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 synthesis using 9-fluorenylmethyloxycarbonyl group (Fmoc)-protected amino acids as described below11. Use Table 2 to determine the Fmoc-compound used, depending on the desired amino acid and stage of synthesis.
<ol>
	<li>Swill 0.2 g of Fmoc-NH-SAL resin with 10 mL of N,N-dimethylformamide (DMF) in a synthesis column.</li>
	<li>Add 5 mL of 30% piperidine in DMF. Rock the solution for 10 min to cleave the Fmoc-group.</li>
	<li>Check Fmoc cleavage with the trinitrobenzenesulfonic acid (TNBS) test12: Add 10 &#181;L of 1% TNBS in DMF and 10 &#181;L of diisopropylethylamine (DIPEA) to a 10 x 75 mm2 glass tube, then transfer a small amount of resin to the glass tube. The Fmoc-cleaved resin should develop an orange color.
	<ol>
		<li>Repeat steps 1.2-1.3 if this result is negative.</li>
	</ol>
	</li>
	<li>Wash the resin three times with 5 mL of DMF.</li>
	<li>Add 5 mL of DMF containing 0.3 mmol of the Fmoc-compound listed in Table 2 (starting with the compound used in stage 1), 0.3 mmol of O-(6-Chloro-1H-benzotriazol-1-yl)-N,N,N&#39;,N&#39;-tetramethyluronium hexafluorophosphate (HCTU), 0.3 mmol of 1-Hydroxy-1H-benzotriazole, monohydrate (HOBt), and 0.6 mmol of diisopropylethylamine (DIPEA). Rock the solution for 30 min to couple it with the resin.
	<ol>
		<li>Check this step with the TNBS test. If the result is positive, this means the reaction has not sufficiently progressed and this step must be repeated.</li>
	</ol>
	</li>
	<li>Wash the resin three times with 5 mL of DMF.</li>
	<li>Repeat the elongation reaction by repeating steps 1.2-1.6, changing the Fmoc-compound used in step 1.5 according to Table 2.</li>
	<li>Add 1 mL of 2.5% water, 1% triisopropylsilane (TIS), and 2.5% ethanedithiol (EDT) in trifluoroacetic acid to cleave peptides from the resin and protection groups. Rock the solution for 60 min.</li>
	<li>Filter, and collect filtrate in a 50-mL tube.</li>
	<li>Add 40 mL of cold diethyl ethanol and store overnight at -20 &#176;C.</li>
	<li>Centrifuge 4,000 x g at -20 &#176;C for 30 min. Collect the pellet.</li>
	<li>Use a desiccator for 3 h to remove the diethyl ether.</li>
	<li>Add 1 mL of 50% acetonitrile to water and dissolve the crude peptides. Lyophilize the solution to remove trifluoroacetic acid. Repeat this step several times.</li>
	<li>Purify the peptides using a C18 cartridge column.
	<ol>
		<li>Activate the resin in the C18 cartridge column using 3 mL of acetonitrile (ACN).</li>
		<li>Equilibrate with 5 mL of 5% ACN, 0.1% TFA in H2O.</li>
		<li>Load the crude synthetic peptides into the C18 cartridge column.</li>
		<li>Wash with 10 mL of 5% ACN, 0.1% TFA in H2O.</li>
		<li>Elute the samples with 3 mL of 60% ACN, 0.1% TFA in H2O.</li>
	</ol>
	</li>
	<li>Check the peptides by measuring their molecular weight using MALDI-TOF mass spectrometry.
	<ol>
		<li>Pipette 1 &#181;L of eluent on a target plate. Immediately add saturated &#945;-cyano-4-hydroxycinnamic acid (CHCA) solution in 50% acetonitrile in 0.1% trifluoroacetic acid</li>
		<li>Crystallize the mixture by air-drying.</li>
		<li>Acquire mass spectra of samples according to the instrument manufacturer&#39;s protocol.
		<ol>
			<li>Manually set the MALDI-TOF mass spectrometer to a positive reflect mode.</li>
			<li>Calibrate the mass spectrometer using CHCA, bradykinin fragment 1-7 (monoisotopic molecular weight = 756.3997 Da), and angiotensin II (monoisotopic molecular weight = 1,045.5423 Da).</li>
			<li>Acquire the mass spectra of synthetic peptides, and then evaluate by comparing it with the theoretical molecular weight.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Preparation of Tissue Extracts
<ol>
	<li>Anesthetize male ICR mice in an isoflurane inhalation chamber and verify anesthesia depth via toe pinch. Euthanize the mice by cervical dislocation.</li>
	<li>Using scissors, make a midline abdominal incision through the skin extending up to the xiphoid process.</li>
	<li>Cut through the diaphragm and collect all lung tissue. Cut the tissue into small pieces.</li>
	<li>Wash the tissue three times with ice-cold 50 mM Tris-buffered saline (pH 7.4).</li>
	<li>Weigh the tissues and transfer about 100 mg of tissues to a 12 x 75 mm2 glass tube.</li>
	<li>Add 0.3 mL of the extraction buffer (50 mM Tris-buffer, pH 7.4, containing 0.1% poly (oxyethylene) octylphenyl ether).</li>
	<li>Homogenize the lung tissue samples using a mixing homogenizer.
	<ol>
		<li>Cool the samples on ice.</li>
		<li>Homogenize the samples at 20,000 rpm for 30 s in a blender. Repeat this step until the samples are completely homogeneous. 
		NOTE: Do not homogenize for more than 1 minute continuously, in order to avoid an increase in temperature.</li>
	</ol>
	</li>
	<li>Transfer 1,000 &#181;L of the homogenate to a 1.5 mL tube and centrifuge for 5 min at 10,000 x g at 4 &#176;C.</li>
	<li>Transfer 500 &#181;L of the supernatant to a new 1.5 mL plastic tube.</li>
	<li>Use 10 &#181;L of the sample to determine protein concentration, using methods such as the bicinchoninic Acid (BCA) or the Coomassie Brilliant Blue (CBB) method.</li>
	<li>To prevent freezing, add 80% glycerol to the samples for a final concentration of 50% glycerol.</li>
	<li>Store the samples at -80 &#176;C until further use.</li>
</ol>
3. Digestion of Substrates in Model Protease and Tissue Extracts
<ol>
	<li>Add 10 &#181;L of 0.1 &#181;g/&#181;L of N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)-trypsin, 0.1 &#181;g/&#181;L of Staphylococcus aureus V8 protease, or 10 &#181;g/&#181;L of tissue extracts into 890 &#181;L of a digestion buffer.
	<ol>
		<li>To clarify the difference in protease specificity depending on the pH, use buffers adjusted to pH 3, 5, 7, and 9. The composition of the digestion buffer is shown in Table 3.</li>
		<li>Carry out a negative control using inactive protease in tissue extracts, prepared by incubation in boiling water (or heating at 100 &#176;C) for 10 min.</li>
	</ol>
	</li>
	<li>Add 10 &#181;L of iminobiotin-labeled substrates (dissolved in dimethyl sulfoxide (DMSO), final concentration = 100 pmol/10 &#181;L).</li>
	<li>Incubate for 3 hours at 37 &#176;C to digest iminobiotin-labeled substrates.</li>
	<li>After incubation, stop the digestion of substrates with boiling water (or by heating at 100 &#176;C) for 10 min.</li>
	<li>Centrifuge the digested substrates for 5 min at 10,000 x g at 4 &#176;C.</li>
	<li>Transfer the supernatant into a new 1.5 mL tube.</li>
</ol>
4. Purification of the Iminobiotin-labeled Substrate
<ol>
	<li>Add 50 &#181;L of 50% streptavidin sepharose beads in 1 M Tris-buffer (pH 9) containing 0.1% poly(oxyethylene) octylphenyl ether.
	<ol>
		<li>Use pH test paper to ensure that the solution is around pH 9. If the pH of the solution is low, adjust it to approximately pH 9 by adding 1 M Tris-buffer (pH 9) containing 0.1% poly(oxyethylene) octylphenyl ether. This step is important, as it involves binding of the iminobiotin-labeled substrate to streptavidin.</li>
	</ol>
	</li>
	<li>Incubate overnight at 4 &#176;C with continuous gentle mixing on a rotator wheel to bind the iminobiotin-labeled substrates to the streptavidin. The beads should remain suspended throughout the incubation.</li>
	<li>Centrifuge for 1 min at 10,000 x g at 4 &#176;C. Remove the supernatant.</li>
	<li>Wash the beads five times as follows:
	<ol>
		<li>Add 1,000 &#181;L of a wash buffer (50 mM Tris-buffer, pH 9) to the beads.</li>
		<li>Wash for 10 min at 4 &#176;C with continuous gentle mixing on a rotator wheel. The beads should remain suspended throughout the incubation.</li>
		<li>Centrifuge for 1 min at 10,000 x g at 4 &#176;C and remove the supernatant.</li>
	</ol>
	</li>
	<li>Add 100 &#181;L of 0.2% trifluoroacetic acid to the beads.
	<ol>
		<li>Use pH test paper to ensure that the solution is below pH 2. If the pH of the solution is high, adjust the pH to below 2 by adding 1% trifluoroacetic acid.</li>
	</ol>
	</li>
	<li>Incubate for 30 min at room temperature with continuous gentle mixing on a rotator wheel.</li>
	<li>Centrifuge for 1 min at 10,000 x g at 4 &#176;C, then transfer the supernatant to a new 1.5 mL tube.</li>
</ol>
5. Sample Preparation and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF Mass Spectrometry)
NOTE: All solutions in the following steps should be prepared with high performance liquid chromatography (HPLC)-grade or amino acid sequencing-grade water.
<ol>
	<li>Pipette 500 &#181;L of acetonitrile onto a C18 resin to activate it. Remove the used acetonitrile. Repeat this step three times.</li>
	<li>Equilibrate the C18 resin with 100 &#181;L of 0.1% trifluoroacetic acid. Remove the eluent. Repeat this step three times.</li>
	<li>To load the samples, bind the samples to the resin using a pipette.</li>
	<li>Wash the resin with 20 &#181;L of 0.1% trifluoroacetic acid. Repeat this step five times.</li>
	<li>Elute the samples with 3 &#181;L of 60% acetonitrile in 0.1% trifluoroacetic acid. Pipette the eluent on a target plate for MALDI-TOF mass spectrometry. Immediately add saturated &#945;-cyano-4-hydroxycinnamic acid (CHCA) solution in 50% acetonitrile in 0.1% trifluoroacetic acid.</li>
	<li>Crystallize the mixture by air-drying.</li>
	<li>Acquire mass spectra of samples according to instrument manufacturer&#39;s protocol.
	<ol>
		<li>Manually set the MALDI-TOF mass spectrometer to a positive reflect mode.</li>
		<li>Calibrate the mass spectrometer using CHCA, bradykinin fragment 1-7 (monoisotopic molecular weight = 756.3997 Da), and angiotensin II (monoisotopic molecular weight = 1,045.5423 Da).</li>
		<li>Acquire the mass spectra of samples, paying close attention to the mass spectra from 700 to 900 Da for the cleaved form of the biotin-labeled substrates.</li>
	</ol>
	</li>
	<li>Identify the detected peaks using Table 1. Detection of the appropriate molecular weight indicates cleavage at the C-terminus of the relevant amino acid.</li>
</ol>

<ol>
	<li>Neurath, H., Walsh, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+proteolytic+enzymes+in+biological+regulation+(a+review).">Role of proteolytic enzymes in biological regulation (a review).</a> Proc Natl Acad Sci U S A. 73 (11), 3825-3832 (1976).</li><li>Surinov, B. P., Manoilov, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+activity+of+proteolytic+enzymes+by+means+of+azocasein.">Determination of the activity of proteolytic enzymes by means of azocasein.</a> Vopr Med Khim. 11 (5), 55-58 (1965).</li><li>Fernandez-Resa, P., Mira, E., Quesada, A. 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. L., 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=Identification+of+protease+specificity+by+combining+proteome-derived+peptide+libraries+and+quantitative+proteomics.">Identification of protease specificity by combining proteome-derived peptide libraries and quantitative proteomics.</a> Mol Cell Proteomics. 15 (7), 2515-2524 (2016).</li><li>Yamamoto, H., Saito, S., Sawaguchi, Y., Kimura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+protease+specificity+using+biotin-labeled+substrates.">Identification of protease specificity using biotin-labeled substrates.</a> Open Biochem J. 11, 27-35 (2017).</li><li>Hofmann, K., Wood, S. W., Brinton, C. C., Montibeller, J. A., Finn, F. 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. T., 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=Recombinant+NeutraLite+avidin:+A+non-glycosylated,+acidic+mutant+of+chicken+avidin+that+exhibits+high+affinity+for+biotin+and+low+non-specific+binding+properties.">Recombinant NeutraLite avidin: A non-glycosylated, acidic mutant of chicken avidin that exhibits high affinity for biotin and low non-specific binding properties.</a> FEBS Lett. 467 (1), 31-36 (2000).</li><li>Gravel, R. A., Lam, K. F., Mahuran, D., Kronis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+human+liver+propionyl-CoA+carboxylase+by+carbon+tetrachloride+extraction+and+monomeric+avidin+affinity+chromatography.">Purification of human liver propionyl-CoA carboxylase by carbon tetrachloride extraction and monomeric avidin affinity chromatography.</a> Arch Biochem Biophys. 201 (2), 669-673 (1980).</li></ol>

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 ...

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JoVE Journal

Biochemistry

6.7K Views.  Nihon Pharmaceutical University. Here, we present a protocol to determine protease specificity in crude mouse tissue extracts using MALDI-TOF spectrometry.

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

Species Determination and Quantitation in Mixtures Using MRM Mass Spectrometry of Peptides Applied to Meat Authentication

We describe a simple protocol for identifying and quantifying the two components in binary mixtures of species possessing one or more similar proteins. Central to the method is the identification of 'corresponding proteins' in the species of interest, in other words proteins that are nominally the same but possess species-specific sequence differences. When subject to proteolysis, corresponding proteins will give rise to some peptides which are likewise similar but with species-specific variants. These are 'corresponding peptides'. Species-specific peptides can be used as markers for species determination, while pairs of corresponding peptides permit relative quantitation of two species in a mixture. The peptides are detected using multiple reaction monitoring (MRM) mass spectrometry, a highly specific technique that enables peptide-based species determination even in complex systems. In addition, the ratio of MRM peak areas deriving from corresponding peptides supports relative quantitation. Since corresponding proteins and peptides will, in the main, behave similarly in both processing and in experimental extraction and sample preparation, the relative quantitation should remain comparatively robust. In addition, this approach does not need the standards and calibrations required by absolute quantitation methods. The protocol is described in the context of red meats, which have convenient corresponding proteins in the form of their respective myoglobins. This application is relevant to food fraud detection: the method can detect 1% weight for weight of horse meat in beef. The corresponding protein, corresponding peptide (CPCP) relative quantitation using MRM peak area ratios gives good estimates of the weight for weight composition of a horse plus beef mixture.

We present a protocol for identifying and quantifying the components in mixtures of species possessing similar proteins. Mass spectrometry detects peptides for identification, and gives relative quantitation by ratios of peak areas. As a tool food for fraud detection, the method can detect 1% horse in beef.

We describe a simple protocol for identifying and quantifying the two components in binary mixtures of species possessing one or more similar proteins. ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Analysis of Histone Antibody Specificity with Peptide Microarrays

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The mass production and distribution of antibodies specific to histone PTMs has greatly facilitated research on these marks. As histone PTM antibodies are key reagents for many chromatin biochemistry applications, rigorous analysis of antibody specificity is necessary for accurate data interpretation and continued progress in the field. This protocol describes an integrated pipeline for the design, fabrication and use of peptide microarrays for profiling the specificity of histone antibodies. The design and analysis aspects of this procedure are facilitated by ArrayNinja, an open-source and interactive software package we recently developed to streamline the customization of microarray print formats. This pipeline has been used to screen a large number of commercially available and widely used histone PTM antibodies, and data generated from these experiments are freely available through an online and expanding Histone Antibody Specificity Database. Beyond histones, the general methodology described herein can be applied broadly to the analysis of PTM-specific antibodies.

This manuscript describes methods for applying peptide microarray technology to specificity profiling of antibodies that recognize histones and their post-translational modifications.

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of reactive oxygen species, and more. These fluctuations can lead to a widespread protein unfolding, aggregation, and cell death. Therefore, cells have evolved a dynamic and stress-specific network of molecular chaperones, which maintain a "healthy" proteome during stress conditions. ATP-independent chaperones constitute one major class of molecular chaperones, which serve as first-line defense molecules, protecting against protein aggregation in a stress-dependent manner. One feature these chaperones have in common is their ability to utilize structural plasticity for their stress-specific activation, recognition, and release of the misfolded client. 
In this paper, we focus on the functional and structural analysis of one such intrinsically disordered chaperone, the bacterial redox-regulated Hsp33, which protects proteins against aggregation during oxidative stress. Here, we present a toolbox of diverse techniques for studying redox-regulated chaperone activity, as well as for mapping conformational changes of the chaperone, underlying its activity. Specifically, we describe a workflow which includes the preparation of fully reduced and fully oxidized proteins, followed by an analysis of the chaperone anti-aggregation activity in vitro using light-scattering, focusing on the degree of the anti-aggregation activity and its kinetics. To overcome frequent outliers accumulated during aggregation assays, we describe the usage of Kfits, a novel graphical tool which allows easy processing of kinetic measurements. This tool can be easily applied to other types of kinetic measurements for removing outliers and fitting kinetic parameters. To correlate the function with the protein structure, we describe the setup and workflow of a structural mass spectrometry technique, hydrogen-deuterium exchange mass spectrometry, that allows the mapping of conformational changes on the chaperone and substrate during different stages of Hsp33 activity. The same methodology can be applied to other protein-protein and protein-ligand interactions.

One of the most challenging stress conditions that organisms encounter during their lifetime involves the accumulation of oxidants. During oxidative stress, cells heavily rely on molecular chaperones. Here, we present methods used to investigate the redox-regulated anti-aggregation activity, as well as to monitor structural changes governing the chaperone function using HDX-MS.

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of ...

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have developed a simple non-labeled and non-radio-isotopic method using a matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis for a proofreading study. Here, a DNA polymerase [e.g., the Klenow fragment (KF) of Escherichia coli DNA polymerase I (pol I) in this study] in the presence of all four dideoxyribonucleotide triphosphates is used to process a mismatched primer-template duplex. The mismatched primer is then proofread/extended and subjected to MALDI-TOF MS. The products are distinguished by the mass change of the primer down to single nucleotide variations. Importantly, a proofreading can also be determined for internal single mismatches, albeit at different efficiencies. Mismatches located at 2-4-nucleotides (nt) from the 3' end were efficiently proofread by pol I, and a mismatch at 5 nt from the primer terminus showed only a partial correction. No proofreading occurred for internal mismatches located at 6 - 9 nt from the primer 3' end. This method can also be applied to DNA repair assays (e.g., assessing a base-lesion repair of substrates for the endo V repair pathway). Primers containing 3' penultimate deoxyinosine (dI) lesions could be corrected by pol I. Indeed, penultimate T-I, G-I, and A-I substrates had their last 2 dI-containing nucleotides excised by pol I before adding a correct ddN 5'-monophosphate (ddNMP) while penultimate C-I mismatches were tolerated by pol I, allowing the primer to be extended without repair, demonstrating the sensitivity and resolution of the MS assay to measure DNA repair.

A non-labeled, non-radio-isotopic method for DNA polymerase proofreading and a DNA repair assay was developed by using high-resolution MALDI-TOF mass spectrometry and a single nucleotide extension strategy. The assay proved to be very specific, simple, rapid, and easy to perform for proofreading and repair patches shorter than 9-nucleotides.

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...