Authors: Leonardo G. Oliveira #, Edward Steadham*, Steven M. Lonergan*, Elisabeth Huff-Lonergan*
# EVZ, Universidade Federal de Goiás, Goiânia, GO, Brazil.
* Muscle Biology Group, Department of Animal Science, Iowa State University, Ames, IA 50011, United States
Acknowledgements: Appreciation is extended to the Iowa Muscle Biology group for funding this research and to CAPES-Brazil to provide the scholarship for the first author.
Abstract: Calapastatin is a specific inhibitor of the calcium dependent proteinases m- and µ-calpain and is related to various metabolic process in the live animal and during post mortem tenderization of meat. Composed of four repetitive regions with inhibitory activity (domains1– 4), and an unique domain L located at N-terminal region. Using an anion exchange chromatography to separate calpastatin and sequencial chromatography steps to purify each peak was found in each peak.
During the process to purify the calpastatin peak 1 the activity per mg of protein is greatly increased but lose half activity, for calpastatin peak 2 the purification process, specific activity was increased 139.8 fold and at the end of this process remains 36% of the initial total activity. Bands of approximately 70 kDa are identified of both peaks and the band from peak 2 was a little higher than peak 1. The calpastatin was identified in the second dimension gel in a similar molecular weight to SDS-PAGE gel and western blot and one spots from calpastatin peak 1 and two from calpastatin peak 2 were identified as calpastatin. Sequence of peptides identified in Spot from purified peak 1 as part of the inhibitory domain III and IV and C terminus and from purified peak 2 a sequence of peptides identified as part of the inhibitory domain I, II and III. This results lead us to believe that both peaks, in this case, are products of degradation of the intact molecule and probably the small peptides are loosed during the process. The results of present study shows that is possible the purification of distinct forms of active calpastatin, however the intact form of calpastatin was not present in this purification.
Presence of peptides was not conclusive to determine the origin and composition of each active peak.
Keywords: Calpain system, Ion exchange chromatography, Proteomics
INTRODUCTION
Specific inhibitor of the ubiquitous calcium dependent proteinases m- and µ-calpain, calpastatin, is related to various metabolic process in the live animal and during post mortem tenderization of meat (Geesink et al. 1995; Goll et al., 1998; Geesink 1999, Huff-Lonergan et al., 1995; Huff-Lonergan et al., 1996). Composed of four repetitive regions (inhibitory domains1– 4), each of this have a possibility to inhibit calpain activity, and an unique N-terminal region: domain L(Takano et al., 1986; Maki et al., 1988).
With a widely varying molecular weights have been purified from a number of tissues and different molecular weights are related. Confusion regarding the molecular weight of calpastatin was due to factors like a proteolytic degradation, and some of this fragments of calpastatin retain inhibitory activity (Melgren et al. 1983; Imajoh et al., 1984; Nakamura et al., 1985), because of its unusual amino acid composition, the molecular weight is overestimated using SDS–PAGE (Maki et al., 1988) and due to the asymmetry estimation using gel filtration leading to an overestimation of its molecular weight (2).
Calpastatins from different species, using cDNAs analysis, has shown that the most prominent form found in all tissues has a predicted molecular weight of 72–77 kDa but anomalously migrates on SDS–PAGE with an apparent molecular weight of 115–130 kDa (Killifer & Koohmaraie, 1994).
However, chromatography of the muscle extracts to separate calpains and calpastatin led to extensive fragmentation of calpastatin (Arnold et al. 1995; Geesink et al., 1998) and this fragmentation could difficult to determine the composition of extracted calpastatin from chromatograph column and purify the intact molecule. During the extraction of calpastatin using anion exchange column, calpastatin could be eluted in two distinct peaks (Pontremoli et al., 1992; Cruzem et al., 2013) and the significance still unclear.
The objective of this work is purify each calpastatin peak to characterize it composition using two dimensional gel electrophoresis and mass spectrometry.
MATHERIALS AND METHODS
A commercial animal was slaughtered following standard humane procedures at the Iowa State University Meat Laboratory and a sample (approximately 1.5 kg center cut) of the Longissimus dorsi (LD) from each animal (animal was weighted approximately 100kg at the time of slaughter) was collected within 10 min post-exsanguination, briefly placed on ice and immediately taken to the laboratory for extraction procedures, chromatographic separation of calpastatin peaks. All process was executed at 4 oC in a cold room.
Calpastatin extraction
Was used a methodology proposed by Thompson et al. (2000) to extract calpastatin from muscle and purify. Using a knife to remove visible fat and connective tissue, 900 grams of muscle Longissimus dorsi was finely minced and 100 g of muscle per time was immediately homogenized using with a Polytron PT 3100 (Lucerne, Switzerland) in 6 volumes (w/v) of cold extraction buffer (100 mM Tris–
HCl, 10 mM EDTA, pH 8.3, 4 °C). Before use were added to the buffer 0.1% 2-mercaptoethanol (2-MCE), 2 μM of E-64, and 100 mg/L trypsin inhibitor. The homogenate was centrifuged at 40,000 ×g for 30 min at 4 °C, and the supernatant was filtered through cheesecloth and dialyzed in 40 volumes of TEM (40 mM Tris–HCl, 1 mM EDTA, pH 7.4, 0.1% MCE).
Was added 277 g/L os Ammonium sulfate slowly and stirring during 12 hours. After stirring, the homogenate was divided in centrifuge tubes, centrifuged at 4 oC at 10000 RPM during 30 minutes.
Was discarded the supernatant after centrifugation and the pellet was ressuspended in 4 volumes of TEM buffer (40 mM TRIS, 1 mM EDTA, 0,1% 2 mercaptoetanol, pH 7.4). This solution was dialyzed 12 hours with TE (40mM TRIS, 1mM EDTA, pH 7.4) using 20 times the amount of final volume of ressuspended solution, and repeated 4 times until complete 100 times the volume of ressuspended solution.
After dialysis, were centrifuged at 40,000 ×g for 30 min at 4 °C and the supernatant filtered through cheesecloth. The filtered was loaded onto a 800 mL Q-Sepharose (GE Healthcare Biosciences, Pittsburgh, PA) anion exchange column. The column was previously equilibrated with TEM and after loaded the sample was washed with 1200 mL TEM. Calpastatin was eluted using a flow rate of 2.0 mL/min, fraction volume of 2.5 mL with a linear gradient of 50 to 225 mM KCl in TEM using an ÄKTA™ prime automated liquid chromatography system (Amersham Pharmacia Biotech Inc., Piscataway, NJ).
Calpastatin Activity
To determine calpastatin activity of the fractions were used the caseinolytic method (Koohmaraie,1990) with some modifications. Was used 50 µL of each fraction, brought to 1 mL with TE (40 mM Tris–HCl, 1 mM EDTA, pH 7.4) in a glass tube. One milliliter of casein buffer (100 mM Tris–acetate 7 mg/mL casein, and 1 mM sodium azide, pH 7.5, with 0.2% MCE added just before use) was added, followed by 100 μl of calcium buffer (200 mM CaCl2). For activity determination, was added in each sample approximately 0.40 units of m-calpain previously purified from porcine lung.
For positive control, was used 1 mL of TE (without sample), casein buffer and added approximately 0.40 units of m-calpain from porcine lung, made in triplicate. For blank tubes was used 1 mL of TE (without sample), casein buffer, and made in triplicate. Tubes were briefly vortexed and incubated in a water bath at 25 °C for 1 h.
To stop the reaction were added 2 mL of 5% trichloroacetic acid in each tube and after vortexed.
Samples were centrifuged at 1500 × g for 20 min at 25 °C and determined the absorbance of the supernatant at 278 nm. The reading was compared to blank and positive control samples (Koohmaraie et al., 1995). The first peak of calpastatin activity was eluted between 60 to 90 mM KCl. The second peak of calpastatin was eluted between 120 to 220 mM KCl. An example of elution peaks is in Figure 1.
Fractions that had calpastatin activity for peak 1 was pooled and the same was proceed for peak 2 to follow the purification and determine the total activity of pooled fractions. To determine the total activity of each pool was used crescent amounts of sample starting with 20 µL until 400µL. To calculate the total activity of pooled calpastatin I and II, was used the value of 50% inhibition of lung m-calpain. To discount the amount of protein contained in the pooled fraction of calpastatin fractions, was used in each tube 1 mL of pooled sample, 100 μl of EDTA buffer (200mM EDTA), one milliliter of casein buffer and approximately 0.40 units of m-calpain previously purified from porcine lung and incubated, briefly vortexed and incubated in a water bath at 25 °C for 1 h. One unit of calpastatin activity was defined as the amount required to inhibit 1 unit of porcine lung m-calpain (Koohmaraie, 1990). Protein concentration of polled Calpastatin peaks was determined using Biuret methodology (Lowry et al., 1951).
Calpastatin peaks purification
To purify the pooled calpastatin peak 1, was dialyzed against 40 volumes of TE at 4 oC during 12 hours. After dialysis was added of ammonium sulfate (Sigma, St Louis, MO) until reach concentration 1 mol/L. The solution containing calpastatin peak 1 was loaded onto a Phenyl Sepharose (GE Healthcare Biosciences, Pittsburgh, PA) anion exchange column. The column was previously equilibrated with 3 times the volume of column of TEM with 1 Mol of ammonium sulfate (Sigma, St Louis, MO) and after loaded the sample was washed with the same solution. Calpastatin was eluted using with a flow rate of 1.5 mL/min, fraction volume of 8.0 mL in a linear gradient of 1.0 to 0 M of ammonium sulfate in TEM using an ÄKTA™ prime automated liquid chromatography system (Amersham Pharmacia Biotech Inc., Piscataway, NJ).
Was determined calpastatin activity of fractions from peak 1 eluted of Phenyl Sepharose column (GE Healthcare Biosciences, Pittsburgh, PA) as described protocol, pooled fractions that had activity and protein concentration was determinate . The same process was made with peak 2 and protein concentration of pooled fraction was determined. Calpastatin peak 1 was eluted between 710 to 560
mM of ammonium sulfate. Was proceeded the same process to purify the peak 2 and was eluted between 620 do 460 mM of ammonium sulfate.
Pooled fractions were dialyzed against 40 times the volume of pooled fractions on TEM during 12 hours at 4 oC. Peak 1 was stored at this point and peak 2 follow the purification. Calpastatin peak 2 was loaded onto a Blue Sepharose (GE Healthcare Biosciences, Pittsburgh, PA) column. The column was previously equilibrated with 3 times the volume of column of TEM and after loaded the sample was washed with tree times the column volume with the same solution and eluted using a flow rate of 1.5 mL/min, fraction volume of 10.0 mL in a linear gradient 0 to 500 mM of potassium chloride in TEM using an ÄKTA™ prime automated liquid chromatography system (Amersham Pharmacia Biotech Inc., Piscataway, NJ). Calpastatin activity was determined in each eluted fraction as described previously and active fractions were pooled and dialyzed against 40 times the volume of pooled fractions on TEM during 12 hours at 4 oC. Calpastatin was eluted between 60 to 300 mM of potassium cloride.
Dialyzed pooled fractions from Calpastatin peak 2 was loaded onto a DEAE CAPTO (GE Healthcare Biosciences, Pittsburgh, PA) ion exchange column. The column was previously equilibrated with 3 times the volume of column of TEM and after loaded the sample was washed with tree times the column volume with the same solution and eluted using a flow rate of 1.5 mL/min, fraction volume of 10.0 mL in a linear gradient 0 to 250 mM of potassium chloride in TEM using an ÄKTA™ prime automated liquid chromatography system (Amersham Pharmacia Biotech Inc., Piscataway, NJ). Calpastatin activity was determined in each eluted fraction as described previously and active fractions were pooled and dialyzed against 40 times the volume of pooled fractions on TEM during 12 hours at 4 oC. Calpastatin was eluted between 100 to 1500 mM of potassium cloride. Protein concentration were determined after all dialyze steps to determine the fold of purification.
SDS-PAGE and immunoblotting
For SDS-PAGE and immunoblotting was used samples taken from end of purification. One mL of each purified calpastatin peak was mixed with 0.5 mL of SDS sample buffer (30 mM Tris-HC1, 3 mM EDTA, 3% [w/v] SDS, 30% [vol/vol] glycerol, and 30 pg of pyronin Y /mL, pH 8.0) (Wang, 1982), 0.1 mL MCE and stored at -80 oC until analyse.
An 12.5% polyacrylamide separating gel (acrylamide:N,N′-bis-methylene acrylamide = 100:1 [w/w], 0.1% [w/v] SDS, 0.05% [v/v] TEMED, 0.05% [w/v] ammonium persulfate, and 0.5 M Tris–
HCl, pH 8.8). A 5% polyacrylamide gel (acrylamide:N,N′-bis-ethylene acrylamide = 100:1 [w/w], 0.1% [w/v] SDS, 0.125% [v/v] TEMED, 0.075% [w/v] ammonium persulfate, and 0.125 MTris–HCl, pH 6.8) was used for the stacking gel. Gels (10 cm wide ×8 cm tall ×1.5 cm thick) were loaded with 10 μg protein per lane and run at a constant 20 V in SE 260 Hoefer Mighty Small II (Hoefer, Inc., Holliston, MA) electrophoresis units overnight.
Transfer of protein from SDS-PAGE gels to a PVDF membrane was performed as described by Melody et al. (2004). Membranes were blocked for 1 h at room temperature in PBS with 0.1% Tween-20 and 5% nonfat dry milk and were then incubated in primary antibody over-night at 4 °C. Primary antibody dilution was for calpastatin, 1:5000 dilution (MA3-945, Thermo Scientific, Rockford, IL).
Membrane was washed 3 times in PBS–Tween for 10 min each at room temperature. The membrane was then incubated in a goat anti-mouse horseradish peroxidase (No 2554, Sigma, St. Louis, MO) secondary antibody at 1:10,000 dilution for 1 h at room temperature. After 3 additional 10 min washes in PBS–Tween, blots were developed using SuperSignal West Femto Maximum Sensitivity Substrate (ThermoScientific, Rockford, IL) and imaged using a ChemiImager 5500 (Alpha Innotech, San Leandro, CA) and Alpha Ease FC software (v 3.03 Alpha Innotech).
Two-Dimensional Difference in Gel Electrophoresis.
To determine a protein profile of calpastatin peaks was used two dimensional DIGE technique described by Anderson et al. (2012) with some modifications.
Preparative gels were loaded with 80μg of protein from either a peak. To first dimension proteins are separated on the basis of isoelectric point (pI) was carried out on Immobiline DryStrips (13 cm, pH 4–7, GE Healthcare, Piscataway, NJ) rehydrated with DeStreak Rehydration Solution (GE Healthcare, Piscataway, NJ) containing 2.5 mM DL-dithiothreitol (DTT). Samples are dispersed in a tray, which soaked immobilized pH gradient strip, placed on top of rehydration mix and was left to rehydrate overnight at room temperature in a humidifier chamber. Isoelectric focusing was performed on an Ettan IPGphor isoelectric focusing system (GE Healthcare) for a total of 14,500 V h.
After isoelectric focusing, strips were equilibrated using two sequential 15 min washes with equilibration buffer (50 mMTris-HCl pH 8.8, 6 M urea, 30% glycerol, 2% SDS and a trace of Bromophenol Blue) containing 65 mM DTT first and after in a 135 mM iodoacetamida (Rozanas &
Loyland, 2008).
The equilibrated strips were loaded onto 12.5% SDS-PAGE gels(acrylamide: N,N′-bis-methylene acrylamide=100:1 [wt/wt], 0.1% SDS [wt/vol], 0.05%N,N,N′N-tetramethylethylenediamine(TEMED), 0.05%ammonium persulfate [wt/vol], and 0.5 M Tris–HCl, pH 8.8), using agarose as an overlay and run over-night at 80 V at 4 oC on Ettan DALT SIX system (GE Healthcare).
After second dimension electrophoresis, preparative gels were stained with Colloidal Coomassie Blue solution (1.7% ammoniumsulfate [wt/vol], 30%methanol [vol/vol], 3% phosphoric acid [vol/vol], and 0.1% Coomassie G-250 [wt/vol]). To destain was used distilled and deionized water. All buffers and water used in this process was filtered using Stericup Filter Unit, poresize 0.22 μm (Millipore Corp., Billireca, MA) to minimize potential contamination.
To identify the proteins showed in each gel the selected spot in the gel was excised and sent to the Iowa State University Protein Facility for identification. It was performed the in-gel digestion (via trypsin) using Genomics Solution ProGest (Chelmsford, MA). To dissolve the peptides were used alpha-cyano-4-hydroxycinnamic acid (5 mg/mL in 50% CH3CN/0.1% Trifluoroacetic acid) and deposited to a matrix-assisted laser desorption/ionization (MALDI) plate. Matrix-assisted laser
desorption/ionization mass spectrometry was performed using a QSTAR XL Quadrupole time-of-flight mass spectrometer equipped with an orthogonal MALDI ion source (AB/MDS Sciex, Toronto, Canada). Spectra were processed by MASCOT data-base search version 2.2.07 (MatrixScience, London, UK).
RESULTS AND DISCUSSION
In the protocol for extraction of calpastatin from muscle using anion exchange chromatography, calpastatin is eluted in two distinct peaks (Figure 1) and this pattern was reported before in (Pontremoli et al., 1992; Salamino et al., 1994; Geesink et al., 1998; Averna et al., 2001; Samanta et al., 2010;
Cruzen et al., 2013), even though there are no consensus about the origin of those peaks.
Phosphorylation of calpastatin molecule by protein kinase C was demonstrated in vitro (Pontremoli et al., 1992; Averna et al. 1999; Averna et al., 2001) that could change the ionic charge of the molecule and modify the affinity to the column and could be involved in the regulation of . Other possibility could be an alternative splicing of the gene (Geesink et al 1998; Gaarder et al., 2011). Degradation of calpastatin molecule is another possibility to this separation in two peaks and the possible reason for losing activity during the purification process (Geesink et al., 1998). The range of elution this first calpastatin peak, shows that this peak has less affinity to the column compared to the second peak, therefore it shows difference in ionic charge between them.
To determine the composition of each peak was proceeded the purification of each calpastatin peak and the result are presented in Table 1. The process to purify the calpastatin peak 1 goes until Phenil sepharose anion exchange column and among Q sepharose and Phenil sepharose calpastatin peak 1 lose half activity but the activity per mg of protein is greatly increased. This fact was attributed to a possible degradation during the purification process (Geesink et al,. 1998). Without heat treatment,
some protease is elute with calpastatin and this could cleave the molecule to small peptides tha could not bind to column and loose activity (Geesink et al., 1998).
For calpastatin peak 2, the purification process was extended, passing through Bluesepharose Cibacron Blue 3G Affinity column and DEAE CAPTO anion exchange column (Thompson et al., 2000). Specific activity was increased 139.8 fold from the separation step and at the end of this process remains only 36% of the initial total activity of this peak. Result of a peak eluted in the similar range of KCl was found using similar anion exchange column, even though was used heat treatment before load into the column and the specific activity was 952 units per mg of tissue (Geesink et al., 1998).
In purification of calpastatin from bovine heart Melgren et al. (1983) obtained a specific activity of 4340 units per mg of protein and 13.9% of recovery from the first column.
Same calpastatin separation in two peaks from rat skeletal muscle was described and was reported and verified the having different specificities for each of the m and µ-calpain (Pontremoli et al, 1991). They found that both forms of calpastatin are influenced by postranslation modification.
In another study involving phosphorilation of calpastatin peaks shows an interchangeable efficiency against both calpains. Calpastatin peak 1 was verified more effective against µ-calpain and peak 2 more effective against m-calpain, after phosphorilation peak 1 turn more effective against m-calpain and a dephosphorilation of peak 2 turn this peak more effective against µ-calpain (Pontremoli et al., 1992). This findings and an increase in calpastatin degradation by proteases, suggesting the existence of a different regulatory mechanism for calpastatin forms (Averna et al., 1999).
The protein profile in SDS-PAGE gel of the pooled fractions of calpastatin of Q sepharose column and at the end of purification are presented in Figure 2 and shows that only weak bands are stained. This result agree with specific activity and the fold of each peak purification and the bands stained by calpastatin antibody (Figure 3). The results shows bands of approximately 70 kDa and the peak 2 was a little higher than peak. Those molecular weight are similar to a fragment of intact calpastatin degraded by endogenous proteases (Doumit et al., 1999).
The intact calpastatin molecule are present in range of 125 to 145 kDa in SDS PAGE. Estimated molecular weight based on amino acid sequence of skeletal calpastatin is between 77 to 80 kDa. In SDS-PAGE calpastatin migrates anomalously, and it is difficult to relate a band migrating at a particular molecular weight in SDS-PAGE to a known calpastatin isoform, this anomalously slow migration of calpastatin in SDS-PAGE is a property of the calpastatin polypeptide itself and probably not due to posttranslational modifications (Maki et al., 1988; Goll et al. 2003).
Intact calpastatin molecule has no found in this study, and a possible reason is because the calpastatin molecule is labile to degradation by endogenous proteases producing peptides that could remain inhibitory activity (Emori et al., 1988; Goll et al.; 2003). This peptides coud not bind to the columns during the process being eluted in early fractions or still bounded to column substrate, and this is a possible reason to found that single band in this study.
The 70kDa molecular weight is similar to a calpastatin constructed in a non-fusing porcine skeletal muscle using 1xa promoter and the authors attribute this band to a large proteolitic fragment product of degradation of intact calpastatin containing the N-terminal epitope intact allowing the anti-1xa peptide antibody to detect this large peptide fragment (Parr et al., 2004).
The calpastatin was identified in the second dimension gel in a similar molecular weight to western blot (Figure 4). In the figure 4-A shows all 6 spots from calpastatin peak 1 and figure 4-B five spots sent to identification and the identified spots are the spot number 2, 7 and 8. The peptide sequences are presented in Table 2.
Sequence of peptides identified as part of the inhibitory domain III and IV and C terminus and from purified peak 2 a sequence of peptides identified as part of the inhibitory domain I, II and III (Figure 5). This results lead us to believe that both peaks, in this case, are products of degradation of the intact molecule and probably the small peptides are loosed during the process. This result help us to identify the composition of the peaks during the calpastatin extraction but is not conclusive about the composition, the origin of peaks and the influence in the tenderness development.
CONCLUSIONS
The results of present study shows that is possible the purification of distinct forms of active calpastatin separated using anion exchange column and purifying by sequential chromatography steps, however the intact form of calpastatin was not present in this purification. Presence of peptides was not conclusive to determine the origin and composition of each active peak. More studies are necessary to improve the characterization and the influence in the tenderization process.
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Tables and Figures
10 Figure 1 Calpastatin activity of eluted fractions (Arbitrary units)
5Table 1 – Steps of purification of calpastatin peaks.
Column Calpastatin peak 1
Activity/mL Activity/mg protein Total Activity Lose activity (%)
Q sepharose 1.48 0.69 344.84 -
Phenilsepharose 0.99 164.50 171.738 50.2%
Column Calpastatin peak 2
Activity/mL Activity/mg protein Total Activity Lose activity (%)
Q sepharose 3.89 5.29 1981.86 -
Phenilsepharose 8.84 368.33 1343.68 32.2%
Bluesepharose 6.49 721.11 1298 34.5%
DEAE CAPTO* 34 739.13 714 64.0%
11 Figure 2 – Load check silver stained of initial and final step of calpastatin purification. Lane M) Broad range molecular marker; 1) Pooled fractions of peak 1 activity of calpastatin from Q sepharose column; 2) Pooled fractions of peak 2 activity of calpastatin from Q sepharose column; 3) Pooled fractions of peak 1 activity of calpastatin from Phenil sepharose column; 4) Pooled fractions of peak 2 activity of calpastatin from DEAE CAPTO column.
12 Figure 3 – Western blott stained by calpastatin antibody of purified calpastatin peak 1 (PK1) and calpastatin peak 2 (PK2).
13 Figure 4 – 2D DIGE of calpastatin peaks coomassie stained. A) Calpastatin peak 1; B) calpastatin peak 2. Numbers of collected spots and sent to identification are presented in boxes.
6 Table 2 - Identified spots from calpastatin peaks gels.
Spot
ID Protein Specie Gene Acession pI
Mass
kDa Coverage Identified Peptides 2 Calpastatin Sus
scrofa CAST P12675 5.33 77.1 9.68 KPEAAQDPIDALSGDFDR KLDDALDQLSDSLGQR LDDALDQLSDSLGQR DDTIPPEYR
QPDPDENKPIEDK LGEKEETIPPDYR
7 Calpastatin Sus
scrofa CAST P12675 5.33 77.1 8.7 STGEVLK SLTSSVPAESK SEPELDLSSIK
ESQATAPTPVGEAVSR LSVTGVSAASGKPAETK
8 Calpastatin Sus
scrofa CAST P12675 5.33 77.1 8.84 ESQATAPTPVGEAVSR LSVTGVSAASGKPAETK KSEPELDLSSIK
SLTSSVPAESK
STGEVLK
MNPTETKAIPVSKQLEGPHSPNKKRHKKQAVKTEPEKKSQSTKPSVVHEKKTQEVKP KEHPEPKSLPTHSADAGSKRAHKEKAVSRSNEQPTSEKSTKPKAKPQDPTPSDGKLS
VTGVSAASGKPAETKKDDKSLTSSVPAESKSSKPSGKSDMDAALDDLIDTLGGPEE TEEDNTTYTGPEVLDPMSSTYIEELGKREVTLPPKYRELLDKKEGIPVPPPD TSKPLGP DDA IDALSLDLTCSSPTADGKKTEKEKSTGEVLKAQSVGVIKSAAAPPHEKKRRVEE DTMSDQALEALSASLGSRKSEPELDLSSIKEIDEAKAKEEKLKKCGEDDETVPPEYRL KPAMDKDGKPLLPEAEE KPKPLSESEL IDELSEDFDQSKRKEKQSKPTEKTKESQAT APTPVGEAVSRTSLCCVQSAPPKPATGMVPDDAVEALAGSLGKKEADPEDGKPVED KVKEKAKEEDREKLGEKEETIPPDYRLEEVKDKDGKTLPHKD PKEPVLPLSEDFV L DALSQDFAGPPAASSLFEDAKLSAAVSEVVSQTSAPTTHSAGPPPDTVSDDKKLDDA LDQLSDSLGQRQPDPDENKPIEDKVKEKAEAEHRDKLGERDDTIPPEYRHLLDKD EEGKSTKPPTKK PEAPKKPEAAQDPIDALSGDFDRCPSTTETSENTTKDKDKKTASK SKAPKNGGKAKDSTKAKEETSKQKSDGKSTS
14 Figure 5 – Representative calpastatin molecule aminoacid sequence from Sus scrofa gene: CAST;
713 Aminoacids; Mass (Da):77,124. Inhibitory domains are presented in closed boxes and peptides identified are in uppercase and in yellow are from peak 1 and from peak 2 as identified by grey.