Functional Screening of Serine Protease Inhibitors in the Medical Leech Hirudo medicinalis Monitored by Intensity Fading MALDI-TOF MS

Functional Screening of Serine ProteaseInhibitors in the Medical Leech Hirudomedicinalis Monitored by Intensity FadingMALDI-TOF MS*Oscar Yanes‡, Josep Villanueva§, Enrique Querol, and Francesc X. Aviles¶

The blood-feeding invertebrates are a rich biologicalsource of drugs and lead compounds to treat cardiovas-cular diseases because they have evolved highly efficientmechanisms to feed on their hosts by blocking bloodcoagulation. In this work, we focused our attention on theleech Hirudo medicinalis. We performed, by “intensity fad-ing” MALDI-TOF mass spectrometry, a comprehensivedetection and functional analysis of pre-existent peptidesand small proteins with the capability of binding to tryp-sin-like proteases related to blood coagulation. Combin-ing “intensity fading MS” and off-line LC prefractionationallowed us to detect more than 75 molecules present inthe leech extract that interact specifically with a trypsin-like protease over a sample profile of nearly 2,000 differ-ent peptides/proteins in the 2–20-kDa range. Moreover weresolved 232 individual components from the complexmixture, 13 of which have high sequence homology withpreviously described serine protease inhibitors. Our find-ings indicate that such extracts are much more complexthan expected. Additionally, intensity fading MS, whencomplemented with LC separation strategies, seems tobe a useful tool to investigate complex biological sam-ples, establishing a new bridge between profiling, func-tional peptidomics, and subsequent drug discovery.Molecular & Cellular Proteomics 4:1602–1613, 2005.

Although the task of identifying and characterizing genes inmany sequenced genomes in the last years has been veryintense, it is even more challenging when applied to the muchhigher complex field of the protein world (1). One difficult setof proteins for such analysis are those which we could term as“small” (i.e. below 15–20 kDa) (2) because of their variety anddifficulty to be detected by several established proteomicapproaches such as 2D1 electrophoresis (3) or computationalgenomic scanning (4) among others. Therefore, the develop-

ment of efficient and high throughput technologies for pro-ducing functionally related data of peptides and small pro-teins (2–20 kDa) is increasingly attracting attention. Some ofthese proteins have potential use for diagnostics or therapeu-tics (5–7). They include families of peptide hormones, neu-ropeptides, cytokines, growth factors, and enzyme inhibitorsacting as biochemical messengers or modulators that orga-nize the regulatory processes in all organisms (6). In fact, mostof the important biopharmaceutical products approved fortherapeutic applications are molecules within a molecularmass range of 1–40 kDa (8).

The proteomic strategy focused on the analysis of peptidesand small proteins from complex mixtures has been named“peptidomics” (9, 10). The primary proteomic tool, 2D gelelectrophoresis with mass spectrometry, has been graduallyenhanced, and sometimes replaced, by three main technolo-gies: MALDI MS in combination with LC (off line) (9, 11, 16),ESI MS combined with nano-LC (on line) (12–16), and MALDI-TOF MS for in situ peptide profiling (17–21).

One of the main goals of these technologies is to provide acomprehensive analysis of molecular masses, concentrationlevels, and molecular structures as well as interactions estab-lished by peptides and small proteins in complex biologicalmixtures. The detection of the interactions between biomol-ecules and their targets provides information on the potentialbiological activity of these compounds (22–24) and ultimatelyshortcuts for the development of new drugs for the pharma-ceutical industry (7, 25–27). In this context, we recently re-ported a new approach to detect non-covalent interactionsbetween molecules by direct spectral perturbation, which wecalled “intensity fading” MALDI-TOF mass spectrometry (28,29). It is based on the analysis of the relative intensitiesderived from the MALDI ions (m/z) to study the formation ofcomplexes between biomolecules. Complexes are detectedthrough the decrease (fading) of the relative intensities of them/z signal corresponding to a ligand or mixture of potentialligands (i.e. a biological extract or a peptide library) after theaddition of the target molecule (i.e. a protein). One of ourpresent goals is to apply this procedure to a series of complexbiological systems to validate the procedure and expand itsrange of applicability to high throughput proteomic strategies.

Leeches have been a useful biological source of drugs to

From the Institut de Biotecnologia i de Biomedicina and Departa-ment de Bioquımica, Universitat Autonoma de Barcelona, 08193 Bel-laterra (Barcelona), Spain

Received, May 18, 2005, and in revised form, July 5, 2005Published, MCP Papers in Press, July 18, 2005, DOI 10.1074/

mcp.M500145-MCP2001 The abbreviations used are: 2D, two-dimensional; RP, reversed-

phase.

Research

© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.1602 Molecular & Cellular Proteomics 4.10This paper is available on line at http://www.mcponline.org

treat cardiovascular diseases or as lead compounds in thisfield because they have evolved highly specific mechanismsto feed on their hosts by blocking blood coagulation (30). Aseries of interesting molecules, mainly protease inhibitors,acting at different points in the coagulation cascade or in theinhibition of platelet aggregation have been purified fromthese organisms (31–33).

In this work we describe the application of the intensityfading MS approach to screen protein inhibitors of serineproteases in the saliva of the leech Hirudo medicinalis. Wemodified our original version of the approach (28) by immo-bilization of the protein target on microbeads and incorporat-ing prefractionation steps, which strongly increased the ana-lytical power. This combination allowed the detection of morethan 75 molecules among a complexity of nearly 2,000 mo-lecular species of different molecular mass, in the 2–20-kDarange, that interact specifically with a trypsin-like protease.Moreover we resolved 232 individual components from thecomplex mixture, showing that 16 of them are new putativetrypsin-like inhibitors displaying inhibitory activity againsttrypsin and in most cases high sequence homology with otherserine protease inhibitors.

EXPERIMENTAL PROCEDURES

Materials

Sinapic acid was obtained from Fluka. Extract from H. medicinaliswas supplied by the group of Profs. H. Fritz and C. Sommerhoff(Chirurgischen Klinik Innenstadt, Ludwig-Maximilians-Universitat,Munich, Germany). Trypsin (proteomic sequencing grade), anhydrot-rypsin-agarose, bovine trypsin, and the synthetic trypsin substrateN-benzoyl-L-arginine ethyl ester hydrochloride were purchased fromSigma. Paraffin wax film (Parafilm) was purchased from PechineyPlastic Packaging, Inc.

Pretreatments of H. medicinalis Extract

For direct and fractionated mass profiling analysis by MALDI-TOFMS, the extracts of H. medicinalis were either cleaned up and con-centrated by a simple reversed-phase C18 resin or fractionated byreversed-phase HPLC, respectively.

C18 Resin Protocol to Clean Up and Concentrate Proteins—Lyoph-ilized H. medicinalis extract was dissolved in deionized water at aconcentration of 20 mg/ml. The insoluble residues were removed bycentrifugation at 8,000 � g for 10 min. The supernatant was pro-cessed by a reversed-phase C18 resin-based protocol to clean andconcentrate peptides and small proteins (34). The reversed-phaseresin-bound molecules were eluted with 50% isopropanol, lyophi-lized, and resuspended in 30 �l of 10 mM Tris-HCl, pH 7.5.

Reversed-phase HPLC Fractionation of the Entire Extract—Lyoph-ilized leech extract (�2 mg) was dissolved and subjected to reversed-phase HPLC on a Vydac C18 column using a linear gradient from 10to 50% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 0.5ml/min for 60 min. Fifty fractions were collected, lyophilized, andresuspended in 30 �l of 10 mM Tris-HCl, pH 7.5.

Enzymatic Measurements for the Detectionof Trypsin Inhibitory Activity

The general assay for trypsin inhibition was carried out by prein-cubation of 10 �l of sample (from each of the lyophilized and resus-

pended reversed-phase (RP)-HPLC fractions of the leech extract andsingle purified trypsin ligands) with 50 �l of bovine trypsin (33 �g/mlin 20 mM CaCl2, 0.1 M Tris, pH 8.0) and 790 �l of activity buffer (20 mM

CaCl2, 0.1 M Tris, pH 8.0) for 3 min at room temperature followed bythe addition of 150 �l of the substrate N-benzoyl-L-arginine ethyl esterhydrochloride (3 mM) and measurement of the absorbance change at254 nm. The RP-HPLC fractionation of the extract and inhibitoryactivity assay of each fraction were performed in triplicate ensuringthat the performance and the results reported here were reproducible.

Interaction Experiments

Initial experiments involved the use of a proteomic sequencinggrade trypsin, a chemically modified form resistant to autolysis. How-ever, due to the high concentration of trypsin used in the assay(complexes between trypsin and proteic trypsin inhibitors are 1:1,w/w), residual autolytic reactions and proteolysis of the sample al-tered the spectra, complicating the interpretation of the results. An-hydrotrypsin, a chemically modified form of bovine trypsin with nodetectable catalytic activity but with a strong affinity toward trypsininhibitors (35, 36) was used throughout this work to avoid artifactssuch as the above mentioned.

Preparation of Anhydrotrypsin-Agarose—The buffered aqueoussuspension (50 mM sodium acetate, pH 5.0, containing 20 mM CaCl2and 0.02% sodium azide) of the anhydrotrypsin-agarose was elimi-nated by centrifugation and replaced with 10 mM Tris-HCl, pH 7.5.

Interaction of Anhydrotrypsin-Agarose with the Reversed-phaseHPLC Fractions of the Extract—1 �l of the dissolved reversed-phaseHPLC fractions (see above) was mixed with 1–2 �l of anhydrotrypsin-agarose and incubated for 3 min at room temperature on a smallpiece of Parafilm. In the control sample, the protease was replaced bya neutral buffer (10 mM Tris-HCl, pH 7.5).

Recovery of Ligands—Given the hydrophobic nature of theParafilm surface, the reacting drop (2–3 �l) exhibited an accentuatedsurface tension that allowed pipetting 0.5 �l from the top of the dropand recovering unbound molecules for further MALDI MS analysis.Next the anhydrotrypsin-agarose was washed three times with 5 �l of10 mM Tris-HCl, pH 7.5, by pipetting the agarose up/down 5–10 timesand subsequent elimination of the Tris-HCl solution by capillarity witha thin absorbent paper. Finally 2 �l of 0.1% formic acid were mixedwith anhydrotrypsin-agarose, and after 3 min 0.5 �l of the drop waspipetted to analyze by MALDI MS those molecules initially bound tothe target protein.

Sample Preparation for MALDI-TOFMass Spectrometry Analysis

Both mass profiling and intensity fading MS experiments wereanalyzed in the same way. The sample was mixed with a matrixsolution (1:2, v/v) of sinapic acid (10 mg/ml) containing 30% (v/v)acetonitrile diluted in deionized water (pH 3). 0.5 �l of the mixture wasdeposited on the MALDI target using the dried droplet method (37).

MALDI-TOF Mass Spectrometry

Mass profiles and intensity fading MS interaction experiments wereanalyzed with an Ultraflex MALDI-TOF mass spectrometer (Bruker,Bremen, Germany) equipped with a 337 nm laser, a gridless ionsource, delayed extraction electronics, a high resolution timed ionselector, and a 2-GHz digitizer. For direct and fractionated massprofiling, separate spectra were obtained for two restricted m/zranges, corresponding to peptides and small proteins with molecularmass of 2–8 and 8–20 kDa, under specifically optimized instrumentssettings. Each spectrum was the result of 500 laser shots per m/zsegment per sample delivered in 10 sets of 50 shots distributed in

Functional Screening of Trypsin Inhibitors from Leech

Molecular & Cellular Proteomics 4.10 1603

three different locations on the surface of the matrix spot. Spectrawere acquired in linear mode geometry under 20 kV and with timedion selector deflection of mass ions �1.500 m/z (2–8-kDa segment) or�6.000 m/z (8–20-kDa segment). Delayed extraction was maintainedfor 100 (2–8 kDa) or 330 ns (8–20 kDa) to give appropriate time lagfocusing after each laser shot.

Some peptides were consecutively eluted in two or more adjacentRP-HPLC fractions. A script was designed to eliminate those re-peated m/z ions of adjacent fractions. External calibration was imple-mented to obtain more accurate m/z values of all the componentsanalyzed.

Multidimensional Liquid Chromatography (2D HPLC)

Approximately 0.1 g of lyophilized leech extract was dissolved in 20mM Tris acetate buffer, pH 8.0, and insoluble residues were removedby centrifugation at 13,000 � g for 10 min. After pH equilibration, thesupernatant was loaded onto a preparative anion-exchange column(TSK-DEAE 5PW, 2.5 � 15 cm; Toyo-Soda) connected to an auto-mated liquid chromatography system (�KTA purifier 10/100, Amer-sham Biosciences). Elution was performed using a linear gradientfrom 2 to 100% 0.8 M ammonium acetate in 20 mM Tris acetate at aflow rate of 2 ml/min for 80 min. Thirty fractions of 5 ml were collected,lyophilized, and subjected to reversed-phase HPLC on a Vydac C18

column using a linear gradient from 15 to 50% acetonitrile in 0.1%trifluoroacetic acid at a flow rate of 0.5 ml/min for 60 min. Peaks weremanually collected, and the determination of the molecular mass (m/z)and purity of the isolated molecules from each reversed-phase chro-matographic peak were checked by MALDI-TOF MS.

Preparation of Samples for Edman Degradation

Liquid samples containing acetonitrile and 0.1% trifluoroaceticacid were directly absorbed onto micro TFA filters (Applied Biosys-tems, Foster City, CA). The isolated small protein inhibitors wereanalyzed by automated amino acid sequencing on a Procise proteinsequencing system (Applied Biosystems).

RESULTS

Direct and Fractionated Mass Profiling of H. medicinalis byMALDI-TOF MS—The analytical potential of the intensity fad-ing MS approach to screen complex biological mixtures toidentify new ligands for a specific target protein depends onthe complexity of the molecular ion profile detected in thecontrol mass spectrum. The more ionized molecules dis-played, the better the peptide and small protein representa-tion of the biological sample for further detection of biomo-lecular interactions.

When a direct sample mass profiling of a salivary glandextract of the leech H. medicinalis was performed by MALDI-TOF mass spectrometry, only 146 ion signals appeared in thespectrum (Fig. 1A) probably due to suppression effects (38–40). Signal suppression may be caused by abundant or dom-inant species in a mixture that suppress the ionization anddetection of the less abundant ones. To determine whethersuch negative effects take place and to try to mitigate them, aprevious fractionation of the extract was carried out. Theentire extract of H. medicinalis was fractionated by means ofRP-HPLC. This is a favorable technique for resolving lowmolecular weight proteins and peptides. Most of the serine

protease inhibitors described until now are peptides and smallextracellular proteins with compact structures, rich in disulfidebonds (i.e. aprotinin and hirudin), and resistant to the harshconditions (high amount of acetonitrile and low pH) of RP-HPLC. It is worth remembering that we cannot ensure thatinhibitory activity of all serine protease inhibitors present innature are fully maintained after RP-HPLC and lyophilization(especially those not stabilized by disulfide bridges).

In our case, only 50 HPLC fractions were collected andsubsequently subjected to off-line MALDI-TOF MS analysis.Such analysis revealed a high complexity of ion signals (m/z)for many of the fractions that increased from the previous 146ionized species to 1,953 in the range of 2–20 kDa. To illustratethis complexity, Fig. 1B shows the 50 MALDI-TOF mass spec-tra derived from the 50 RP-HPLC fractions in the 2–8- and8–20-kDa ranges. To represent the differences in complexitybetween direct and fractionated mass profiling, the molecularweight range distribution and the quantitation of all thesepeptides and small proteins (after computational discrimina-tion to delete co-eluted compounds) have been plotted in Fig.1C. Most of the ionized molecules are peptides in the range of2–8 kDa. On the other hand, RP-HPLC fractionation allowedthe detection of 164 small proteins in the 10–20-kDa upperrange not previously displayed with direct mass profiling byMALDI-TOF MS.

Functional Screening of Serine Protease Inhibitors by Inten-sity Fading MALDI-TOF MS—Given the large complexity ofthe sample from H. medicinalis, its analysis was simplified byfocusing on those species with the capability to inhibit (andtherefore bind) serine proteases. All 50 RP-HPLC chromato-graphic fractions were submitted to an inhibitory activity as-say against trypsin using a classical but sensitive spectropho-tometric assay (41) (results not shown). Nineteen fractionsexhibited inhibitory activity and were subsequent subjected tointensity fading MS. With this step, the potential trypsin-ligandpopulation was reduced from nearly 2,000 to 750 speciesapproximately. Fig. 2 shows this analytical process, indicatingthe level of inhibition detected for each chromatographic frac-tion and the subsequent mass spectra of three selected frac-tions of the extract (used as examples) before and after theaddition of anhydrotrypsin (bound to microbeads). Some ionsignals are greatly diminished in the mass spectra after theaddition of the target enzyme. Given that the catalytic (but notbinding) capability of the protease was eliminated by previousconversion in its anhydro derivative, the few ion signals (m/z)affected by the addition of the enzyme should correspond tomolecules that specifically bind to the target protein.

Once it had been shown that some of the ion signals of thespectra were faded and given that the protease was immobi-lized on agarose (see “Experimental Procedures”), an addi-tional step of the analytical assay was performed to assessthe specificity of the assay. Anhydrotrypsin-agarose waswashed with neutral buffer after the binding reaction to dis-card unbound molecules followed by lowering the pH to re-


1604 Molecular & Cellular Proteomics 4.10

FIG. 1. Mass profiling by MALDI-TOF MS of the salivary glands of the leech H. medicinalis. A, MALDI mass spectra derived from theanalysis of a concentrated and cleaned-up sample of the leech extract. B, MALDI mass spectra derived from the reversed-phase HPLCfractionation of the leech extract. C, plot showing the molecular mass distribution of all peptides and small proteins displayed with both directand fractionated mass profiling. Co-eluted peptides and small proteins were deleted to quantify the sample complexity.



FIG. 2. A, reversed-phase HPLC chromatogram of the entire extract of the leech H. medicinalis. Shaded areas indicate regions displayinginhibitory activity against trypsin. B, plot representing the slope of the spectrophotometric inhibitory assay for each one of the 50 reversed-phase HPLC fractions. C, MALDI-TOF mass spectra of three different reversed-phase HPLC fractions from the leech extract showing inhibitoryactivity before (upper) and after (lower) the addition of the trypsin-like protease. Faded m/z ions are indicated by dotted lines. Abs, absorbance.



lease species bound to the protease (see “Experimental Pro-cedures”). Fig. 3 shows the results of the intensity fading MSassay including this additional step for four of the RP-HPLCfractions. By breaking the interactions of the protease with theputative binding molecules, the faded signals after the addi-tion of the protease appear again in the MS spectra. There-fore, these signals are attributed to molecules that specificallybind to the protease.

A triplicate screening of all the chromatographic fractionsshowing inhibitory activity against trypsin was performed fol-

lowing a two-step strategy. First, immobilized protease wasadded to check which molecules of the biological extractwere bound to the protease followed by an acidification of themixture. Only the ion signals presenting a clear mass spec-troscopy difference were considered for further experiments,that is, those that showed a fading or disappearance of itsrelative intensity signal after the addition of the enzyme and areappearance after the acid treatment of the mixture in eachtriplicate assay. This functional screening gave rise to a list ofmore than 75 m/z ions of putative serine protease inhibitors

FIG. 3. MALDI-TOF mass spectra of four different reversed-phase HPLC fractions from the leech extract showing inhibitory activitybefore (upper) and after (middle) the addition of the trypsin-like protease. The recovery of the m/z signal corresponding to trypsininhibitors, after acidification of the sample, is shown (lower).



present in the H. medicinalis extract (Table I). Further analysisallowed characterization of some them (see below).

Multidimensional Liquid Chromatography for the Separationand Identification of Novel Serine Protease Inhibitors—A strat-egy of combining 2D HPLC and MALDI-TOF MS off line wasfollowed using the crude H. medicinalis extract to isolate (insufficient quantity), identify, and characterize the moleculeswith molecular masses present in Table I. For the 2D HPLCapproach we selected two modes characterized by differentseparation selectivities: coupling anion-exchange and re-versed-phase chromatography as the first and second dimen-sion, respectively. Fraction collection of the anion-exchangechromatography followed by the injection of the individualfractions onto the second dimension column resulted in about690 reversed-phase chromatographic peaks. Fig. 4, A and B,shows this preparative process, displaying four examples offractions from reversed-phase chromatography from whichwe isolated five new serine protease inhibitors. Analyzing byMALDI-TOF MS each of the RP-HPLC peaks allowed us tovisualize 232 isolated single components in the range of 2–20kDa. The remainder of the peaks showed two or more co-eluted components.

The resolved single molecules with m/z shown in Table Iwere subjected to automated Edman N-terminal sequencinganalysis (without fragmentation), giving rise to an array ofpartial sequences (see Table II). After a database search usingsuch partial sequences, clear homology relationships withother serine protease inhibitors from leeches or other para-sites for 12 of them were obtained. No homology was foundwith any other sequences in the databases for four of them(see Table II). We also were able to isolate and identify somepreviously described serine protease inhibitors from H. me-dicinalis such as hirustasin (5879 m/z) or bdellin A (6333 m/z)(data not shown).

Validating the Approach—If the small proteins identified byintensity fading MS, purified, and partially sequenced aretrypsin inhibitors, they should display inhibitory activity. Toensure that the molecules isolated and identified in this workare actually trypsin inhibitors, classical enzymology assayswere performed with the purified molecules. Fig. 4C showsthe inhibition data of the RP-HPLC-purified forms, which werelater used for identification by Edman degradation. Within the17 partial sequences shown in Table II, we detected trypsininhibitory activity in 16 of them. The one remaining, with nodetectable inhibitory activity, showed a molecular mass of7,765 Da and 100% sequence similarity with hemoglobin �-1

chain after partial sequencing using Edman degradation. Wechecked the sequence of this protein and verified the pres-ence of an Arg in the C terminus, suggesting that the detectedbinding was not a consequence of its inhibitory activity but asa consequence of the binding characteristics of anhydrotryp-sin with the Arg residue in the C terminus. So far, this is theonly one false positive detected using this approach.

Homology Relationships among the Novel Protease Inhibi-tors Isolated—Most of the new serine protease inhibitorsfound in this work can be located in different families of theleech trypsin inhibitors. However, others show homology re-lationships with other families. For instance, the inhibitor witha molecular mass of 3,128 Da displays a well conservedregion (7DENTPCP13) showing 100% sequence identity withchymotrypsin/elastase isoinhibitor 1 (42) from Ascaris lumbri-coides. In addition, two couples of protein inhibitors withmolecular masses of 6,218/6,227 and 5,463/5,325 Da showidentical sequences within the first 49 and 19 amino acidsfrom the N terminus, respectively, suggesting degradation ofthe proteins in the extract or the presence of isoforms. Alsothe protein with a molecular mass of 7,900 Da showed 100%sequence identity with Eglin C (43), but a difference of 199 Dain its molecular mass suggests that it could be a degradedform of Eglin C or just a new isoform not described previously.

Some of the isolated putative serine protease inhibitors ofthis work display a clear similarity with other inhibitors of theantistasin family previously found in leeches, such as guam-erin (44, 45), piguamerin (46), bdellin A (47), and hirustasin (48)(see Table II). In Fig. 5, the amino acid sequences of fiveinhibitors displaying homology with these four inhibitors of theantistasin family are compared, and amino acids that areconserved in the nine proteins are boxed. The region corre-sponding to the “reactive center” in this type of proteaseinhibitors is located in an exposed binding loop (49, 50) clearlysequenced in this work. The homology there is reduced quitesignificantly (except for a cysteine residue) and breaks downabruptly at the boundaries. Nowhere else in the molecule cana stretch of comparable length approaching this degree ofdivergence be found. Finally the N-terminal sequences ofmolecules with 3,038, 4,014, 5,448, and 4,083 Da revealed nosimilarities with any other sequences in protein sequencedatabases, although they showed trypsin inhibitory activity.

DISCUSSION

Increasing attention is paid nowadays to peptidomic re-search because of its potential use diagnostically or thera-

TABLE IMolecular weights (m/z) of peptides and small proteins that specifically interacts with the trypsin-like protease by intensity fading MS



FIG. 4. A, anion-exchange chromatogram of the entire extract of the leech H. medicinalis indicating the 30 fractions collected at thepreparative level. B, reversed-phase HPLC chromatograms derived from four selected anion-exchange chromatographic fractions. Shadedpeaks correspond to four of the isolated single inhibitors. C, representation of the slope derived from the spectrophotometric inhibitory assayfor each one of the single isolated forms of the trypsin inhibitors assayed prior to the Edman degradation. Abs, absorbance.



peutically. In this sense, the leech is an organism with bio-medical applications that generate outstanding interest.Several anticoagulants are present in the salivary glands ofleeches because these organisms depend on a diet of freshblood and have evolved mechanisms that interfere with thecoagulation of the blood “donor.” These bioactive molecules

are mainly small protease inhibitors of serine proteases in-volved in the coagulation cascade (32).

Peptidomics, like proteomics, has three subareas that arecurrently attracting very active research: profiling, functional,and structural analysis. In this work, we tried to join andcomplement the former two subareas, that is, profiling and

FIG. 5. Comparison of the protein sequences for five fragments of trypsin inhibitors identified in this work (denoted by numbersreferring to their masses) and four previously described leech inhibitors. Identical amino acids for five of the nine proteins are boxed. Asolid line indicates the reactive center region. The P1 residue of guamerin, piguamerin, hirustasin, and bdellin A is marked with an arrow.

TABLE IISome of the new small proteins and peptides identified as trypsin ligands in the leech H. medicinalis by intensity fading MS

A indicates no sequence homology found with any other protease inhibitor.



functional aspects, through the use of the intensity fading MSapproach. We combined LC-MS off line by means of (one-dimensional) RP or (2D) ion-exchange RP-HPLC and MALDI-TOF MS to display the H. medicinalis complexity. In additionwe isolated some of its components and semicharacterizedthem by Edman sequencing. Complementarily the applicationof the intensity fading MS methodology for screening trypsin-like inhibitors among the displayed population of peptides andsmall proteins detected more than 75 different proteins show-ing binding properties for a trypsin-like protease with molec-ular masses ranging from 2,000 to 20,000 Da. This is the mostinteresting mass range for our present purposes and alsobecause this is the spectral range in which the present aver-age MALDI-TOF spectrometer performs its best for peptidesand proteins.

The (one-dimensional) RP-HPLC and MALDI-TOF com-bined strategy substantially mitigated sample suppressioneffects in mass spectrometry and allowed the visualization ofmore than 10-fold ionized species from the H. medicinalispeptidome with respect to direct mass profiling strategy.However, separation based on one single parameter like re-versed-phase HPLC does not provide the required resolutionand capacity to resolve individual components of an ex-tremely complex biological mixture like the H. medicinalisextract. For this reason, in a second step the strategy wasimproved by using off-line 2D LC based on a strong anion-exchange column as a first dimension and reversed-phase liq-uid chromatography as the second separation. Despite the 2DHPLC strategy, it was not feasible to resolve the nearly 2,000individual components detected by mass spectrometry, andsome components were co-eluted in the second dimension.

The use of immobilized anhydrotrypsin to generate spectralperturbations following the intensity fading MS assay allowedus to overcome the problems derived from the autolytic andcatalytic activity toward possible substrates of trypsin andminimized potential contaminations from the added target; italso permitted us to have additional control over the putativeinhibitors through their isolation and by monitoring their com-plex dissociation. One of the limitations of the approach is thefact that anhydrotrypsin can selectively bind peptides withArg, Lys, or S-aminoethylcysteine residues at the C terminus,resulting in some possible false positives. To get around thislimitation, a complementary spectrophotometric assay on thepurified species (see “Experimental Procedures”) ensuredtheir trypsin inhibitory activity.

Some of the isolated serine protease inhibitors of this workdisplay a clear similarity among them and with several trypsininhibitors previously reported from leeches, such as guam-erin, piguamerin, bdellin A, and hirustasin (see Table II) (44–48). These four serine protease inhibitors belong to the anti-stasin family, and they were isolated from H. medicinalis andHirudo nipponia. Despite having a high similarity, guamerin,piguamerin, bdellin A, and hirustasin have different proteasespecificities (see Table II). Based on these data, it is quite

possible that the trypsin inhibitors isolated in this work showmore than one specificity for different serine proteases likethrombin, plasmin, kallikrein, chymotrypsin, acrosin, or ca-thepsin G. The difference in specificity between these fourproteins has been attributed to the high degree of non-ho-mology within the short stretch of amino acids of the reactivecenter region and particularly to the resulting change in the P1

amino acid (32). As initially postulated by Hill and Hastie (51)for the serpin family, we suggest that all these small serineprotease inhibitors pertaining to the antistasin family are anexample of accelerated evolution in the reactive center regionof serine protease inhibitors (see Fig. 5) (52). The proteinsequence of the reactive center region has undergone muchmore rapid evolution than the rest of the molecule as a con-sequence of the selective forces proceeding from extrinsicproteases of the host. Because leeches are exclusively fed byblood of poikilothermic and homeothermic animals, fast ad-aptation to this kind of nutrition has resulted in acceleratedevolutionary selection and fixation of very specific interactionsbetween proteinase inhibitors of the leech and proteases ofthe host to prevent clotting of the sucked blood.

Given the specific recognition by proteases of definedamino acid sequences, it may be possible to inhibit enzymesinvolved in pathological processes. Potent inhibitors have thepotential to be developed as new therapeutic agents. In thelast decade, it became obvious that invertebrates have beenshown to be truly useful models in drug discovery for manycardiovascular, tumoral, and inflammatory diseases. Leechesprovide a source of new candidate molecules for drug dis-covery, especially serine protease inhibitors, that can be ex-ploited by the medical industry, i.e. to treat emphysema,coagulation, inflammation, dermatitis, and cancer (30–32, 53).

In conclusion, the array of new serine protease inhibitorsidentified in this work together with those previously de-scribed from the leech H. medicinalis confirms its potentialityas a biological source of lead compounds for the medicalindustry, especially for those diseases in which serine prote-ase are involved. Our good results confirm the intensity fadingMS approach as a new robust strategy for the functionalscreening of complex biological samples, particularly theones containing proteases and inhibitors of interest. The ap-proach takes advantage of the low cost and rapid perform-ance of MALDI MS technology together with the capability tocheck affinity properties of the analyzed compound.

Acknowledgments—We are indebted to Profs. H. Fritz and C.Sommerhoff (Chirurgical Clinic, Munich, Germany) for providing usthe leech extract. We thank F. Canals for helpful discussions.

* This work was supported in part by Ministerio de Ciencia y Tec-nologıa, Spain (MCYT) Grants BIO2004-05879 and GEN2003-20642-C09-05 and by the Centre de Referencia en Biotecnologia (CERBA)de la Generalitat de Catalunya. The costs of publication of this articlewere defrayed in part by the payment of page charges. This articlemust therefore be hereby marked “advertisement” in accordance with18 U.S.C. Section 1734 solely to indicate this fact.



‡ Supported by a fellowship from MCYT.§ To whom correspondence may be addressed: Memorial Sloan-

Kettering Cancer Center, 1275 York Ave., New York, NY 10021. Tel.:212-639-6676; Fax: 212-717-3604; E-mail: [email protected].

¶ To whom correspondence may be addressed: Institut de Biotec-nologia i de Biomedicina, Universitat Autonoma de Barcelona, 08193Bellaterra (Barcelona), Spain. Tel.: 34-93-581-1315; Fax: 34-93-581-2011; E-mail: [email protected].

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Functional Screening of Serine Protease Inhibitors in the Medical Leech Hirudo medicinalis Monitored by Intensity Fading MALDI-TOF MS

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