Monitoring of Protease Catalyzed Reactions by Quantitative MALDI

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Monitoring of Protease Catalyzed Reactions by Quantitative MALDI MS Using Metal Labeling Barbara Gregorius,† Thomas Jakoby,† Dirk Schaumlöffel,‡ and Andreas Tholey*,† †

Institute for Experimental Medicine − Div. Systematic Proteome Research, Christian-Albrechts-Universität, 24105 Kiel, Germany Laboratoire de Chimie Analytique Bio-Inorganique et Environnement/IPREM, Université de Pau et des Pays de l’Adour/CNRS UMR 5254, Helioparc, 2, av. Pr. Angot, 64053 Pau, France

‡

S Supporting Information *

ABSTRACT: Quantitative mass spectrometry is a powerful tool for the determination of enzyme activities as it does not require labeled substrates and simultaneously allows for the identification of reaction products. However, major restrictions are the limited number of samples which can be measured in parallel due to the need for isotope labeled internal standards. Here we describe the use of metal labeling of peptides for the setup of multiplexed enzyme activity assays. After proteolytic reaction, using the protease trypsin, remaining substrates and peptide products formed in the reaction were labeled with metal chelators complexing rare earth metal ions. Labeled peptides were quantified with high accuracy and over a wide dynamic range (at least 2 orders of magnitude) using MALDI MS in case of simple peptide mixtures or by LC-MALDI MS for complex substrate mixtures and used for the monitoring of time-dependent product formation and substrate consumption. Due to multiplexing capabilities and accuracy, the presented approach will be useful for the determination of enzyme activities with a wide range of biochemical and biotechnological applications.

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the reaction mixture. Further, multiplexing is challenging due to overlapping absorption or fluorescence of the labels.3 Due to these restrictions in recent years mass spectrometry (MS) based approaches for the determination of protease substrate and product concentrations gained increasing interest. Together with electrospray ionization (ESI) MS-based approaches,4−7 matrix assisted laser desorption/ionization (MALDI) MS was successfully applied to study the kinetics of proteolytic reactions and for screening purposes.8−11 Since MALDI MS in many cases suffers from poor correlations between peptide amount and their signal intensities, the use of internal standards (IS)12,13 is recommended for peptide quantification. Sample preparation methods improving sample homogeneities, e.g. the use of ionic liquid matrices, also enabled direct readout of quantitative information.11 Internal standards and tags for the relative quantification of different samples must fulfill several requirements. First, they should have a high molecular similarity with the analyte, thus

he cleavage of peptide bonds by hydrolysis, catalyzed by proteases, is an important post-translational protein modification, which is responsible for cellular protein turnover and protein quality control and thus apparently involved in numerous pathological processes. In addition proteases are a useful tool for analytical chemistry, in particular in proteome analysis, and as biocatalysts in organic chemistry and biotechnology. For a deeper understanding of proteolytic reactions, the profiling and screening for novel proteases and substrates, the development of inhibitors and optimization of reaction conditions, the knowledge of protease activities is an indispensable prerequisite. Most commonly, proteolytic activities are determined by spectrophotometric protease assays using chromophoric or fluorophoric substrates. A major drawback of these methods is that they are usually restricted on a single substrate. In order to introduce suitable chromophoric or fluorophoric groups into the peptides, in many cases a derivatization of the substrate prior to enzymatic reaction is necessary, which can tremendously impact the properties of the substrate.1,2 Misleading results can also derive from disturbing buffers or cofactors in © 2013 American Chemical Society

Received: February 20, 2013 Accepted: April 15, 2013 Published: April 15, 2013 5184

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Analytical Chemistry

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(30 s, 60 s..., 60 min) 20 μL aliquots of digests were removed and subsequently quenched in 40 μL of stop-solution (250 μM PMSF (phenylmethane-sulfonylfluorid), 17 μM AEBSF (4-(2Aminoethyl)-benzensulfonylfluorid)). For derivatization 40 μL of NHS-DOTA solution (12.5 mM NHS-DOTA in 75% ACN + 25% 200 mM HEPES (pH > 7.5)) was added followed by incubation for 2 h at room temperature. 50 μL of lanthanide (Ln) salt solution (Tm3+, Ho3+, Dy3+, Tb3+, Gd3+, Sm3+, Pr3+, or La3+; 5 mM in 100 mM triethylammonium acetate, pH 5.0) was added to 20 μL of derivatized digests for complexation with 2.5 molar excess of lanthanide to derivatization reagent. For the generation of internal product standard (ISP), Neurotensin (15 μM) was digested with 12 ng of trypsin for 16 h. For the generation of internal substrate standard (ISS), Neurotensin (15 μM) was incubated with 25 mM TEAB instead of enzyme. The subsequent quenching, derivatization, and complexation was done as described above. Sample Preparation for Multisubstrate Protease Assay. In 2 mg of a mixture of 20 proteins, cysteines were reduced and alkylated as described previously.19,24 To generate shorter peptide fragments as substrates for the later trypsin multisubstrate assay, proteins were first digested with endoprotease Glu-C in 100 mM HEPES pH 7.5 for 16 h at 37 °C. Resulting peptides were separated from undigested proteins and the protease via 10 kDa cut off filters (Amicon Ultra-0.5 mL centrifugal filters), and the peptide containing flow-through was diluted with 25 mM TEAB to an estimated total protein concentration of ß1 = 1.9 μg/μL or ß2 = 0.85 μg/ μL, assuming a 100% recovery of the applied starting material. Multisubstrate Protease Assay. For the time-course analysis 0.6 μg of trypsin was added to 180 μL aliquots of each substrate concentration (ß1, ß2). Quenching, derivatization, and complexation was performed as described for singlesubstrate assay. Two enzyme catalyzed reactions were performed for each concentration, representing the biological replicate. All samples for the time-course experiments of tryptic activity were incubated with terbium (ß1-A), dysprosium (ß1-B, biological replicates), holmium (ß2-A), or thulium (ß2-B, biological replicates), respectively. For reverse labeling ß1-A was incubated with thulium, ß1-B with holmium, ß2-A with terbium, and ß2-B with dysprosium. Internal standards were also generated analogical to the single substrate procedure using the higher concentration (ß1). As control, aliquots of each substrate concentrations were incubated with buffer instead of enzyme solution, representing the 0 min time point. Sample Preparation for MALDI MS Analysis. All samples, generated by pooling of each 20 μL of each channel, were subsequently acidified with 1 μL of 10% TFA. Samples from single-substrate assay were 5-fold diluted with 5 mg/mL of CHCA and spotted in 1 μL aliquots -8 replicates per sample - on a 384well Opti-TOF MALDI Insert (AB SCIEX, Darmstadt, Germany). Nano Ion-Pair Reversed-Phase High-Performance Liquid Chromatography- (Nano IP RP HPLC)-Separation. All samples were separated via nano IP RP HPLC on a U3000 nano-HPLC system (Dionex, Idstein Germany) combined with a Probot microfraction collector (LC Packings, Amsterdam, The Netherlands). For peptide purification and concentration 20 μL of each sample was loaded over a 50 μL loop on an Acclaim PepMap100 C18 trap column (5 μm, 0.3 × 10 mm; Dionex, Idstein, Germany) with a flow rate of 30 μL/min 0.1% aqueous TFA, 3% ACN. Peptides were then flushed into an Acclaim PepMap100 C18 separation column (3 μm, 75 μm ×

comparable mass spectrometric properties; isotope-labeled analogues are best suited for this purpose. Second, in order to perform multiplexed analysis, e.g. time-course analyses or the parallel investigation of different reaction conditions, a suitable number of different labels must be available. For absolute or relative quantification in MS/MS-mode, isobaric tags like iTRAQ (isobaric tag for relative and absolute quantitation)14 and TMT (tandem mass tags)15 have been developed, offering 4 and 8 channels (iTRAQ), respectively, or 6 channels (TMT) to be measured in parallel. Recently a novel strategy encompassing labeling of peptides by lanthanide (Ln) metals using suitable chelators was introduced.16−19 The labeled peptides can be quantitatively determined via their metal content by inductively coupled plasma-mass spectrometry (ICP MS) with high accuracy, sensitivity and matrix robustness.20 Based on this approach a protease assay using element-tagged substrates was established combining high sensitivity and applicability as quadruplex assay, which allows the simultaneous quantification of four differentially tagged substrates;21,22 but as ICP MS provides no molecular information about the analytes, this implies, in the case of protease assays, that the cleavage site specificity or possible byproducts have to be already characterized or remain unknown during analysis. Therefore, a combination of ICP MS for relative or absolute quantification of peptides and proteins and their identification with molecular mass spectrometry, in particular ESI or MALDI MS, was suggested.17,18,20,23 Recently, we showed that quantitative MALDI MS encompassing DOTA derivatized peptides chelating lanthanide ions can be performed with high accuracy over a dynamic range over more than 2 orders of magnitude.24 The goal of the present study was to investigate the suitability of this metal labeling strategy to quantify proteolytic conversions directly via relative peptide quantification by MALDI MS and LC-MALDI MS. In a first experiment, a multiplexed time-course analysis of tryptic activity using Neurotensin as substrate was performed demonstrating the evolution of products and simultaneously the degradation of substrates. In a second experiment, the method was applied to a digest of a mixture of 20 proteins representing a multisubstrate mixture. The aim was to illustrate the ability of the method to quantify proteolytic cleavage in a time-dependent manner in complex mixtures.

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EXPERIMENTAL SECTION Materials. Trypsin was purchased from Promega (Madison, MI, USA), complete, mini, EDTA-free protease inhibitor cocktail from Roche Diagnostics (Basel, Switzerland), DOTANHS-ester from CheMatech (Dijon, France), isotope enriched samarium- ( 152Sm 96.5%), dysprosium- ( 162Dy 94.4%), gadolinium (156Gd 95.5%), and ytterbium-oxide (172Sm 95%) from Eurisotop (Saint-Aubin Cedex, France). Other lanthanides were obtained from Sigma-Aldrich (Taufkirchen, Germany) as lanthanide-(III)-chloride hexahydrate salts as well as endoprotease GluC, Neurotensin, all standard proteins, and other chemicals. Deionized water (18.2 MΩ*cm) was generated with an arium611 VF system (Sartorius, Göttingen, Germany). Single-Substrate Protease Assay. For time-course analysis the peptide Neurotensin was diluted with 25 mM TEAB (triethylammonium bicarbonate) buffer to eight different substrate concentrations from 15 μM to 9.75 μM. To 180 μL aliquots of each substrate concentration 12 ng of trypsin in 20 μL of 25 mM TEAB was added. After defined time intervals 5185

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Figure 1. Workflow of single-substrate protease assay. (A) For time-course analysis different concentrations of neurotensin were incubated with equal amounts of trypsin. After certain time points, small amounts of reaction mixture were inhibited using protease inhibitors. After NHS-DOTA derivatization and lanthanide complexation, each aliquot, representing a discrete time point and/or a certain substrate concentration, was mixed with equal amounts of internal standards for products (ISP) and substrates (ISS). (B) For the generation of ISP, Neurotensin (highest concentration) was incubated with trypsin derivatized and loaded with ytterbium. For the generation of ISS, Neurotensin was incubated with reaction buffer, followed by derivatization and lutetium complexation. All samples were finally analyzed by MALDI-TOF-MS. Asterisk (*) denotes labeling sites; [Sx]: substrate concentration at time x, [P] product concentration.

150 mm; Dionex, Idstein, Germany) and separated with a flow rate of 0.3 μL/min. Samples derived from multisubstrate analysis (100 ng each) were separated using a gradient of 5− 50% B (80% ACN, 0.04% TFA (serving as ion-pair additive), 20% deionized water (v/v/v)) over 96 and 5 min 50−95% B to elute peptides to the microfraction collector. From minute 20− 101 eluting peptides were directly mixed with matrix (3 mg/mL CHCA in 70% ACN, 0.1% TFA, 5 nM Glu1-fibrinopeptide B) in a ratio of 1:3 (v/v) and collected in 20 s intervals on stainless steel MALDI plates (OptiTof). MALDI MS Analysis. MALDI MS was performed on an AB SCIEX TOF/TOF 5800 mass spectrometer (AB SCIEX, Darmstadt, Germany) accumulating 2000 shots over an m/zrange from 800 to 4000. MS-calibration was done as default calibration for single peptide measurements and for LC MS data additional internal calibration on the signals of Glu1fibrinopeptide B and a matrix cluster (877.034 m/z). Fragment spectra were acquired with a medium CID pressure of 2 × 10−6 Torr. Up to 40 precursors per fraction were fragmented in the multisubstrate assay using dynamic exit algorithm starting from the most to the less intense precursor. Minimum signal-to-noise filter of 10, precursor mass tolerance between spots of ±200 ppm, with a minimum chromatogram peak width of 1 were used as parameters. A detailed description of parameters used for peptide identification and quantification is given in the Supporting Information.

avoids aforementioned drawbacks of many protease assays, where the introduced labeling can disturb the reaction. Lathia et al. describe the negative influence of the bulky DOTA-tag at their substrate, that prevents chymotryptic proteolysis.21 The solution of this problem was the application of an artificial substrate that exhibits a spacer between labeling and cleavage site, but this strategy restricts the choice of substrate. In a postdigestion strategy the enzyme can be kept in an almost native environment, and every peptide or protein and even proteomic samples can serve as substrate. The detailed workflow of the procedure is given in Figure 1. Single-Substrate Proteolytic Time-Course Analysis. Trypsin as model enzyme and Neurotensin as model substrate were used in order to evaluate the metal-NHS-DOTA labeling as a quantification tool for determining product and substrate concentrations. The workflow is divided into two parts: (i) the proteolytic time-course investigation (Figure 1A) and (ii) the generation of internal standards to determine substrate (ISS) or product concentration (ISP) (Figure 1B). In order to record a time-dependent proteolysis, first the reaction must be quenched quantitatively after certain time intervals, and second the product and substrate concentrations must be determined at this time points (Figure 1A). The method applied for enzyme deactivation depends on the enzyme applied but has in every case to be irreversible and compatible with the following derivatization. In our experiments a combination of the protease inhibitors PMSF and AEBSF (molar ratio: 14.7:1) was applied to inactivate the serine protease trypsin. The later inactivator is more stable against hydrolysis than PMSF but contains a free amino-group which could potentially interfere with NHS-activated metal labeling and was therefore only applied in low substoichiometric amounts. However, this inhibitor mixture sufficiently inhibited enzyme activity (see

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RESULTS AND DISCUSSION General Strategy. The aim of our study was to design an analytical workflow to study the protease catalyzed proteolytic cleavage of natural substrates, in which the derivatization is performed after the reaction. This postdigestion derivatization 5186

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Figure 2. MALDI MS spectrum of Neurotensin after 1 min of tryptic digestion (A). The signal at 1216.50 m/z represents the C-terminal, the signal at 1585.62 m/z the N-terminal cleavage product of ytterbium labeled ISP. Lutetium labeled ISS: 2231.02 m/z, underivatized Neurotensin 1672.92 m/z. (B) shows a detailed view of remaining substrate signals after 1 min reaction time, derived from 8 different initial substrate concentrations, NHS-DOTA derivatized and complexed with Ln and lutetium labeled ISS. (C) shows a detailed view of the evolving N-terminal and lysine labeled product pGluLYENK*PR, and (D) shows the C-terminal cleavage product *RPYIL, which is N-terminally labeled.

The lutetium labeled ISS showed an intense signal at 2231.02 m/z (monoisotopic) in the MS spectra for every time point, exemplarily shown for 1 min of digestion in Figure 2. A signal at 1585.62 m/z, representing the N-terminal cleavage product pGlu-LYENK*PR with ytterbium-DOTA-labeled lysine, and a signal at 1216.50 m/z, representing the C-terminal cleavage product with N-terminal ytterbium-DOTA-tag *RPYIL, were detected in all MS spectra. A detailed list of the expected m/z values for the different labeling products is given in Table S-1. The remaining lanthanide labeled substrates after 1 min of digestion compared to the lutetium labeled ISS are shown in Figure 2. In order to investigate whether trypsin activity was completely inhibited and ISP production was complete (thus the time for full cleavage of the peptide was sufficient), all spectra were further investigated for ytterbium-labeled substrate signals (m/z = 2228.01) and lutetium-labeled product signals. The absence of lutetium-labeled product signals in all spectra and the absence of ytterbium-labeled substrates (Figure 2) showed that both criteria were fulfilled. In several spectra recorded in this study we observed signals for the unlabeled peptides (e.g., Figure 2; 1672.92 m/z), indicating that the derivatization reaction was not quantitative; this was described earlier19 and is not an artifact of a loss of the label in MS but can be clearly accounted to the incompleteness of the reaction itself. The amount of remaining underivatized Neurotensin is small (about 3−5% according to peak intensities), but it sums up after pooling the eight substrate concentrations plus ISP and ISS. However, this does not

below), and virtually no interference with postdigestion derivatization was observed. Generally parameters to be accounted for choosing enzyme deactivation are defined by the needs of the subsequent NHSDOTA derivatization, which requires a pH above 7.0, and the further avoidance of free amines in any solutions was required. The usage of EDTA or other protease inhibitors that act as chelating agents are unlikely to impede significantly the subsequent complexation of the DOTA-macrocycle (only when extremely high molar excess of the chelator is used), as the KD-values for DOTA-trivalent metal complexes are far above those for EDTA-complexes. For the proteolytic time-course Neurotensin stock solutions were diluted in 8 different substrate concentrations and subjected to tryptic digestion. At defined time points, 20 μL aliquots of the reaction mixture were added to the enzyme inhibitor solution, followed by derivatization and complexation of the tagged peptides. Each sample deriving from the same initial substrate concentration was loaded with the same metal in order to compare product or substrate amount at a certain time point. The differentially labeled samples were finally mixed together with ISS and ISP, spotted, and analyzed by MALDITOF MS. ISS was produced by incubating the substrate in reaction buffer without enzyme followed by derivatization and lutetiumcomplexation, while for ISP an aliquot of Neurotensin was completely digested with trypsin and labeled with ytterbiumNHS-DOTA (Figure 1B). 5187

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concentrations. Additionally, more samples should be analyzed at the beginning of the reaction to enable a more accurate determination of the initial velocities. Nevertheless, the results shown clearly demonstrate the potential of this approach for simultaneous analysis and comparison of different inhibitors or inhibitor concentration during a proteolytic reaction. The identification and characterization of the formed products is an advantage of mass spectrometry based enzyme assays as every enzymatic reaction can produce unwanted byproducts due to suboptimal reaction conditions or contaminations in substrate or enzyme solutions. Therefore, MS/MS for pGlu-LYENK*PR (Figure 4) and the C-terminal product *RPYIL were measured, which verified the expected sequences and additionally the expected cleavage site.

disturb the time-course analysis itself, because the derivatization solution was identical for all ten reactions, which are merged to represent a single time point. Figure 3 illustrates the time-dependent degradation of substrate and the formation of products for an initial

Figure 3. Time-dependent formation of the Tm-labeled N-terminal product pGluLYENK*PR (-■-) and C-terminal product *RPYIL (-○-) from initial substrate concentration of 15 μM. Substrate concentrations, calculated from Tm/Lu-ratios are indicated by (-▼-). Curves were drawn by nonlinear regression using the equation y = ax/ (b+x). Asterisk (*) denotes labeling sites.

Neurotensin concentration of 15 μM. The applied quantification strategy is precursor ion based, and MS peak areas (or intensities) serve to determine peptide concentrations relative to the internal standards. Product and substrate concentrations can be calculated from thulium-labeled peptide peak as the concentrations of internal standard peak areas are known. As to be expected for proteolytic reactions leading to two reaction products, N-terminal and C-terminal products developed similar over time. In some cases we observed apparent moderate differences between both product concentrations (deviations