This work was supported in part by the Nihon Pharmaceutical University Research Grant from Nihon Pharmaceutical University (2016) and the MEXT KAKENHI Grant Number JP17854179.

Animal experimental protocols were approved by the Nihon Pharmaceutical University&#39;s ethics committee and performed in accordance with the guidelines for the Care and Use of Laboratory Animals of the Nihon Pharmaceutical University. The protocol has been adjusted for tissue extracts derived from ICR&#160;mice. ICR mice were purchased from Nippon SLC Ltd. (Shizuoka, Japan)
1. Preparation of the Iminobiotin-labeled Substrate
The substrate undergoes solid-phase synth...

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+detection+of+casein+zymography+of+matrix+metalloproteinases.">Enhanced detection of casein zymography of matrix metalloproteinases.</a> Anal Biochem. 224 (1), 434-435 (1995).</li><li>Erlanger, B. F., Kokowsky, N., Cohen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+preparation+and+properties+of+two+new+chromogenic+substrates+of+trypsin.">The preparation and properties of two new chromogenic substrates of trypsin.</a> Arch Biochem Biophys. 95, 271-278 (1961).</li><li>Smith, R. E., Bissell, E. R., Mitchell, A. R., Pearson, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+photometric+or+fluorometric+assay+of+proteinases+using+substrates+containing+7-amino-4-trifluoromethylcoumarin.">Direct photometric or fluorometric assay of proteinases using substrates containing 7-amino-4-trifluoromethylcoumarin.</a> Thromb Res. 17 (3-4), 393-402 (1980).</li><li>Latt, S. A., Auld, D. S., Vallee, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescende+determination+of+carboxypeptidase+A+activity+based+on+electronic+energy+transfer.">Fluorescende determination of carboxypeptidase A activity based on electronic energy transfer.</a> Anal Biochem. 50 (1), 56-62 (1972).</li><li>Timmer, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+constitutive+proteolytic+events+in+vivo.">Profiling constitutive proteolytic events in vivo.</a> Biochem J. 407 (1), 41-48 (2007).</li><li>Biniossek, M. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iminobiotin+affinity+columns+and+their+application+to+retrieval+of+streptavidin.">Iminobiotin affinity columns and their application to retrieval of streptavidin.</a> Proc Natl Acad Sci U S A. 77 (8), 4666-4668 (1980).</li><li>Merrifield, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid+Phase+Peptide+Synthesis.+I.+The+synthesis+of+a+tetrapeptide.">Solid Phase Peptide Synthesis. I. The synthesis of a tetrapeptide.</a> J. Am. Chem. Soc. 85, 2149-2154 (1963).</li><li>Hancock, W. S., Battersby, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+micro-test+for+the+detection+of+incomplete+coupling+reactions+in+solid-phase+peptide+synthesis+using+2,4,6-trinitrobenzenesulphonic+acid.">A new micro-test for the detection of incomplete coupling reactions in solid-phase peptide synthesis using 2,4,6-trinitrobenzenesulphonic acid.</a> Anal. Biochem. 71, 260-264 (1976).</li><li>Marttila, A. 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The authors declare no conflict of interest.

This protocol uses MALDI-TOF mass spectrometry to identify protease specificity in crude samples derived from tissue extracts and may easily be scaled up for multiple sample processing. In particular, we manipulated pH to cause a change in substrate specificity.
The avidin-biotin complex (ABC) method, which has been widely used in biochemistry due to its binding specificity, was utilized in our protocol. Due to the strong affinity of ABC, the binding of avidin to biotin is almost irreversible....

Proteases regulate physiological processes by cleaving proteins, and the initiation of protease activity at specific times and locations is regulated1. It is therefore important to identify the expression and activity of tissues that are locally regulated, and to develop a novel method to detect proteases. The method proposed in this study aims to identify protease specificity in parallel to detecting proteases.
There are some methods for detecting protease specificity. The azocasein method is well-known for its use in the detection of proteases2 but is limited in its ability to obtain further information. Zymography is another comprehensive protease detection method that can be used to determine the molecular weight of proteases3, but it cannot be used to examine substrate specificity. Protease specificity can be determined by spectrometric assays, using substrates such as p-nitroanilide4, and fluorometric assays, using coumarin substrates5 or Fluorescence Resonance Energy Transfer (FRET) assays6. These methods enable single-specificity detection of substrates contained in a single well. In the present study, the protease specificity of tissue extracts is examined under various conditions, as these extracts contain multiple proteases. Protease activity is regulated by several factors, including pH, coenzymes, prosthetic groups, and ion strength. Recently, proteomics-based methods have been used to identify protease specificity; for example, the method reported by Biniossek et al.7 uses the proteome to determine substrate specificity even in crude extracts and allows accurate determination of the cleavage activity of proteases that recognize multiple amino acid sequences8. However, this method is not suitable for the analysis of numerous samples. In contrast, our method enables the simultaneous processing of multiple samples, and the use of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry for sample analysis facilitates rapid and easy detection of the cleaved substrates.
This paper outlines a method to evaluate protease specificity by using MALDI-TOF mass spectrometry to measure the molecular weight of unique substrates. The molecular forms of cleaved substrates, along with their theoretical molecular weights, are shown in Figure 1 and Table 1. Previous studies have used substrates containing polyglycine spacers and biotin9; however, these substrates are flawed because the polyglycine sequence may be cleaved by enzymes that recognize the glycine sequence. Additionally, the high affinity between avidin and biotin may lead to a low recovery rate. To improve upon these drawbacks, in this study we synthesized a unique substrate, which was composed of polyethylene glycol (PEG), iminobiotin, and an amino acid that could identify the cleavage site (Figure 1). To discriminate between amino acids of similar molecular weights, D-serine was added between the PEG spacer and the amino acid at the cleaved site.
The N-terminal of the substrate was labeled with iminobiotin, which enables affinity purification from crude samples. The use of iminobiotin instead of biotin is critical; biotin has a strong affinity with avidin, which results in low recovery rates of biotin-labeled substrates from avidin resin, while iminobiotin&#39;s affinity for avidin can be altered by pH. Iminobiotin-labeled substrates will bind to avidin in conditions above pH 9, while avidin releases iminobiotin-labeled substrates in conditions below pH 4. Therefore, iminobiotin was used for affinity purification10. In summary, we describe a detailed protocol for detecting protease specificity using unique substrates.

<table>
	<tbody>
		<tr>
			<td>Fmoc-NH-SAL Resin(-[2,4-Dimethoxyphenyl-N-(9-fluorenylmethoxycarbonyl)aminomethyl]phenoxy resin)</td>
			<td>Watanabe Chemical Co., Ltd.</td>
			<td>A00102</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-dimethylformamide (DMF)</td>
			<td>Watanabe Chemical Co., Ltd.</td>
			<td>A00185</td>
			<td></td>
		</tr>
		<tr>
			<td>piperidine</td>
			<td>Watanabe Chemical Co., Ltd.</td>
			<td>A00176</td>
			<td></td>
		</tr>
		<tr>
			<td>trinitrobenzenesulfonic acid (TNBS)</td>
			<td>WAKO Chemical</td>
			<td>209-1483</td>
			<td></td>
		</tr>
		<tr>
			<td>O-(6-Chloro-1H-benzotriazol-1-yl)-N,N,N&#39;,N&#39;-tetramethyluronium hexafluorophosphate (HCTU)</td>
			<td>Watanabe Chemical Co., Ltd.</td>
			<td>A00067</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Hydroxy-1H-benzotriazole,monohydrate (HOBt)</td>
			<td>Watanabe Chemical Co., Ltd.</td>
			<td>A00014</td>
			<td></td>
		</tr>
		<tr>
			<td>diisopropylethylamine (DIPEA)</td>
			<td>Watanabe Chemical Co., Ltd.</td>
			<td>A00030</td>
			<td></td>
		</tr>
		<tr>
			<td>triisopropylsilane (TIS)</td>
			<td>Watanabe Chemical Co., Ltd.</td>
			<td>A00170</td>
			<td></td>
		</tr>
		<tr>
			<td>ethanedithiol (EDT)</td>
			<td>Watanabe Chemical Co., Ltd.</td>
			<td>A00057</td>
			<td></td>
		</tr>
		<tr>
			<td>trifluoroacetic acid (TFA)</td>
			<td>Millipore</td>
			<td>S6612278 403</td>
			<td></td>
		</tr>
		<tr>
			<td>acetonitrile</td>
			<td>Kokusan Chemical</td>
			<td>2153025</td>
			<td></td>
		</tr>
		<tr>
			<td>C18 cartridge column</td>
			<td>Waters</td>
			<td>WAT051910</td>
			<td></td>
		</tr>
		<tr>
			<td>poly(oxyethylene) octylphenyl ether</td>
			<td>WAKO Chemical</td>
			<td>168-11805</td>
			<td>Triton X-100</td>
		</tr>
		<tr>
			<td>mixing homogenizer</td>
			<td>Kinematica</td>
			<td>PT3100</td>
			<td>polytron homogenizer</td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue (CBB) -G250</td>
			<td>Nakarai Chemical</td>
			<td>094-09</td>
			<td></td>
		</tr>
		<tr>
			<td>glycerol</td>
			<td>Nakarai Chemical</td>
			<td>170-18</td>
			<td></td>
		</tr>
		<tr>
			<td>N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)-trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>T1426</td>
			<td></td>
		</tr>
		<tr>
			<td>dimethyl sulfoxide (DMSO)</td>
			<td>WAKO Chemical</td>
			<td>043-07216</td>
			<td></td>
		</tr>
		<tr>
			<td>streptavidin sepharose</td>
			<td>GE Healthcare</td>
			<td>17-511-01</td>
			<td></td>
		</tr>
		<tr>
			<td>ZipTipC18</td>
			<td>Millipore</td>
			<td>ZTC18S096</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-cyano-4-hydroxycinnamic acid (CHCA)</td>
			<td>Sigma-Aldrich</td>
			<td>C2020</td>
			<td></td>
		</tr>
		<tr>
			<td>MALDI-TOF mass spectrometry</td>
			<td>Bruker Daltonics, Germany</td>
			<td>autoflex</td>
			<td></td>
		</tr>
		<tr>
			<td>2-iminobiotin</td>
			<td>Sigma-Aldrich</td>
			<td>I4632</td>
			<td></td>
		</tr>
		<tr>
			<td>Fmoc-amino acids</td>
			<td>Watanabe Chemical Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

the determination of protease specificity in mouse tissue extracts by maldi-tof mass spectrometry: manipulating ph to cause specificity changes

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important for revealing protease function. The method proposed in this study determines protease specificity by measuring the molecular weight of unique substrates using Matrix-assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry. The substrates contain iminobiotin, while the cleaved site consists of amino acids, and the spacer consists of polyethylene glycol. The cleaved substrate will generate a unique molecular weight using a cleaved amino acid. One of the merits of this method is that it may be carried out in one pot using crude samples, and it is also suitable for assessing multiple samples. In this article, we describe a simple experimental method optimized with samples extracted from mouse lung tissue, including tissue extraction, placement of digestive substrates into samples, purification of digestive substrates under different pH conditions, and measurement of the substrates&#39; molecular weight using MALDI-TOF mass spectrometry. In summary, this technique allows for the identification of protease specificity in crude samples derived from tissue extracts using MALDI-TOF mass spectrometry, which may easily be scaled up for multiple sample processing.

Evaluation of the method for identification of protease specificity by model protease
The efficiency of this system was evaluated using TPCK-trypsin and V8 protease, whose substrate specificities were obtained. TPCK-trypsin and V8 protease were estimated to cleave the amino acid sequence at the C-terminal of the lysine and arginine residues, and at the carboxy side of the aspartic acid and glutamic acid residues, respectively. <str...

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The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes

Here, we present a protocol to determine protease specificity in crude mouse tissue extracts using MALDI-TOF spectrometry.

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important ...

The overall goal of this experiment, is to determine protease specificity in multiple crude extracts. This method can help answer key questions to determine protease specificity. The main advantage of this technique, is that it can identify protease specificity of multiple samples in parlay.Cool lung tissue samples obtained from male ICR mice in ice. Homogenize the samples at 20, 000 RMP for 30 seconds in a blender. Repeat this step until the samples are completely homogenous.Do not homogenize for more than one minute continuously in order to avoid an increase in temperature. Transfer 1, 000 microliters of the homogenate to a 1.5 milliliter tube and centrifuge for five minutes at 10, 000 times G at four degrees Celsius. Transfer 500 microliters of the supernatant to a new 1.5 milliliter plastic tube.Use 10 microliters of the sample to determine the protein concentration. Using methods, such as the bicinchoninic acid assay or the Bradford assay. To prevent freezing, add 80%glycerol to the samples for a final concentration of 50%glycerol.To clarify the difference in protease specificity depending on the pH, use digestion buffers adjusted to pH three, five, seven, and nine. Into 890 microliter of a digestion buffer, add either 10 microliters of 0.1 microgram per microliter or TPCK trypsin, 0.1 microgram per microliter of staphylococcus aureus V8 protease, or 10 micrograms per microliter of tissue extracts. Prepare negative controls by inactivating protease in tissue extracts by incubation and boiling water for 10 minutes.Next, add 10 microliters of MNO biotin label substrates to the samples and incubate for three hours at 37 degrees Celsius to digest. After incubation, stop the digestion of substrates with boiling water for 10 minutes. Centrifuge the digested substrates for five minutes at 10, 000 times G at four degrees Celsius.Transfer the supernatant into a new 1.5 milliliter tube. Add 50 milliliter of 50%streptavidin sepharose beads in one molar tris buffer, containing 0.1%polyoxyethylene octyl phenyl ether. Use pH test paper to ensure that the solution is around pH nine.It is important to adjust the proper pH to nine or more. Amino biotin cannot bite a beading unless it is pH nine or higher. If the pH of the solution is low, adjust it to approximately pH nine by adding more buffer.Additionally one molar tris buffer can be added to bring up the pH if needed. Incubate the suspension overnight at four degrees Celsius with continuous gentle mixing on a rotator wheel to bind the MNO biotin labeled substrates to the streptavidin. The beads should remain suspended throughout the incubation.The following day, centrifuge the samples for one minute at 10, 000 times G at four degrees Celsius. After removing the supernatant, wash the beads by adding 1, 000 microliters of a wash buffer. Then, incubate for 10 minutes at four degrees Celsius with continuous gentle mixing on a rotator.Centrifuge the beads for one minute at 10, 000 times G at four degrees Celsius. Remove the supernatant and repeat this wash four times. Next, add 100 microliters of 0.2%trifluoroacetic acid to the beads.Use pH test paper to ensure that the solution is below pH two. If the pH of the solution is high, adjust the pH to below two by adding 1%trifluoroacetic acid. Incubate the suspension for 30 minutes at room temperature with continuous gentle mixing on a rotator wheel.After centrifuging the beads as before, transfer the supernatant to a new 1.5 milliliter tube. Pipet 20 microliters of acetonitrile onto a C18 resin to activate it. Remove the used acetonitrile and repeat this activation step three times.Then, equilibrate the C18 resin with 20 microliters of 0.1%trifluoroacetic acid. Remove the eluant and repeat this equilibration step three times. to load the samples, bind them to the resin using a pipet.Wash the resin with 20 microliters of 0.1%trifluoroacetic acid. Repeat this step five times. Elute the samples with three microliters of 60%acetonitrile in 0.1%trifluoroacetic acid.Pipet the eluant on a target plate for MALDI-TOF mass spectromitry. Immediately add CHCA solution, saturated in 50%acetonitrile in 0.1%trifluoroacetic acid. Crystallize the mixture by air drying.Acquire mass spectra of samples, according to the instrument manufacturer's protocol, paying close attention to the mass spectra from 700 to 900 daltons for the cleaved form of the biotin labels substrates. Mass spectra of synthetic substrates are shown here. Peaks corresponding to the precursor molecular weight were detected between 850 and 1, 050.The cleaved fragments are detected in the range of 700 to 850. Shown here are the spectra for MNO biotin labeled substrates treated with TPCK trypsin. The 839.81 dalton and 796.76 dalton peaks correspond to the fragments cleaved from the C terminus of lysine and arginine respectively.Shown here are the mass spectra of synthetic substrates digested with V8 protease. The 769.78 dalton and 755.62 dalton peaks correspond to the fragments cleaved from the C terminus of glutamic acid and aspartic acid respectively. The spectra for MNO biotin labels substrates treated with a lung tissue extract at various pH conditions are shown here.In the acidic condition, at pH five, the molecular weight of a cleaved fragment was 770.13 daltons and 826.66 daltons, indicating cleavage at the C terminus of glutamic acid and tryptophan. In the neutral and basic condition, at pH seven and nine, the molecular weight of a cleaved fragment was increased at the peak of 767 daltons and 840 daltons, indicating cleaved at the C terminus of arginine and lysine. Conversely, the peaks of the glutamic acid and tryptophan were decreased.In addition, this method cannot detect cleaved fragments with inactivated extracts. Once mastered, this technique can be done in two days if it is performed properly, except for the synthesis of substrate. Additionally, once the substrate is synthesized, it can be measured in several samples.Why?Attempting this procedure, it's important to remember to operate at the buffer pH, about nine. Generally, the reaction of the avidin amino biotin complex. Following this procedure, it is possible to quantify enzyme activity by performing a CMS.This method will allow researchers in the proteon field to pave the way for exploring protease expression in several conditions.

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6.7K Views.  Nihon Pharmaceutical University. The overall goal of this experiment, is to determine protease specificity in multiple crude extracts. This method can help answer key questions to determine protease specificity. The main advantage of this technique, is that it can identify protease specificity of multiple samples in parlay.Cool lung tissue samples obtained from male ICR mice in ice. Homogenize the samples at 20, 000 RMP for 30 seconds in a blender. Repeat this step until the samples are completely homogenous.Do...