also
infused on days 3 and 7 postimplantation. After completion of the
dialysate collection on day 7, the probes and surrounding tissue
was surgically explanted. Tissue far away from the probe was also
explanted as a control.
MMP Activity of Blank PBS Dialysates. The MMP activities
in the collected blank PBS samples were tested to see whether
activated MMPs were collected into the microdialysis sampling
probe. After collection, the MMP-1 and -9 substrate mixture
solution (50 µM each) was added to the blank PBS samples to
achieve an initial substrate concentration of 20 µM for each
substrate. The reaction mixtures were kept dark at 25 °C for 12 h,
and the production of product was determined using LC/MS/
MS.
Zymography. Tissue lysates were prepared by homogenization
in RIPA (radio immunoprecipitation assay) buffer (50 mM Tris,
pH 7.4, 150 mM NaCl, 12.7 mM deoxycholate, 25 mM glycero-
phosphate, 1% SDS, and 1% Triton X-100 containing protease
inhibitors); tissue was sonicated briefly to decrease viscosity and
cleared of insoluble material by ultracentrifugation (1 h × 100 000g,
4 °C).
Protein concentration in the tissue lysates was determined by
BCA assay using a bovine serum albumin (BSA, Sigma, St. Louis,
MO) standard curve (0-300 µg/mL). Lysates were diluted to
obtain readings on the standard curve. Samples were incubated
at 60 °C for 30 min in a water bath. Absorbance was read at 540
nm using a plate reader. The protein concentration was deter-
mined using the standard curve.
Protein (10 µg per well) was loaded onto the gelatin zymog-
raphy gels; 20 µg per well was used for casein zymography; 20
µg of HT15 media containing MMP-1, -2, and -9 was used as
positive control. Electrophoresis was performed according to
manufacture’s directions (Invitrogen, Carlsbad, CA) using a XCell
Surelock Mini-Cell system at 125 V constant voltage for about 90
min or until the tracking dye reached the bottom of the gel. After
the electrophoresis, MMPs were renatured in-gel using the
zymogram renaturing buffer (30 min, room temperature with
gentle agitation). Following renaturation, the gels were equili-
brated in zymogram developing buffer and incubated overnight
at 37 °C. During this time the renatured MMPs degraded either
the gelatin or casein, clearing the region of the gel containing
the MMP of protein. By staining the gels for protein with
SimplyBlue SafeStain (1 h, room temperature), the presence of
MMPs could be detected as clear bands against the blue
background. The gels were scanned and dried using the DryEase
Mini-Gel drying system.
LC/MS/MS in product ion monitoring mode was used for the
detection scheme. The peak area of the EIC of all the product
ions was used to construct the calibration curves and to quantify
the analytes in the dialysates. Generally for peptide quantitation,
MS/MS in SRM (selective reaction monitoring) mode is preferred
due to its high sensitivity and selectivity by fragmenting the parent
ion followed by quantification of a specific product ion. However,
peptides undergo fragmentation in MS/MS and form a series of
product ions from the N- or C-terminal loss of amino acids. Several
product ions may show comparable signal intensity, and a specific
product ion signal is much lower compared to the sum of product
ion signals. In this case, the sum of the product ion signals is
sometimes used for quantitation.33,34
Upon enzymatic cleavage, the substrates will produce both N-
and C-terminal peptides. Both of these peptides could be used
for quantitation purposes. However, the C-terminal peptides for
both substrates were poorly retained on the chromatographic
column compared to the N-terminal peptides as well as the
substrates. Additionally, the C-terminal peptides produced ap-
proximately 10-fold lower signal intensities as compared to the
N-terminal peptides at equimolar concentrations. For these
reasons, the N-terminal peptides were chosen as the products to
quantify in the dialysate samples. Figure 2 shows the MS/MS
spectra obtained for the protonated ions for each of the different
analytes. The corresponding fragment ions are labeled noting that
in Figure 2A for the MMP-1 substrate m/z 755.3 and 670.2 and in
Figure 2B for the MMP-2/-9 substrate m/z 775.2 and 690.2 are
unassigned.
Within the calibration range of 5-50 µM the calibration curves
were linear for both the MMP-1 substrate and MMP-2/-9 substrate
(5-50 µM) based on the EIC of MS/MS of m/z 992 and MS/MS
of m/z 1012 versus concentration, respectively. The limit of
quantitation (LOQ) was used as the lowest concentration standard
for the calibration curve and was achieved at the concentration
that yields a signal-to-noise ratio (S/N) of 10:1; and the limit of
detection (LOD) was achieved at the concentration that yields a
S/N of 3:1. For both substrates, LOD was 1 µM and LOQ was 5
µM. For MMP-1 NTP and MMP-2/-9 NTP, linear calibration
curves (1-50 µM) were obtained based on the EIC of MS/MS of
m/z 466 and MS/MS of m/z 452 versus concentration, respec-
tively. For both peptide products, LOD was 0.2 µM and LOQ was
1 µM.
The intraday and interday relative standard deviations (RSD%)
were determined and were found to be 4.9% (intraday) and 10.8%
(interday) for GM 6001, 1.4% (intraday) and 9.5% (interday) for
the MMP-1 substrate, 3.1% (intraday) and 4.2% (interday) for the
MMP-2/-9 substrate, 4.7% (intraday) and 8.8% (interday) for the
MMP-1 NTP, and 4.1% (intraday) and 7.3% (interday) for the MMP-
2/-9 NTP.
In Vitro Microdialysis Sampling. The EE% values for the
different substrates were determined under quiescent as well as
well-stirred conditions at 37 °C. For the MMP-1 substrate the EE%
was 58.0 8.1 (quiescent) and 69.1 2.1 (well-stirred), n ) 4.
RESULTS AND DISCUSSION
Analytical Method Performance. At 4 °C, the MMP-1
substrate, MMP-2/-9 substrate, and their N-terminal products
(NTPs) at 50 µM concentrations were found to be stable for at
least 7 days with less than 10% variance in analyte concentration.
At room temperature, this set of analytes (50 µM initial concentra-
tions) was found to be stable for at least 24 h with less than 10%
RSD.
For the MMP-2/-9 substrate the EE% values were 64.5
7.6
(quiescent) and 73.5 1.6 (well-stirred), n ) 4. For GM 6001, its
(33) John, H.; Walden, M.; Scha¨fer, S.; Genz, S.; Forssmann, W. Anal. Bioanal.
Chem. 2004, 378, 883–897
(34) Prokai, L.; Zharikova, A. D.; Jana´ky, T.; Prokai-Tatrai, K. Rapid Commun.
Mass Spectrom. 2000, 14, 2412–2418
.
.
Analytical Chemistry, Vol. 81, No. 24, December 15, 2009 9965

Figure 2. Full scan product ion spectra for analyte characterization: (A) MS/MS of m/z 992 for MMP-1 substrate, (B) MS/MS of m/z 1012 for
MMP-2/-9 substrate, (C) MS/MS of m/z 466 for MMP-1 NTP, and (D) MS/MS of m/z 452 for MMP-2/-9 NTP; peaks are labeled with the
corresponding fragment ions.
9966 Analytical Chemistry, Vol. 81, No. 24, December 15, 2009

Figure 3. Time-dependent MMP-1 NTP and MMP-2/-9 NTP formation: (A) microdialysis with inhibitor included in the sample medium with
activated MMPs and (B) microdialysis with inhibitor included in the perfusate with substrates. Control denotes MMP-1 substrate (50 µM) and
MMP-2/-9 substrate (50 µM) being perfused to the sample medium containing activated MMP-1 and MMP-9, with no inhibitor present. IN10,
IN20, IN50 denote inhibitor concentrations of 10, 20, 50 µM present in the sample medium or perfusate. Data represent mean ( SD; n ) 4.
EE% was determined for different concentrations (10, 20, and 50
µM) and was approximately 70% with no variation among the
different concentrations that were perfused through the microdi-
alysis sampling probe. Additionally, there was no change in either
the substrate EE% values or the GM 6001 EE% values when
compounds were coperfused.
The time-dependent formation of MMP-1 NTP and MMP-2/-9
NTP during the localized infusion of the MMP substrates under
control and inhibited in vitro conditions is shown in Figure 3. The
formation of the N-terminal peptide products increased during the
entire collection period. To test the effectiveness of the broad-
spectrum MMP inhibitor, GM 6001, it was added at concentrations
of 10, 20, and 50 µM to the vial containing the enzyme solution.
In separate experiments, GM 6001 was included in the perfusion
fluid at concentrations of 10, 20, and 50 µM. For all concentrations
of GM 6001 and under both experimental conditions, i.e., delivered
through the dialysis probe or included in the external solution,
the formation of products was completely inhibited at all time
Table 2. Percentage Inhibition of MMP-1 and MMP-9 (in
Vitro) with Different Concentrations of Inhibitor in the
Systema
microdialysis with
inhibitor in the
sample medium
microdialysis with
inhibitor in the
perfusate
inhibitor
concentration
(µM)
MMP-1
MMP-2/-9
MMP-1
MMP-2/-9
10
20
50
96.1 1.0
96.7 0.5
96.7 0.7
99.1 0.3
99.0 0.2
99.3 0.2
95.2 1.8
96.7 0.9
97.1 0.9
97.9 0.7
98.9 0.3
99.6 0.1
a The percentage inhibition was calculated the difference of product
concentration in control and inhibition experiment divided by the
control. Data represent mean SD; n ) 4.
points as shown in Figure 3. Table 2 shows the calculated
inhibition at the 210 min collection point. Nearly 100% inhibition
was obtained for all the inhibitor concentrations. Additionally, the
Analytical Chemistry, Vol. 81, No. 24, December 15, 2009 9967

Figure 4. LC/MS/MS chromatograms of in vivo dialysates: (A) dialysate with substrate perfusion and (B) PBS blank sample. Peaks were
labeled with the corresponding analyte. Full scan product ion spectra of the [M + H]+ ions were monitored in different segments including
MS/MS of m/z 1012 for MMP-2/-9 substrate (10-13.8 min), MS/MS of m/z 452 for MMP-2/-9 NTP (13.8-16.3 min), MS/MS of m/z 466 for
MMP-1 NTP (16.3-20 min), and MS/MS of m/z 992 for MMP-1 substrate (20-25 min).
Table 3. Protease Activity Found in Vivo in Blank Dialysatesa
MMP-1
day 3
MMP-2/-9
day 3
day 0
day 7
day 0
day 7
substrate ratio
NTP ratio
103.8 2.6
0.7 0.2
97.7 2.1
2.8 1.6
46.2 0.4
15.4 5.9
106.1 2.7
0.7 0.2
100.0 6.4
2.7 1.0
40.6 6.5
27.0 3.4
a Substrate ratio denotes substrate concentration formed in blank dialysates divided by substrate concentration in a control solution. NTP ratio
denoted NTP concentration formed in blank dialysate divided by corresponding substrate concentration in a control solution. Data represent mean
SD; n ) 4.
EE% for the substrates was not altered when the inhibitor was
added to the perfusion fluid (data not shown).
vivo dialysates. Dialysates obtained during perfusion with the
MMP substrates as well as blanks (no substrates) were compared
(Figure 4). For the dialysate obtained when the MMP substrates
were perfused, the peaks corresponding to both substrates and
products are shown, indicating the presence of active MMPs
outside the probe, thus converting the substrates to products that
were recovered in the dialysates. For the blank dialysates (those
collected that had no substrates included), no peaks that coeluted
with either the MMP substrates or their products were obtained,
indicating that no peptide or other endogenous component with
the same retention time, structure, or fragmentation pattern similar
to the MMP substrates or their products was present in the
blanks.
Microdialysis sampling was performed to determine if MMP
enzymes in the sample medium would be excluded by the PAES
20 kDa MWCO membrane. The MMP substrate mixture was
spiked into the dialysate. The results indicated no significant
difference between the substrate concentration in the dialysate
sample and control substrate mixture sample (less than 5%
variance). In addition, no product peaks were obtained in the
dialysate sample. These results suggest that, under these experi-
mental conditions, the activated MMPs cannot pass through PAES
probes having a 20 kDa MWCO membrane.
LC/MS/MS Method Validation and Analyte Quantitation.
The method used for the analysis of the dialysates collected in
vitro was applied to analysis of in vivo dialysates with a few
changes. The eluate from the first 5 min of the chromatographic
run was directed to waste, and the entire run time was shortened
to 25 min. Figure 4 shows a typical LC chromatogram for the in
Blank dialysates were also tested for MMP activity to deter-
mine whether active MMPs or other proteolytic enzymes would
diffuse through the probe membrane. Spikes of the MMP
substrates into these blank dialysates obtained on the implantation
day resulted in substrate concentrations that were 103.8% 2.6%
9968 Analytical Chemistry, Vol. 81, No. 24, December 15, 2009

Figure 6. EE% of MMP-1 substrate and MMP-2/-9 substrate in the
control dialysate vs inhibition dialysate on day 0 of implantation. The
control dialysates were obtained by perfusing the MMPs substrate
mixture (MMP-1 substrate 50 µM and MMP-2/-9 substrate 50 µM in
PBS) through the implanted probes at 1 µL/min. The inhibition
dialysates were obtained by perfusing MMP inhibitor GM 6001 50
µM in the above substrates mixture through the implanted probes.
Data represents mean ( SD; n ) 4 rats.
significantly lower than those obtained in vitro (15.6 1.7 µM
MMP-1 NTP and 17.1 2.4 µM for MMP-2/-9 NTP, n ) 4) and
may be caused by lower activity of MMPs in vivo since the
concentration as well as the distribution of MMPs within the tissue
site surrounding the dialysis probe is not known. Additionally,
there are mass transport differences caused by the tissue space
as compared to the in vitro setting. Since the substrate EE% values
were between both the in vivo and in vitro experiments (nearly
70%), it is unlikely that differences in the product concentrations
obtained are due to small differences in EE% between the two
experiments.
Figure 5 shows the time-dependent MMP-1 NTP and MMP-
2/9 NTP formation in both the control and GM 6001-containning
dialysate on different collection days. Similar to the control
dialysates, MMP-1 NTP and MMP-2/9 NTP formation in the GM
6001-containing dialysate showed an increase in product formation
that then stabilized. Lower amounts of products were obtained in
the GM 6001-containing dialysate, indicating the MMP activities
outside the probe can be partially inhibited by the inhibitor
coperfusion with the substrates. The average concentration of the
individual products in control and inhibition experiments were
obtained from the dialysate data collected between 90 and 210
min, and the percentage inhibition can be calculated by the
difference between the concentrations obtained from the control
and GM 6001-containing dialysate divided by the control concen-
tration.
Figure 5. MMP-1 NTP and MMP-2/-9 NTP formation in the control
dialysate vs inhibition dialysate on day 0 of implantation. The control
dialysates (closed symbols) were obtained by perfusing the MMPs
substrate mixture (MMP-1 substrate (50 µM) and MMP-2/-9 substrate
(50 µM) in PBS) through the implanted probes at 1 µL/min. The
inhibition dialysates were obtained by perfusing MMP inhibitor GM
6001 (50 µM) in the above substrates mixture through the implanted
probes (open symbols). Data represents mean value; n ) 4 rats.
(n ) 4) in the blanks versus controls for the MMP-1 substrate
and 106.1%
2.7% (n ) 4) for the MMP-2/-9 substrate. This
indicates that the blank dialysates after implantation did not
contain additional proteases, which has been recently reported
as a potential problem with microdialysis of peptides.35 Similar
results were observed for dialysates collected on day 3 (Table 3).
However, on day 7, significant protease activity was observed in
the collected blanks. The source of these proteases is unknown
at this time and is being further investigated.
In Vivo Microdialysis Sampling through Long-Term Im-
planted Microdialysis Probes: Control. The time-dependent
formation of products for the control experiment is illustrated in
Figure 5. The concentrations of both the MMP-1 NTP and MMP-
2/-9 NTP increase and stabilize around 90 min. The maximum
concentration obtained for the MMP-1 NTP was 2.6 µM, and for
the MMP-2/-9 NTP it was 3.1 µM. These concentrations were
The broad-spectrum MMP inhibitor GM 6001 was added to
the microdialysis perfusion fluid to determine if MMP activity can
be effectively inhibited by this assay scheme. GM 6001 is a potent,
cell-permeable, broad-spectrum hydroxamic acid inhibitor of
MMPs (Ki ) 400 pM for MMP-1, Ki ) 500 pM for MMP-2, Ki
) 27 nM for MMP-3, Ki ) 100 pM for MMP-8, and Ki ) 200
pM for MMP-9) that can be used to indicate the presence of
(35) Li, Q.; Zubieta, J.-K.; Kennedy, R. T. Anal. Chem. 2009, 81, 2242–2250
.
Analytical Chemistry, Vol. 81, No. 24, December 15, 2009 9969

Table 4. MMP-1 NTP and MMP-2/-9 NTP Formation and Percentage Inhibition on Different Collection Daysa
MMP-1 NTP
MMP-2/-9 NTP
concn control
concn inhibition
percentage
inhibition (%)
concn control
concn inhibition
percentage inhibition(%)
(µM)
(µM)
(µM)
(µM)
day 0
day 3
day 7
2.6 0.5
1.7 0.2
1.5 0.3
1.8 0.4
1.2 0.1
1.0 0.2
28.5
31.0
31.7
3.1 0.8
2.1 0.9
1.8 0.5
2.4 0.6
1.5 0.2
1.4 0.3
22.3
27.5
24.2
a The control dialysates were obtained by perfusing the MMPs substrates mixture (MMP-1 substrate 50 µM and MMP-2/-9 substrate 50 µM
in PBS) through the implanted probes. The inhibition dialysates were obtained by perfusing MMP inhibitor GM 6001 50 µM in the above substrates
mixture through the implanted probes. Data represents mean SD; n ) 4 rats.
Figure 7. Zymogram of tissue around implanted microdialysis probes vs normal tissue: (A) 12% gelatin gel; (B) 10% casein gel. Lanes 1 and
2, normal tissue; lanes 3-6, tissue around the implanted microdialysis probe; lane 7, HT-15 medium containing MMP-2 and MMP-9.
,30,36
active MMPs.29
The use of the inhibitor is important as it
enough to reflect the inhibition of MMPs external to the probe.
This indicates that other removal processes in the tissue contribute
more to the EE% than enzymatic activity. However, decreased
product formation was observed with inhibited enzyme activities
outside the probe despite the small or no change in EE% of
substrate and inhibitor. Although at this point this methodology
is not able to give quantitative information regarding the enzymatic
activities surrounding the dialysis probes, the difference in the
amount of product formed under control and inhibited conditions
provides information regarding the presence of MMPs external
to the microdialysis sampling probes.
Table 4 shows the product concentrations obtained for the
infused substrates on days 3 and 7. As compared to the initial
implantation day, there is no significant difference among the
concentrations obtained and the percentage of inhibition with the
infusion of the GM 6001 MMP inhibitor. Again, the use of the
inhibitor partially blocked product formation but did not com-
pletely inhibit it. Additional experiments beyond the scope of this
initial work are necessary to determine the MMP activities as well
as their localization external to the dialysis probes.
allows us to rule out the activity of other proteases that might
cleave the peptide substrates.
The percentage inhibition for MMP-1 was approximately 29%,
and for MMP-2/-9 it was approximately 22%, which is much lower
compared to the complete inhibition observed with the in vitro
experiment. There are several possible explanations for this
apparent disparity. The first could be that not enough GM 6001
reached the tissue space to inhibit the enzymes. Another is that
GM 6001 was tested using human MMPs; rat MMPs may be
slightly different in terms of their Ki values. This is unlikely as
GM 6001 is used as an inhibitor for MMPs across different
species including rat..37 Additionally, endogenous inhibitors of
MMPs known as TIMPs from drosophila can inhibit mam-
malian MMPs suggesting conservation of activity across many
species.38 Finally, the most likely explanation is that other GM
6001-insensitive proteases are activated in vivo that cleave the
substrates. These different possibilities are being addressed
in our laboratory.
Figure 6 illustrates the EE% for substrates from the probes
implanted with and without the inclusion of the GM 6001 inhibitor.
There was no significant difference for the EE% of substrates
between the controls and the inhibited infusions. These results
indicated that the EE% of substrates and inhibitor are not sensitive
Confirmation of MMP Activity via Zymography. Figure 7
shows the zymograms of normal tissue versus that of encapsulated
tissue around the probes that have been implanted for a week. In
the gelatin gel, the pro-MMP-9 (92 kDa), pro-MMP-2 (72 kDa),
and active MMP-2 (62 kDa) were shown as white bands against
the blue background, indicating the presence of these enzymes
in the lysates obtained from the tissue that encapsulated the probe.
(36) Grobelny, D.; Poncz, L.; Galardy, R. E. Biochemistry 1992, 31, 7152–7154
(37) Amantea, D.; Russo, R.; Gliozzi, M.; Fratto, V.; Berliocchi, L.; Bagetta, G.;
Bernardi, G.; Corasaniti, M. T. Int. Rev. Neurobiol. 2007, 82, 149–169
.
.
(38) Wei, S.; Xie, Z.; Filenova, E.; Brew, K. Biochemistry 2003, 42, 12200–12207
.
9970 Analytical Chemistry, Vol. 81, No. 24, December 15, 2009

Another band at around 220 kDa was also present, and it may be
a complex of MMP-9.39 Additional bands can be seen which
correspond to active MMP-9 (86 kD) and active MMP-2 (67 kDa).
It should be noted that MMP-1, MMP-8, and MMP-13 can also
cleave the gelatin substrate, but typically these bands are weak
as gelatin is not their preferred substrate. For the normal tissue,
only very light bands corresponding to the pro-MMP-2 (72 kDa)
and active MMP-2 (62 kDa) were present, indicating a very low
concentration of MMP-2 in the normal tissue; no MMP-9 was
detected. In the 10% casein gel, a very light band corresponding
to active MMP-1 (45 kDa) was present. Bands corresponding to
pro-MMP-9 (92 kD) were also present as MMP-9 can also cleave
the casein substrate. All the bands are very light in comparison
to the gelatin gel even with double the protein amount loaded, as
the casein zymograms are much less sensitive than their gelatin
counterparts. For the normal tissue, no MMP bands were
detected. The above zymogram results clearly indicated that the
encapsulation tissue around the probe showed higher MMPs
activity in comparison to the normal tissue, suggesting induction
of MMP-1, MMP-2, and MMP-9 during the long-term microdialysis
probe implantation.
CONCLUSIONS
An in vitro and in vivo assay has been developed to simulta-
neously detect MMP-1 and MMP-9 activity using microdialysis
sampling and LC/MS/MS. The perfusion of appropriate MMP
substrates, combined with recovery of products, allows in situ
detection of MMPs. A broad-spectrum MMP inhibitor was also
included into the system either in the sample medium containing
activated enzyme or in the perfusate along with substrate coper-
fusion. Regardless of the method of delivery, complete inhibition
was shown for in vitro product formation, suggesting the product
formation can provide valuable information regarding changes in
the enzymatic reaction outside the probe. In addition, the assay
scheme for inclusion of inhibitor in the perfusate has the potential
to be applied in the further in vivo MMPs activities detection as
a validation for the presence of MMPs activities around the probe.
ACKNOWLEDGMENT
NIH EB 001441 supported this work. The Agilent MSD Trap
Instrument was purchased under Grant NSF CHE-0091892.
Received for review July 30, 2009. Accepted October 30,
2009.
(39) Pucci-Minafra, I.; Minafra, S.; La Roccaa, G.; Barrancaa, M.; Fontanaa, S.;
Alaimoa, G.; Okadac, Y. Matrix Biol. 2001, 20, 419–427
.
AC901703G
Analytical Chemistry, Vol. 81, No. 24, December 15, 2009 9971