Functional proteomics-aided selection of protease inhibitors for herbivore insect control

Studies have reported the potential of protease inhibitors to engineer insect resistance in transgenic plants but the general usefulness of this approach in crop protection still remains to be established. Insects have evolved strategies to cope with dietary protease inhibitors, such as the use of proteases recalcitrant to inhibition, that often make the selection of effective inhibitors very challenging. Here, we used a functional proteomics approach for the ‘capture’ of Cys protease targets in crude protein extracts as a tool to identify promising cystatins for plant improvement. Two cystatins found to differ in their efficiency to capture Cys proteases of the coleopteran pest Leptinotarsa decemlineata also differed in their usefulness to produce transgenic potato lines resistant to this insect. Plants expressing the most potent cystatin at high level had a strong repressing effect on larval growth and leaf intake, while plants expressing the weakest cystatin showed no effect on both two parameters compared to untransformed parental line used for genetic transformation. Our data underline the relevance of considering the whole range of possible protease targets when selecting an inhibitor for plant pest control. They also confirm the feasibility of developing cystatin-expressing transgenics resistant to a major pest of potato.


Studies have reported the potential of protease inhibitors to engineer insect resistance in transgenic plants but the general usefulness of this approach in crop protection still remains to be established.
Insects have evolved strategies to cope with dietary protease inhibitors, such as the use of proteases recalcitrant to inhibition, that often make the selection of effective inhibitors very challenging. Here, we used a functional proteomics approach for the 'capture' of Cys protease targets in crude protein extracts as a tool to identify promising cystatins for plant improvement. Two cystatins found to differ in their efficiency to capture Cys proteases of the coleopteran pest Leptinotarsa decemlineata also differed in their usefulness to produce transgenic potato lines resistant to this insect. Plants expressing the most potent cystatin at high level had a strong repressing effect on larval growth and leaf intake, while plants expressing the weakest cystatin showed no effect on both two parameters compared to untransformed parental line used for genetic transformation. Our data underline the relevance of considering the whole range of possible protease targets when selecting an inhibitor for plant pest control. They also confirm the feasibility of developing cystatin-expressing transgenics resistant to a major pest of potato.
Three papers have described, almost 30 years ago, the potential of plant genetic transformation to implement insect resistance into crop genomes. Two of those papers, by Vaeck et al. 1 and Fischhoff et al. 2 , reported on the potential of Cry toxin-encoding genes from the soil bacterium Bacillus thuringiensis (Bt) to produce transgenic plant lines resistant to the tobacco hornworm Manduca sexta. The third paper, by Hilder et al. 3 , discussed the potential of a trypsin inhibitor from cowpea to produce transgenic lines resistant to another lepidopteran pest, the tobacco budworm Heliothis virescens. These three seminal papers were followed by hundreds of reports addressing various questions on insect-resistant transgenic crops, notably related to the large-scale deployment and durable use of 'Bt crops' worldwide [4][5][6] or to the basic reasons for the mitigated efficiency and still limited use of protease inhibitors in plant protection 7,8 .
One explanation for the commercial success of Bt crops over protease inhibitor-expressing crops most likely lies in the different modes of action and pesticidal efficiency of the expressed proteins in agricultural contexts 9 . A second explanation is the natural ability of herbivore pests to elude the effects of protease inhibitors shortly after consumption, as a result of a long co-evolutionary history with their plant hosts that use these proteins as a defensive strategy against predation [9][10][11] . Whereas Cry toxins show strong toxicity towards relatively specific pests and allow for an effective insecticide-like effect in the field, protease inhibitors interfere with dietary protein digestion and lead, in the most potent cases, to amino acid shortage and a detrimental overexpression of digestive proteases causing growth delays and eventual death of the herbivore 12 . Most importantly, herbivorous insects have evolved a range of strategies to cope with dietary protease inhibitors, typically involving the secretion of complex midgut protease complements, the overexpression of inhibitor-sensitive proteases to outnumber the ingested inhibitors and the up-regulation of protease isoforms weakly sensitive to inhibition 8 . Nevertheless, and despite numerous unsuccessful attempts to use protease inhibitors in pest control, a number of promising cases have been reported recently [13][14][15][16][17][18][19][20] that still remind the importance of digestive proteases in herbivorous pests and the possible relevance of these enzymes as effective targets for crop improvement [21][22][23][24] .
Protein engineering efforts have been made over the years to enhance the protective effects of plant protease inhibitors, notably involving fusion protein constructs to integrate complementary inhibitor domains into single polypeptides or the rational design of inhibitor variants with improved activity towards animal or plant protease models 25 . A practical challenge at present is to develop strategies for the selection of potent inhibitor candidates in such a way as to limit compensatory responses in the herbivore upon ingestion. Herbivorous arthropod genomes encode large families of digestive protease genes 26-28 that allow the herbivores to produce protease isoforms with a wide range of affinity spectra towards dietary protein substrates and protease inhibitors 11,[29][30][31][32][33] . Considering this, the most effective way to select useful inhibitors among a collection of available candidates may not be to test their inhibitory potency against one or a few model proteases, but to compare their effective binding range against the whole complement of possible protease targets in the pest midgut. In this study, we used a functional proteomics approach for the 'capture' and tandem mass spectrometry (MS/MS) analysis of protease inhibitor-susceptible proteases in crude biological extracts 33 as a decision tool for the rational selection of a protease inhibitor useful to engineer resistance to the major coleopteran pest Colorado potato beetle (Leptinotarsa decemlineata) in potato, Solanum tuberosum. Unsuccessful attempts to implement resistance to this pest in potato varieties using recombinant protease inhibitors have been associated with the onset of multiple adaptive responses including increased leaf consumption to counterbalance the loss of digestive functions and overexpression of inhibitor-insensitive proteases to sustain basic protein digestion rates [34][35][36] . Here we show such compensatory responses to be compromised in larvae fed potato plants engineered to express a Cys protease inhibitor -or cystatin 37 -variant efficient in capturing midgut Cys proteases of this insect under our proteomics setup.

Results and Discussion
Tomato SlCYS8 variant P2V as a promising inhibitor for L. decemlineata control. Tomato cystatin SlCYS8 38 and single functional variants of this protein 39 bearing an isoleucine (P2I), a leucine (P2L) or a valine (P2V) in place of the original proline at position 2, or an arginine (T6R) in place of the original threonine at position 6, were considered as possible candidates for potato transformation (Fig. 1a). L. decemlineata digestive Cys proteases in theory sensitive to cystatin inhibition, the so-called 'intestains' 40 , are categorized into six functional families referred to, respectively, as intestains A (IntA), IntB, IntC, IntD, IntE and IntF 33,40 . Our proteomic approach consists of capturing cystatin-sensitive intestains in midgut extracts after binding biotinylated versions of the cystatins produced in Escherichia coli to an avidin-linked matrix for affinity enrichment 33    positive correlation between the number of captured peptides and the inhibitory range of the cystatin variant against specific intestain families or the whole range of intestains 33,41 .
We used this approach recently to address basic questions on the evolution and structure/function determinants of intestain-cystatin interactions in L. decemlineata 11,42 . An MS/MS peptides dataset generated during these studies was here reassessed to compare the ability of our SlCYS8 variant candidates to bind the insect intestains, assimilating the complement of captured proteases to the binding range of each variant. Thirty unique intestain peptides were detected overall following MS/MS, that could each be assigned to a single functional family (Supplementary Table S1). IntB-, IntC-and IntD-specific peptides were detected for all cystatin variants while no IntA-, IntE-or IntF-specific peptides were detected, presumably due to low numbers of intestains from these families in midgut extracts or to the reported insensitivity of some intestains to cystatin inhibition 40 . As inferred from total spectral counts, mean numbers of captured peptides differed from one variant to another, giving for instance mean numbers with P2V-and P2L-biotin six to seven times the peptide numbers obtained with the original SlCYS8 inhibitor (Fig. 1b). By comparison, T6R-biotin captured a smaller number of peptides, similar to the number captured with SlCYS8. These observations pointed overall to the usefulness of our functional proteomics approach to discriminate cystatin variants based on their effective binding range towards the whole complement of possible target Cys proteases in midgut extracts. They suggested, in practice, the potential of this approach to identify potent inhibitors, such as P2V in the present case, to implement insect resistance in planta.
SlCYS8 variants P2V and T6R expressed in potato show differential effects on L. decemlineata larvae. P2V and T6R were used as candidate inhibitors for potato transformation (Fig. 2) to confirm a possible relationship between protease capture efficiency under our proteomics setup, insect resistance or susceptibility of transgenic plant lines expressing these inhibitors, and the relative ability of the target herbivore to mount an effective compensatory response upon leaf consumption. P2V was selected as an 'effective inhibitor' candidate based on the broad binding range of P2V-biotin towards intestains (see Fig. 1b) and previously reported data suggesting a strong inhibitory potency for this variant against L. decemlineata Cys proteases 39 . T6R was selected as a 'weak inhibitor' (negative control) candidate based on a narrow intestain binding range of T6R-biotin similar to the binding range of SlCYS8-biotin (Fig. 1b), despite previously reported inhibitory data with synthetic peptide substrates suggesting a greater potency of the single variant in vitro 39 . Potato lines engineered to express either inhibitors along with the antibiotic selection marker neomycin phosphotransferase II were regenerated on kanamycin-containing growth medium following genetic transformation with the appropriate gene constructs (Fig. 2a). In vitro clones produced from independent calli were acclimated in greenhouse and PCR-tested for the selection marker transgene in genomic DNA using appropriate DNA primers. A ~500-base-long 'nptii' amplicon was amplified from the DNA of all tested plants, confirming that all clones regenerated on kanamycin had been genetically transformed by the Agrobacterium transgene vector. Immunodetections were performed with polyclonal IgG raised in rabbit against SlCYS8 to compare the relative amounts of recombinant P2V or T6R in transgenic leaves. As expected given the random insertion of transgenes in the genome of Agrobacterium-infected cells, immunoblot signals differed from one clone to another, from weak to moderate in leaves of low-expressing clones (such as P2V3) to strong or very strong in leaves of clones (such as T6R1, T6R3 or P2V10) expressing the cystatin at mean levels approximately seven times the mean level in line P2V3 (anova; F (3,8) = 35.0, P = 0.001) (Fig. 2b). In agreement with studies reporting stress-related pleiotropic effects for recombinant cystatins in different plant species 7 and the constitutive expression of naturally inducible pathogenesis-related (PR) proteins in potato lines expressing corn cystatin II 43 , ß-glucanases of the PR-2 family were up-regulated in leaves of cystatin-expressing clones compared to their steady-state levels in healthy leaves of parental line K (Fig. 2c). These observations confirmed the successful expression of T6R and P2V under an active form inducing pleiotropy in leaf tissue. They also pointed, given the accumulation of ß-glucanases at comparable rates among transgenic clones (see Fig. 2c), to the possible usefulness of T6Rand P2V-expressing lines as models to differenciate the expected protease inhibitory-mediated effects of P2V on L. decemlineata from eventual indirect effects via pleiotropic alterations of the endogenous defense system altering leaf tissue composition.
A feeding assay was conducted to compare the impacts of ectopically expressed P2V and T6R on growth and leaf consumption rates of L. decemlineata larvae (Fig. 3). Potato plants expressing either inhibitors were provided to 4 th instars over 72 h, a period of time sufficient for the larvae to adjust their protease complement to dietary protease inhibitors 36 . Short-term effects on foliage intake after 24 h were observed among larvae fed the different lines (anova; F (4,25) = 78.0, P < 0.001) (Fig. 3a), giving relative consumption rates (RCR) for those larvae fed line T6R1, line T6R3 or line P2V3 about two times the RCR determined for larvae fed untransformed plants (post-anova Tukey's HSD; P < 0.05). RCR values still differed after 72 h (anova; F (4,25) = 219.5, P < 0.001) but increased RCR relative to control larvae at this time point were restricted to larvae fed the P2V-expressing line (post-anova Tukey's HSD; P < 0.05). Similar trends were observed overall for larval growth after both 24 h (anova; F (4,25) = 69.4, P < 0.001) and 72 h (anova; F (4.25) = 409.1, P < 0.001) (Fig. 3b). As for consumption rates, relative growth rates (RGR) of larvae fed the two T6R-expressing lines were similar to the RGR of control larvae after 72 h (post-anova Tukey's HSD; P > 0.05) but different from the RGR of larvae fed the P2V-expressing lines (P < 0.05). Diverging alterations of both the RCR and the RGR were observed for larvae fed the two P2V lines (post-anova Tukey's HSD; P < 0.05) (Fig. 3a,b). As observed earlier with larvae given transgenic potato lines engineered to express the moderately efficient inhibitor oryzacystatin at high concentration 34,35 , line P2V3 (expressing P2V at low level) induced overcompensatory responses leading to increased larval growth and leaf consumption compared to control larvae. In sharp contrast, line P2V10 (expressing P2V at high concentration) had strong negative effects on the larvae, altering leaf consumption and compromising growth shortly after intake. These data indicated a strong, dose-dependent detrimental effect of P2V on L. decemlineata larvae and the practical usefulness of cystatin activity-based functional proteomics as a tool for the selection of protease inhibitors eventually useful in plant protection. They also suggested a limited impact of cystatin-mediated pleiotropic effects on the target insect, as inferred by the null effects of lines T6R1 and T6R3 on larval growth and food intake despite ß-glucanase inductions in leaves similar to those observed in the P2V lines.
T6R-and P2V-expressing potato lines differentially alter digestive protease profiles in L. decemlineata. Protease assays were performed in vitro with family-specific fluorigenic peptide substrates to detect an eventual adjustment of the midgut protease complement in larvae fed cystatin-expressing lines, and to determine whether P2V and T6R had differential effects on Cys (e.g. intestain) and non-Cys protease activities after 72 h (Fig. 4). Cathepsin L-like Cys protease activity differed depending on the plant line provided  36 . These observations pointed overall to a broad compensatory adjustment of the midgut protease complement involving protease forms of at least two mechanistic classes in larvae fed the transgenic lines, including a cystatin variant-specific adjustment of cathepsin L-like (intestain) activity. The nature and the amplitude of protease adjustments were roughly similar in larvae fed the P2V3 or P2V10 lines despite the diverging effects of these two lines on growth or leaf consumption rates (see Fig. 3). This observation, although surprising at first sight, may simply reflect the priority given to digestive proteases in herbivorous insects and the ability of these organisms to readily adjust their protease complement upon ingestion of antidigestive compounds to sustain basic digestive functions. A shotgun proteomics procedure was used to compare intestain profiles in P2V-and T6R-line fed larvae and to determine whether altered Cys protease activities after transgenic line ingestion were associated with a general effect on the intestains or, instead, with the targeted induction of specific intestain families ( Fig. 5 and Table 1). The procedure was similar to the procedure described above for cystatin-captured intestains, except that  Supplementary Table S2 for details on intestain peptides). (b) Total intestain, IntA-specific, IntB-specific and IntD-specific spectral counts for larvae fed control line K or cystatin-expressing line P2V10. Data are expressed as total spectral counts overall (Total) or total spectral counts for each intestain family (see Table 1 and Supplementary Table S2 for details on unique peptide counts). Each bar is the mean of three independent (insect replicate) values ± se. Asterisks indicate statistically different values between diet treatments (post-anova Tukey's HSD; P < 0.05). no capture step was carried out prior to migration and recovery of the protein bands in Coomassie blue-stained gels. One hundred and thirty-two unique intestain peptides were detected following MS/MS, that could each be assigned to a single intestain family (Supplementary Table S2). Intestain members of the six functional families were found at similar relative rates in control and cystatin-fed larvae after 72 h, with IntA, IntB and IntD isoforms being by far the most abundant regardless of the plant line provided (Fig. 5a). In accordance with the above-described protease assays showing plant line-dependent alterations of cathepsin B-and cathepsin L-like protease activities, total numbers of intestain peptide spectral counts differed from line to another (anova; F (4,10) = 4.87, P = 0.023), with midgut extracts of P2V10 line-fed larvae containing the largest number of peptides overall (post-anova Tukey's HSD; P < 0.05) ( Table 1). Peptide spectral counts for all intestain families, except the IntE family (anova; F (4,10) = 2.02, P = 0.168), were influenced to some extent by the plant line, with levels in midgut extracts of larvae fed the T6R1 line often lower than those of P2V line-fed larvae (post-anova Tukey's HSD; P < 0.05) ( Table 1). More specifically, total spectral counts and spectral counts specific to the abundant IntA, IntB and IntD isoforms in larvae fed the P2V10 line were about twice the spectral counts observed in line K-fed control insects (Fig. 5b). These data suggested a general, intestain family-independent up-regulation of the intestain complement after ingestion of the P2V-expressing lines. They also supported the hypothesis of a link between protease inhibitor (e.g. cystatin) detrimental effects and digestive protease (e.g. intestain) overexpression upon inhibitor intake, as proposed earlier for lepidopteran pests given Ser protease inhibitors at high concentration 12 .

Conclusion
Our main goal in this study was to assess the potential of a cystatin activity-based functional proteomics approach to identify cystatin variants eventually useful in plant protection. Current procedures to compare the potency of protease inhibitors against herbivore digestive proteases generally rely on in vitro protease inhibitory assays with synthetic peptide substrates to determine dissociation constants (K d ) towards one or a few model proteases, or to estimate threshold inhibitory concentration values (e.g. IC 50 values) towards specific protease functional families in midgut extracts 44 . Such measurements give useful information about the relative potency of different inhibitors against specific proteases or protease subsets, but they say little about the actual inhibitory range of these proteins towards the whole complement of possible protease targets in the herbivore pest. As importantly, diagnostic peptide substrates assumed to be specific to a given protease family may sometimes be resistant to protease isoforms of this family or, on the contrary, be susceptible to protease isoforms of alternative families. By comparison, functional proteomics approaches such as the activity-based procedure adopted herein to capture inhibitor-sensitive proteases provide a more realistic picture of protease-inhibitor interactions that take place in source extracts, with no masking or confounding effects causing an over-or underestimation of protease binding ranges. An obvious case of discrepancy for a same set of samples characterized using the two experimental approaches was here provided with T6R and P2V. These two cystatin variants were suggested previously to present a similar inhibitory range towards midgut Cys proteases of L. decemlineata larvae, based on in vitro assay data produced with synthetic peptide substrates 39 . In sharp contrast, the P2V-biotin fusion captured almost five times more intestain peptides than its T6R counterpart (see Fig. 1), pointing in fact to significantly different protease binding ranges for the two cystatins and a likely overestimation of protease targets for T6R as assessed with commonly used in vitro assays.
The high potency of P2V in capturing intestains in L. decemlineata midgut extracts was associated with significant short-term effects of transgenic potato lines expressing this inhibitor on larval growth and leaf consumption rates. Studies assessing the long-term detrimental effects of P2V-expressing potato lines on L. decemlineata throughout its life cycle 34,35 and studies with alternative inhibitors of Cys proteases (e.g. ref. 45), host plants and herbivorous pests will be welcome in coming years to further confirm the potential of functional proteomics as a predictive tool for the selection of protease inhibitors useful in crop protection. Studies will also be welcome to explore the potential of inhibitor activity-based procedures for the selection of effective Ser protease inhibitors. Many papers have described the diversity of midgut chymotrypsin-and trypsin-like enzymes in lepidopteran insects 26 , and the hurdles still to be overcome to confirm the general usefulness of Ser protease inhibitors in plant protection 8 .  P2V3 and P2V10).
Data are expressed as total spectral counts for each intestain family (IntA to IntF) or total spectral counts overall (Total) (see Supplementary Table S2 for details on unique peptide counts). Each value is the mean of three independent (insect replicate) counts ± se. On each line, data with the same letter(s) are not significantly different (post-anova Tukey's HSD; α = 0.05). were produced by Agrobacterium tumefaciens-mediated transformation of 'line K' axenic plantlets 46 and selected in vitro using the neomycin phosphotransferase II selection marker for kanamycin resistance, as described earlier 47 . Gene constructs for transformation consisted of either cystatin-encoding sequences 39 introduced between the NcoI and BsrGI cloning sites of a modified pUC19 vector (Fermentas Life Science) harbouring a duplicated version of the Cauliflower mosaic virus (CaMV) 35 S promoter, a tobacco etch virus enhancer sequence and the CaMV 35 S terminator sequence. The resulting constructs were transferred into the pCambia 2300 vector (CAMBIA) for plant genetic transformation (see Fig. 2a). Regenerated plantlets were acclimated for 14 days in a growth chamber under a 24°/21 °C day/night temperature cycle, a 12:12 h light to dark photoperiod, a light intensity of 175 μ mol.m −2 .s −1 and a relative humidity of 60%, before their transfer in greenhouse for multiplication and further analysis. Integration of the nptii selection transgene in kanamycin-resistant plants was confirmed by PCR using DNA extracted from the fifth leaf of 30-cm tall potato plants [down from the apex] according to Edwards et al. 48 . The following primers were used for amplification: 5′ -ACTGA AGCGG GAAGG GACTG GCTGC TATTG, and 3′ -GATAC CGTAA AGCAC GAGGA AGCGG TCAG. The transgene amplicon was visualized by ethidium bromide staining after resolving the PCR products (~500 bases) in 1% (w/v) agarose gels.
Cystatins and ß-glucanases in leaves. T6R, P2V and ß-glucanases (PR-2 proteins) were immunodetected in total soluble protein extracts prepared from the fifth leaf of 30 cm-tall plants, down from the apex. Leaf soluble proteins were extracted in mild conditions as described earlier 49 and resolved by 12% (w/v) SDS-PAGE using the Bio-Rad Mini Protean III Electrophoresis Unit ™ (Bio-Rad). The proteins were electrotransferred onto Hybond ECL nitrocellulose sheets (GE Healthcare) using the Bio-Rad Mini-transfer Unit ™ , according to the supplier's instructions. T6R and P2V were immunodetected with commissioned polyclonal IgG raised in rabbits against SlCYS8 (AgriSera), ß-glucanases with commercial polyclonal IgG raised in rabbits against tobacco PR-2 proteins (AgriSera), and primary IgG with goat anti-rabbit IgG conjugated to alkaline phosphatase. Protein-antibody complexes were visualized using the alkaline phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium for colour development (Life Technologies). Densitometric analysis of the protein signals on nitrocellulose sheets was performed with the Phoretix 2D Expression software, v. 2005 (NonLinear USA) after scanning the immunoblots with an Amersham Image Scanner digitalizer (GE Healthcare). All immunodetections involved three independent (biological) replicates to allow for statistical analyses.
Insect feeding assay. Synchronized 4 th instars were used for the insect feeding assay, derived from a laboratory colony collected on field-grown potato plants at Laval University experimental station near Québec City QC, Canada. The experimental setup was kept at 20 °C and 65% relative humidity in a PGw36 growth chamber (Conviron), under a 16 h daily photoperiod. Three plants of each line were distributed randomly in the growth chamber and two larvae were assigned to each plant, i.e. one on the 5 th leaf and one on the 6 th leaf. Individual larvae (n = 6) and leaves (n = 6) were monitored after 24 h (1 d) and 72 h (3 d) to estimate larval relative growth rates (RGR) and relative consumption rates ×(RCR) 34 , based on the following equations: where W 0 is the mean fresh weight of each group of six larvae at time 0 (mg) and time the number of days (d) after starting the assay. Individual larvae were weighed using an MT5 microbalance (Mettler Toledo). Foliage consumption corresponded to total leaf surface eaten (mm 2 ) as estimated using a 1-cm 2 paper disc guide 36 . Larvae were collected and dissected at the end of the 3-d assay, and their midgut frozen at -80 °C until use for enzymology and proteomic analyses.
Insect midgut proteins. Midgut proteins for protease assays and proteomic profiling were extracted in 100 mM citrate phosphate extraction buffer, pH 6.0, containing 10% (v/v) ethylene glycol. Snap-frozen insect samples were first ground to a fine powder in liquid nitrogen and kept on ice for 10 min in three volumes of extraction buffer. Protein mixtures were clarified by centrifugation at 15,000 g for 10 min at 4 °C, and the supernatant used as source material for further analysis. Protein content in the extracts was adjusted to 2 μ g/μ l by the addition of extraction buffer, after assaying soluble proteins according to Bradford 50 with bovine serum albumin as a protein standard.
Protease assays. Protease activities were determined by the monitoring of fluorigenic peptide hydrolysis progress curves as described earlier 39 . Cathepsin B-like activity was measured at pH 6.5 in 100 mM sodium phosphate containing 10 mM l-cysteine, with Z-Arg-Arg-methylcoumarin (MCA) (Peptides International) as a substrate. Cathepsin L-like activity was measured in the same buffer with Z-Phe-Arg-MCA (Peptides International) as a substrate. Cathepsin D-like activity was measured at pH 3.0 in 50 mM citrate phosphate with the substrate MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-d-Arg-NH 2 (Peptides International). Mass spectrometry. Intestains were identified by liquid chromatography (LC)-MS/MS analysis of intestain bands recovered from Coomassie blue-stained polyacrylamide slab gels. Thirty μ g of midgut protein was resolved by 12% (w/v) SDS-PAGE and gel slices encompassing protein bands in the ~25-32-kDa range were carefully excised 33 . The gel slices were destained, reduced in 10 mM dithiothreitol, alkylated in 55 mM iodoacetamide and hydrolyzed for 1 h at 58 °C with 125 nM TrypsinGold (Promega) using a MassPrep Workstation robot (Waters-Micromass) 51 . Peptides in the gel matrix were extracted in 2% (v/v) acetonitrile (Acn):1% (v/v) formic acid and then washed several times in 50% (v/v) Acn:1% (v/v) formic acid. The extracts were pooled, vacuum centrifuged and resuspended in 7 μ l of 0.1% (v/v) formic acid, from which 2 μ l was taken for LC-MS/MS analysis. The peptides were resolved by reversed-phase nanoscale capillary LC and then submitted to electrospray MS. A Thermo Surveyor MS pump was used, connected to an LTQ linear ion trap mass spectrometer equipped with a nanoelectrospray ion source (ThermoFisher). Peptide separation was performed on a PicoFrit column (NewObjective) packed with Jupiter 5 μ C18 300 A bulk packing (Phenomenex), at 200 nl/min [obtained by flow splitting] over 30 min along a linear gradient going from 2 to 50% (v/v) Acn:0.1% (v/v) formic acid. MS/MS data were acquired under the data-dependent acquisition mode using the Xcalibur software, v. 2.0 (Thermo Scientific).
The seven most intense ions in the 400-2,000 m/z range were selected for collisional induced fragmentation, with the dynamic exclusion function enabled, an exclusion duration of 30 s and relative collisional fragmentation energy set at 35.
Protease identification. MS 53 . Search parameters for protein matching were as follows: a fragment ion mass tolerance of 0.5 Da, a parent ion tolerance of 2.0 Da, iodoacetamide derivatives Cys residues as fixed modification, oxidized Met residues as variable modification, and a maximum of two missed trypsin cleavages allowed. MS/MS based peptide and protein identifications were validated with Scaffold, v. 3.6.1 (Proteome Software). Peptide identifications were accepted if they could be established at greater than 95% probability as specified by the Peptide Prophet algorithm 54 . Protein identifications were accepted if they included at least two identified unique peptides and could be established at greater than 95% probability using the Protein Prophet algorithm 55 . Proteins that contained similar peptides and could not be differentiated based on MS/MS spectra were grouped to satisfy the principle of parsimony.
Spectral count analyses. Quantitative analysis of MS spectra was done using spectral count sampling statistics 56 on those counts corresponding to peptides that were specific to an intestain functional family 33 . Spectra obtained from the individual bands were combined for each repetition, and only those belonging to peptides specific to an intestain family were included in the quantitative analysis. The differential rates of identified intestains were discriminated statistically with a significance threshold of 5%, taking into account spectral count mean values greater than 5 for at least one treatment 57 .