In situ and Multi-Substrate Detection of Elastase Enzymatic Activity External to Microdialysis Sampling Probes using LC-ESI-MS

Abstract

Extracellular proteases play significant roles in mammalian development and disease. Enzymatic activity external to a microdialysis sampling probe can be determined by infusing judicious choices of substrates followed by collecting and measuring the products. Porcine pancreatic elastase was used as a model enzyme with two substrates possessing different cleavage sites, N-methoxysuccinyl-Ala-Ala-Pro-Val-7-amino-4-methylcoumarin (FL-substrate) and N-Succinyl-Ala-Ala-Ala-p-nitroanilide (UV-substrate). These substrates were infused through the microdialysis sampling probe to a solution containing elastase. The resulting four products and the remaining two substrates were collected into the dialysate and were subsequently analyzed offline using LC-MS with electrospray ionization. All analytes were identified using extracted ion chromatograms of m/z 628 (FL-substrate); m/z 452 (UV-substrate); m/z 471 (N-methoxysuccinyl-Ala-Ala-Pro-Val, FL-NTP); m/z 332 (N-Succinyl-Ala-Ala-Ala, UV-NTP); m/z 176 (7-amino-4-methylcoumarin, AMC) and m/z 139 (p-nitroaniline, pNA). FL-NTP and FL-substrate exhibited 10-fold higher ion production as compared to AMC with equimolar standards. Microdialysis sampling combined with LC-ESI-MS detection allowed for in situ determination of the enzymatic activity of a protease external to the microdialysis probe when using different peptide-based substrates.

Extracellular proteinases play essential roles in the degradation of extracellular proteins allowing for multicellular organs to develop and function normally. ¹ Matrix metalloproteinases (MMPs), a family of zinc-dependent proteinases, have been recognized for their ability to cleave extracellular matrix (ECM) structural proteins including collagen, laminin, and fibronectin.² MMPs regulate cell behavior via cleavage of cell surface molecules and other pericellular non-matrix proteins including proteinases, proteinase inhibitors, clotting factors, chemotactic molecules, latent growth factors, growth factor binding proteins, cell surface receptors, and cell-cell and cell-matrix adhesion molecules.³ MMPs have known involvement with numerous disease states including arthritis, cancer, and cardiovascular disease. Elucidating the roles that MMPs play in vivo via chemical analysis is challenging since their endogenous substrates are typically insoluble proteins. The multiplicity of MMP substrates makes it difficult to effectively measure the activity of a single enzyme.⁴ Additionally, once MMPs cleave their targeted substrate, the resulting product can be cleaved by additional MMPs or extracellular proteases. Many analytical methods have been described to assay the clinically relevant MMPs ex vivo from tissue biopsies.⁵ Immunochemical methods give quantitative information of total enzyme concentrations, but they cannot discriminate between zymogens and active enzymes.⁶ MMPs are synthesized as inactive zymogens (inactive proteins) and require enzymatic activation that is tightly regulated via tissue inhibitors of metalloproteinases (TIMPs). Zymography is a common method to separate and quantify the concentrations between the pro-enzyme and the active enzyme.⁷ Although zymography allows visualization of enzyme activities, it is a tedious method and is difficult to quantify.⁸ A variety of fluorescence and colorimetric assays have been applied to quantify the active enzyme based on changes in spectroscopic properties of the resulting products after substrate cleavage.^9–13

Few methods are available to determine in vivo MMP activity. Assessing MMP enzymatic activity is important for identification and localization of activated MMPs during various disease states. Additionally, such assays would be useful in pharmaceutical studies designed to inhibit targeted MMPs. Considerable efforts have been made in the areas of fluorescent-based imaging techniques for in vivo assessment of MMP activity.^14–16 However, the target specificity for these approaches is presently limited by the number of fluorophores available that would provide non-overlapping emission bands for the enzymatic products.

Microdialysis sampling is a well-established separation technique for continuous collection of analytes, such as neurotransmitters, drugs and their metabolites, both in vivo and in vitro.^17,18 It is accomplished by using a probe consisting of a semi-permeable hollow-fiber dialysis membrane affixed to inlet and outlet tubing. A perfusion solution is passed through the probe at μL/min flowrates and collected for analysis. Analytes that are smaller than the membrane pores can diffuse freely through the membrane and are carried to the outlet by the perfusion fluid. Larger analytes will be rejected by the membrane pores that their recovery is negligible. Microdialysis sampling can also be used to deliver certain substances with a molecular weight smaller than the membrane molecular weight cutoff (MWCO) to the fluid outside the probe thus allowing localized metabolism to be monitored,¹⁹ including MMP-2 and MMP-9 activity in breast cancer,²⁰ phenol metabolism from multiple sites in the liver,²¹ and substance P metabolism at the blood-brain barrier. ²² During microdialysis sampling, sampling effectiveness is determined via the bi-directional extraction efficiency (EE), which can be calculated as shown in eq 1,

$$\text{EE}(\%)=\frac{{\mathrm{C}}_{\mathrm{d}}-{\mathrm{C}}_{\mathrm{i}}}{{\mathrm{C}}_{\mathrm{e}}-{\mathrm{C}}_{\mathrm{i}}}\times 100$$ (1)

where C_d, C_e, C_i stand for the analyte concentration in dialysate, external sample medium and perfusion fluid, respectively.²³ This equation allows the calculation of extraction for a delivered substrate that is locally infused through the dialysis probe. Additionally, when the analyte concentration in the perfusion fluid is zero, the equation simplifies to the percentage of analyte concentration in the dialysate divided by the analyte concentration in the external sample medium, and is usually termed relative recovery (RR). Here, loss of substrate from the probe will be termed EE_loss and recovery will be termed EE_rec.

In this work, in vitro microdialysis sampling and LC-ESI-MS was applied to determine the activity of porcine elastase external to a microdialysis sampling probe. Elastase exhibits similar substrate specificity to neutrophil elastase (MMP-12) at a substantially reduced cost. Since many of the enzymatic substrates for different MMPs contain similar colorimetric or fluorescent products, we chose to focus on using the N-terminal peptide as an analytical target. Peptides are easily ionized using ESI conditions and the use of chromatographic separation prior to the detection allows quantitative measurements for each individual compound. Such an approach would ultimately allow multiplexed analysis of various MMP or protease activities at their site of action. An advantage for using LC-MS is that it allows simultaneous monitoring of multiple analytes in a single experiment as long as the substrate and product species exhibit mass to charge ratios (m/z) that can be spectroscopically resolved.^24,25 LC-MS analysis of peptide products for various in vitro studies of enzymatic activity and profiles have been reported.^26–29 Recently, an LC-MS approach has been described for peptide biomarker studies of MMP-13 activity in synovial fluid and urine.³⁰

To determine the elastase enzymatic activity external to the microdialysis probe, a fluorogenic elastase substrate and a colorimetric elastase substrate with different N-terminal peptides were co-perfused and delivered through the dialysis probe as shown in Figure 1. The organic and N-terminal peptide products were detected and quantified along with the substrates using LC-ESI-MS.

Figure 1.

Microdialysis sampling schematic. FL-substrate and UV-substrate diffuse to the surrounding medium react with elastase forming products that diffuse back into the probe.

EXPERIMENTAL SECTION

Chemicals

Fluorescent series chemicals: self-quenched fluorogenic elastase substrate V, N-methoxysuccinyl-Ala-Ala-Pro-Val-7-amino-4-methylcoumarin (MeOSuc-AAPV-AMC, FL-substrate) was obtained from Calbiochem (San Diego, CA); N-methoxysuccinyl-Ala-Ala-Pro-Val (MeOSuc-AAPV; Fluorescent substrate N-terminal peptide, FL-NTP) was purchased from Bachem (Bubendorf, Switzerland) and 7-amino-4-methylcoumarin (AMC) was purchased from Sigma (St. Louis, MO, USA).

Colorimetric series chemicals: N-Succinyl-Ala-Ala-Ala-p-nitroanilide (Suc-AAA-pNA, UV-substrate) and p-nitroaniline (pNA) were both purchased from Sigma (St. Louis, MO, USA).

Porcine pancreatic elastase (Type I, aqueous suspension) was purchased from Sigma (St. Louis, MO, USA). One unit elastase will hydrolyze 1.0 μmole of Suc-AAA-pNA per minute, pH 8.0 at 25 °C. All other chemicals were reagent grade or better. HPLC grade water was purchased from Fisher Scientific. Stock solutions (2 mM) of FL-substrate, FL-NTP, AMC and UV-substrate were prepared in DMSO and stored at 4 °C. All other solutions were prepared with HPLC grade water.

HPLC-mass spectrometry system (LC-MS)

For separation and detection, an Agilent 1100 series HPLC with the MSD 1100 Ion Trap mass analyzer (Agilent Technologies, CA, USA) operating in the electrospray ionization (ESI) mode was used. All samples were injected using an autosampler with an injection volume of 2 μL. An Alltima 5 micron C18 column (150×4.6 mm ID) with guard column was employed to separate the substrates and products. A binary pump was used to deliver the solvents at 0.5 mL/min flowrate. Solvent A contained 0.2% formic acid in water and solvent B contained 0.2% formic acid in acetonitrile. A linear gradient with solvent B changing from 25% (v:v) to 40% over a 20-min period was used to separate the analytes.

Mass spectrometric measurements were performed in the positive ion mode. Mass spectra were recorded over the range from m/z 100 to 700. The ESI source conditions were as follows: capillary voltage 4.5 kV, nebulizer gas (N₂) pressure 25.0 psi, dry gas (N₂) flow rate 10 L/min, dry temperature 350 °C.

Calibration curves and analytes quantitation

Standard solutions of FL-substrate (0.5 to 50 μM), FL-NTP (1 to 20 μM), AMC (2 to 20 μM) and UV-substrate (0.5 to 50 μM) were prepared by appropriately diluting the corresponding 2 mM stock solution with water. Standards (2 μL) were analyzed by the LC-ESI-MS system in triplicate. Peak areas of extracted ion chromatograms (EIC) of m/z 628 (FL-substrate), 471 (FL-NTP), 176 (AMC), 452 (UV-substrate) vs. concentration were used to create calibration curves.

Samples (2 μL) were analyzed in duplicate by the LC-ESI-MS system. Base peak chromatograms (BPC) of m/z 100 to 700 and EIC m/z 628 (FL-substrate), 471 (FL-NTP), 452 (UV-substrate), 332 (UV substrate N-terminal peptide, UV-NTP), and 139 (pNA) were monitored. Each peak was characterized by their specific mass spectrum. Peak areas from the above extracted ion chromatogram were used for quantitation.

Analyte stability

The stability of FL-substrate, FL-NTP and AMC were tested by storing FL-substrate 50 μM, FL-NTP 20 μM and AMC 20 μM (diluted from 2 mM stock solution by water, respectively) in the dark at room temperature for various time periods. Samples were analyzed by LC-ESI-MS using the above conditions and quantified by using the calibration curves.

HPLC-fluorescence system (LC-FL)

In order to compare with the LC-MS detection, separation and quantitation were also performed on a LC-FL system, which includes a Shimadzu SIL-10ADvp autoinjector, LC-10ADvp pumps, SCL-10vp system controller and RF-551 fluorescence HPLC detector. The same column, flowrate, injection volume, and gradient were used as in the LC-MS system. Fluorescence detection at an excitation (Ex) wavelength of 370 nm and emission (Em) wavelength of 460 nm was chosen for AMC detection.

Microdialysis sampling

Microdialysis sampling was performed with a 1 mL Hamilton syringe (Hamilton Company, Reno, NV, USA) with plastic tip and a CMA-102 syringe pump (CMA/Microdialysis, North Chelmsford, MA, USA). CMA 20/04 microdialysis probes with a 10-mm 20-kDa molecular weight cutoff (MWCO) polycarbonate/polyether (PC) membrane (CMA/Microdialysis, North Chelmsford, MA, USA) were used in all experiments. All experiments were performed in quiescent solutions at 37 °C maintained by a sand bath (Fisher Scientific, USA).

The extraction efficiency for the FL-series substrate and products in the absence of enzyme was obtained at different flow rates. For AMC and FL-NTP, microdialysis was performed in recovery mode with water perfused through a probe to a solution containing 15 μM AMC or 15 μM FL-NTP, respectively. For the FL-substrate, microdialysis was performed in delivery mode with 15 μM FL-substrate perfused through a probe immersed into water. Dialysates (60 μL) were collected in triplicate at different flowrates (0.5, 1.0, 2.0, 3.0 μL/min) and analyzed by the LC-ESI-MS.

Next, enzyme was included in the microdialysis system. The solution external to the microdialysis probe contained 0.5 units/mL elastase in water. In the single-substrate microdialysis assay, the perfusion fluid passing through the dialysis probe contained 50 μM FL-substrate in HPLC-grade water. For the multi-substrate microdialysis assay, the perfusion fluid contained 50 μM FL-substrate and 50 μM UV-substrate in HPLC-grade water. The microdialysis sampling perfusion fluid flowrate was 1 μL/min. Dialysate collection was initiated 25 minutes post probe immersion into the enzyme solution to allow for product concentrations to reach detectable levels. Samples were collected for 60 minutes. Three replicate samples were collected during single substrate experiments. Four replicate samples were collected during multi-substrate experiments. Statistical analysis required pooling the data due to differences in replicate sample numbers.

Due to the presence of enzyme outside the probe, the extraction efficiency of products may differ from the values obtained when there is no enzyme present. Standard addition experiments were performed to test the EE_rec of FL-NTP and AMC in the presence of enzyme. In this experiment, water was perfused to microdialysis probes at 1 μL/min flowrate. Elastase was added to the FL-substrate solution to achieve a concentration of 0.5 units/mL elastase in 25 μM FL-substrate, and the enzymatic reaction was initiated. This reaction mixture was vortexed and incubated at 37°C for 15 min. Fluorescence at Ex 360 nm, Em 465 nm was monitored to ensure reaction completion. Then microdialysis probes were immersed in this reaction mixture, and 10 μL dialysate was collected. Next, FL-NTP solution was added to the reaction mixture to achieve an increase of FL-NTP concentration of 20 μM. The reaction mixture was well mixed and 10 μL dialysate was collected. Then AMC solution was added to the reaction mixture to achieve an increase of AMC concentration of 20 μM. The reaction mixture was well mixed and 10 μL dialysate was collected. All dialysates were subsequently analyzed by LC-ESI-MS.

Kinetics

A Tecan SPECTRAFluor plate reader (Tecan Group Ltd., Männedorf, Switzerland) was used for all the kinetics measurements. The K_M, k_cat and k_cat/K_M values for both substrates were determined by plotting 1/v vs. 1/[S] in a Lineweaver-Burk Plot.

Elastase kinetics for the UV-substrate was determined by adding 50, 63, 83, 100, 125, 167, 250, and 333 μM UV-substrate to 0.01 units/mL elastate. The reaction progress was monitored at 405 nm every 30 seconds for 8 min using a p-NA calibration curve between 0 and 400 μM. At 405 nm, the product of the reaction, pNA, has significant molar absorbtivity and the UV-substrate exhibits no absorbance.

Kinetics of the FL-substrate in the presence of elastase were determined by adding 67, 75, 86, 100, 120, 150, and 200 μM FL-substrate to an enzyme concentration of 0.2 units/mL. The production of AMC from the cleavage reaction was measured using a 360 nm for excitation and 465 nm for emission every 30 second for 8 minutes. The calibration curve of AMC concentration ranging from 0 to 250 μM was obtained using fluorescence Ex 360 nm, Em 465 nm.

RESULTS AND DISCUSSION

Analyte stability and method optimization

At room temperature, the FL-substrate was found to be stable for 24 hrs; the FL-NTP was stable for greater than 12 hrs and was reduced to 96% of its initial concentration after 24 hrs. AMC was found to be the least stable. It was stable for at least 10 hrs and was reduced to 90% of its initial concentration after 24 hrs at room temperature. Collection followed by detection of dialysates was often less than 24 hrs suggesting that no more than 10% variation in sample concentration would be expected under these conditions.

Figure 2 shows a typical LC chromatogram and mass spectra for the single-substrate dialysate analyzed by LC-ESI-MS. Separation was achieved on a C18 column with FL-NTP, AMC and FL-substrate eluting at 5.4, 14, and 19 min, respectively. The retention behavior of the FL-substrate and products was consistent with a reversed-phase separation mechanism as the N-terminal peptide is more hydrophilic than AMC. ESI-MS spectra showed peaks of [M+H]⁺ ions of FL-NTP (m/z 471), AMC (m/z 176) and FL-substrate (m/z 628). Sodium ion adducts, [M+Na]⁺ were observed for FL-NTP (m/z 493) and FL-substrate (m/z 650) despite the addition of formic acid to suppress sodium-adduct formation; however, their intensities are much lower compared to the ions of the protonated molecules. Only the EIC peak area of [M+H]⁺ ions was used for quantitation.

Figure 2. LC chromatograms and mass spectra for the FL-substrate and its products, FL-NTP and AMC.

(A) Extracted ion chromatogram (EIC) of FL-NTP (m/z 471), AMC (m/z 176) and FL-substrate (m/z 628).

(B) Mass spectra of FL-NTP, AMC and FL-substrate in full scan mode from m/z 100–700. Peaks at m/z 493 and 650 are Na⁺ adducts and were not quantified.

Linear calibration curves were achieved for FL-substrate (0.5 to 50 μM), FL-NTP (1 to 20 μM) and AMC (2 to 20 μM) (figures not shown), based on the peak area from EIC of [M+H]⁺ ions vs. concentration. 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.

For equimolar concentrations of all three analytes, the signal of AMC was about 10 times lower and it exhibited poor S/N due to the low ionization efficiency compared to the peptides.^31,32 Several methods were applied to optimize AMC signal, including tuning the capillary voltage to a relatively high value of 4.5 kV;^31,32 using a narrow scan range (m/z 170~190), or using selected-reaction monitoring (SRM) for m/z 176. A narrow scan range can relatively increase the sensitivity but the S/N is poor; SRM can significantly increase S/N though it did not cause an obvious signal increase. Additionally, MS/MS detection (FL-NTP m/z 471→ m/z 215, AMC m/z 176→ m/z 117, FL-substrate m/z 628→ m/z 443) can reach significantly lower LOQ for all analytes: 100 nM for FL-NTP, 500 nM for AMC and 50 nM for FL-substrate. Yet, the calibration curve for AMC using MS/MS did not exhibit a linear concentration relationship above a concentration of 5 μM, but was linear for the FL-substrate and FL-NTP up to 20 μM. Extracted ion chromatograms obtained from the mass spectra recorded over the range from m/z 100 to 700 were used for quantitation of dialysates.

The difference in ionization efficiency between the peptide product and AMC also suggests the advantage for using peptide substrates for the protease analysis due to the high ionization efficiency typically obtained for peptides. While two different peptide products may exhibit differences in their ionization efficiency because of their different amino acid sequences; generally their ESI signal will be much higher than typically observed for organic products such as AMC that are used in conventional protease assays.^31,32

Microdialysis sampling extraction efficiency without Elastase

In vitro probe calibration for all three analytes without elastase was performed and the determined EE values are shown in Table 2. At 1 μL/min, the EE_loss of FL-substrate in water was 48.5 ± 2.6% (n=3) and the UV-substrate was 42.8±2.1 (n=3). The in vitro EE_rec of FL-NTP at 1 μL/min flowrate was 11.5±0.2 (n=3), which is much lower than the EE_loss for the FL-substrate. FL-NTP standard prepared in water instead of from the 2 mM DMSO standard and diluted to an external concentration of 15 μM gave an EE_rec of 17.5±0.4 (n=3), indicating that use of the DMSO solvent did not cause the significantly lower EE_rec for the FL-NTP compared to the FL-substrate. Microdialysis sampling was also performed in delivery mode where 15 μM FL-NTP was included in the perfusion fluid and perfused to water at 1 μL/min flowrate. In the delivery mode, the EE_loss of FL-NTP was 14.7±3.6 (n=3) indicating that FL-NTP mass transport characteristics to and from the microdialysis probe are similar as would be expected.³³

Table 2.

FL-substrate, FL-NTP and AMC Extraction Efficiencies

Flowrate (μL/min)	AMC EErec%	FL-NTP EErec%	FL-substrate EEloss%
0.5	83.7±1.3	18.3±0.3	63.6±5.5
1.0	76.3±1.7	11.5±0.2	48.5±2.6
2.0	68.2±8.3	8.7±1.8	32.8±3.9
3.0	56.0±1.8	7.6±0.2	23.0±0.2

EE_rec% denotes extraction efficiency in recovery mode. EE_loss% denotes extraction efficiency in delivery mode. All experiments were performed in quiescent solutions at 37°C. Data represent mean ± S.D., n=3.

Single-substrate microdialysate analysis in presence of enzyme

The flowrate-dependent FL-substrate EE_loss to a solution containing elastase and the collected AMC concentrations are determined by LC-FL and shown in Figure 3. As the perfusion flowrate increased, both the concentration of AMC collected into the probe and the FL-substrate EE_loss decreased. The time-dependent delivery of FL-substrate and collected FL-NTP concentrations are determined by LC-MS and shown in Figure 4. Through the duration of the collection time, the FL-substrate EE_loss remained constant and the recovered concentration of FL-NTP increased. These results are consistent with microdialysis sampling mass transport principles. It is well established that mass transport rates in the dialysate are affected by the analyte aqueous solution diffusion coefficient and the volumetric flowrate (Q_d) of the microdialysis perfusion fluid, an increase in Q_d leads to a decrease in the microdialysis EE.³⁴ With a localized infusion combined with product collection, the concentration of products collected would be expected to increase with an increasing infusion into a quiescent system. Additionally, the enzymatic reaction external to the probe causes the FL-substrate to reach an EE_loss steady state.

Figure 3.

FL-substrate delivery and collected AMC concentrations as a function of perfusion fluid flow rate. The probes were perfused with 50 μM FL-substrate to a quiescent 0.5 units/mL elastase solution at 37°C. Symbols and error bars denote mean ± S.D., n=3.

Figure 4.

Time-dependent delivery of FL-substrate and FL-NTP recovery during single-substrate dialysis. FL-substrate (50 μM) was perfused at 1 μL/min to a quiescent solution containing 0.5 units/mL elastase at 37°C. FL-NTP concentrations in the 10 and 20 min dialysate samples were below the LOQ. Symbols and error bars denote mean± S.D., n=3.

To determine approximate EE_rec values of the products in the presence of elastase (EE_enzyme) values were determined using a standard addition experiment. Table 3 shows that while AMC EE_rec values decreased under these experimental conditions from 76.3% (water) to 49.5% (enzyme solution), the FL-NTP EE_rec slightly increased in the presence of the enzyme to 22.3%. While it is possible that AMC binds nonspecifically to the elastase enzyme causing a reduction in its EE_rec, it is not clear why the FL-NTP has an increased EE_rec value under these conditions.

Table 3.

Estimated Extraction Efficiencies in Elastase.

	Dialysate Product Concentration (μM)	Product Concentration (μM) after Spike to External Solution	EEenzyme (%)
FLNTP	4.8±0.7	9.3±1.8	22.3
AMC	12.4±2.4	22.3±2.9	49.5

Microdialysis samples (1 μL/min) were collected after 25 μM FL-substrate was incubated with 0.5 units/mL at 37°C for 15 min. Then FL-NTP and AMC were spiked such that the external concentration was increased by 20 μM. EE_enzyme was calculated by dividing the difference of recovered product concentration by 20 μM. Data represent mean ± S.D., n=3 for 10 μL dialysates.

In the single-substrate microdialysis experiment, FL-substrate (50 μM or 100 μM) was perfused through the probe immersed in a medium containing 0.5 units/mL elastase. The EE data for the FL-substrate and collected product concentrations are listed in Table 4. With a 50 μM FL-substrate infusion, the EE_loss was 66.6±4.6% (n=4) using the LC-MS detection method, which is comparable to 69.4±2.2% (n=3) using LC-fluorescence detection method (p < 0.05 level). The FL-substrate EE_loss is greater for probes immersed in an elastase solution as compared to solutions with no enzyme (48.5%) as would be expected since the enzyme removes the substrate as it diffuses from the probe. When either 50 or 100 μM FL-substrate was perfused, the FL-substrate delivered outside the probe was 33.3±2.3 pmole/min and 62.1±0.9 pmole/min. The FL-NTP collected in the dialysate was 4.3±0.7 pmole/min and 11.0±0.5 pmole/min. Combining this information, it shows a higher percentage conversion of substrate to product outside the probe when 100 μM FL-substrate was perfused as compared to 50 μM FL-substrate was perfused. This increase is expected considering the enzyme kinetics, that when more substrate is present, the reaction will proceed at a faster rate and thus within a certain collection time more substrate is converted to products and the conversion rate is higher.

Table 4.

Single-substrate vs. multi-substrate comparison.

Perfusate	EEenzyme of FL-substrate (%)	Delivered FL-substrate (pmoles/min) outside probe	Recovered FL-NTP conc. (μM) in the dialysate	Recovered FL-NTP (pmoles/min) in the dialysate
FL-substrate 50 μM	66.5±4.6*	33.3±2.3*	4.3±0.7*	4.3±0.7*
FL-substrate 100 μM	62.1±0.9*	62.1±0.9	11.0±0.5	11.0±0.5
FL-substrate 50 μM & UV-substrate 50 μM	67.3±3.6*	33.7±1.8*	5.6±0.1*	5.6±0.1*

^*Denotes no statistical significance difference at the 95% confidence level between each designated value and the FL-substrate 50 μM value.

Microdialysis was performed at 1 μL/min with quiescent conditions at 37 °C. Sample collection (60 μL) was initiated 25 minutes post probe insertion. Data represent mean ± S.D., n=3 for single-substrate experiments and n=4 for multi-substrate experiments.

Multi-substrate Microdialysate Analysis in the Presence of Elastase

Based on all the previous MS setup, a multi-substrate microdialysis assay scheme was developed to determine the potential for multiple substrate infusion followed by product detection. FL-substrate (50 μM) and UV-substrate (50 μM) were co-perfused through the dialysis probe to a surrounding solution containing 0.5 units/mL elastase. Four different products, FL-NTP, AMC, UV-NTP and pNA diffused back to the probe combined with the two substrates remaining in the dialysate. Using the same HPLC gradient, all six analytes can be resolved and quantified based on the EIC of their [M+H]⁺ ions. Figure 5 shows the LC-ESI-MS chromatograms and mass spectra obtained during the multi-substrate infusion to elastase.

Figure 5. LC chromatograms and mass spectra of multi-substrate dialysate experiments. FL-substrate (50 μM) and UV-substrate (50 μM) were co-perfused at 1.0 μL/min to a quiescent medium containing 0.5 units/mL elastase at 37 °C.

(A) EIC of FL-NTP (m/z 471), AMC (m/z 139), FL-substrate (m/z 628) and UV-NTP (m/z 332), pNA (m/z 139), UV-substrate (m/z 452).

(B) Mass spectra of FL-NTP, AMC, FL-substrate and UV-NTP, pNA, UV-substrate in full scan mode from m/z 100–700. Peaks at m/z 354, 474, 493 and 650 are Na⁺ adducts and were not quantified.

When a single enzyme acts on two different substrates, each substrate will work as a competitive inhibitor to the other.³⁵ The two substrates will compete for the binding site and the enzyme exhibits kinetics that make it appear as if the K_M increases for each substrate. Comparing the kinetics data shown in Table 1, elastase has a weaker affinity to the FL-substrate (K_M 79.62 μM) than to the UV-substrate (K_M 65.78 μM), and the k_cat is slower for the FL-substrate. Considering the k_cat/K_M value, which allows the direct comparison of the effectiveness of the enzymatic conversion toward different substrates, it is much more effective for elastase to cleave the UV-substrate than to cleave the FL-substrate.

Table 1.

FL-substrate and UV-substrate Kinetic Parameters

	FL-substrate†	UV-substrate††
KM (μM)	79.62	65.78
kcat (min−1)	1.59	22.74
kcat/KM (min−1μM−1)	0.02	0.35

^†FL-substrate kinetics were measured using 0.2 units/mL elastase.

^††UV-substrate kinetics were measured using 0.01 units/mL elastase.

The FL-substrate and FL-NTP were quantified by comparing with the calibration curves. The EE_loss and product amounts recovered between the multi-substrate vs. the single-substrate dialysate are shown in Table 4. There is no significant difference (p<0.05 level) between the EE_loss value of FL-substrate in the multi-substrate dialysate and in the single-substrate dialysate, when either 50 μM or 100 μM FL-substrate was perfused. This indicates that the EE value is not as sensitive to minor changes in kinetics external to the probe. The recovered concentrations of FL-NTP during the multi-substrate vs. the single-substrate infusion exhibited no significant differences (p<0.05 level). Despite the higher k_cat/K_M value of UV-substrate as compared to the FL-substrate, there appeared to be no potential inhibition of the elastase catalytic activity towards the FL-substrate. This is most likely due to the higher enzyme concentrations used in these studies. Finally, the collected recovered concentrations of FL-NTP were doubled when 100 μM FL-substrate was perfused.

CONCLUSIONS

Enzymatic activity external to a microdialysis sampling probe can be detected by infusing proper enzymatic substrates through the dialysis probe followed by analysis of the products in the dialysate. Compared to LC-FL detection, the use of LC-ESI-MS in enzymatic bioassay offers many distinct advantages. The main advantage with the LC-MS approach is that selected peptide substrates can be used allowing for a multiplexed approach towards determining enzymatic activity.