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)&lt;br/&gt;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&#039;,N&#039;-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>&amp;alpha;-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...

Watch this Scientific Journal Video about The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes at JoVE.com

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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Universidad de Cartagena UDEC

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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

Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass determination allows one to evaluate the correctness of protein primary sequences, the presence of mutations and/or post-translational modifications, the possible protein degradation, the sample homogeneity, and the degree of isotope incorporation in case of labelling (e.g. 13C labelling).
Electrospray ionization (ESI) mass spectrometry (MS) is widely used for mass determination of denatured proteins, but its efficiency is affected by the composition of the sample buffer. In particular, the presence of salts, detergents, and contaminants severely undermines the effectiveness of protein analysis by ESI-MS. Matrix-assisted laser desorption/ionization (MALDI) MS is an attractive alternative, due to its salt tolerance and the simplicity of data acquisition and interpretation. Moreover, the mass determination of large heterogeneous proteins (bigger than 100 kDa) is easier by MALDI-MS due to the absence of overlapping high charge state distributions which are present in ESI spectra.
Here we present an accessible approach for analyzing proteins larger than 100 kDa by MALDI-time of flight (TOF). We illustrate the advantages of using a mixture of two matrices (i.e. 2,5-dihydroxybenzoic acid and &#945;-cyano-4-hydroxycinnamic acid) and the utility of the thin layer method as approach for sample deposition. We also discuss the critical role of the matrix and solvent purity, of the standards used for calibration, of the laser energy, and of the acquisition time. Overall, we provide information necessary to a novice for analyzing intact proteins larger than 100 kDa by MALDI-MS.

Matrix-assisted Laser Desorption/Ionization Time of Flight MALDI-TOF Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Accurate mass measurement represents an important step during the investigation of proteins. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) can be used for such determination and its key advantage is the tolerance to salts, detergents and contaminants. Here we illustrate an accessible approach for the analysis of proteins larger than 100 kDa by MALDI-MS.

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass ...

Chemistry

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 (Fig.1). A 12 &mu;m thin tissue section is covered with a MALDI matrix, which facilitates desorption and ionization of intact peptides and proteins that can be detected with a mass analyzer, typically using a MALDI TOF/TOF mass spectrometer. Generally hundreds of peaks can be assessed in a single rat brain tissue section. In contrast to commonly used imaging techniques, this approach does not require prior knowledge of the molecules of interest and allows for unsupervised and comprehensive analysis of multiple molecular species while maintaining high molecular specificity and sensitivity2. Here we describe a MALDI IMS based approach for elucidating region-specific distribution profiles of neuropeptides in the rat brain of an animal model Parkinson's disease (PD). 
PD is a common neurodegenerative disease with a prevalence of 1% for people over 65 of age3,4. The most common symptomatic treatment is based on dopamine replacement using L-DOPA5. However this is accompanied by severe side effects including involuntary abnormal movements, termed L-DOPA-induced dyskinesias (LID)1,3,6. One of the most prominent molecular change in LID is an upregulation of the opioid precursor prodynorphin mRNA7. The dynorphin peptides modulate neurotransmission in brain areas that are essentially involved in movement control7,8. However, to date the exact opioid peptides that originate from processing of the neuropeptide precursor have not been characterized. Therefore, we utilized MALDI IMS in an animal model of experimental Parkinson's disease and L-DOPA induced dyskinesia. 
MALDI imaging mass spectrometry proved to be particularly advantageous with respect to neuropeptide characterization, since commonly used antibody based approaches targets known peptide sequences and previously observed post-translational modifications. By contrast MALDI IMS can unravel novel peptide processing products and thus reveal new molecular mechanisms of neuropeptide modulation of neuronal transmission. While the absolute amount of neuropeptides cannot be determined by MALDI IMS, the relative abundance of peptide ions can be delineated from the mass spectra, giving insights about changing levels in health and disease. In the examples presented here, the peak intensities of dynorphin B, alpha-neoendorphin and substance P were found to be significantly increased in the dorsolateral, but not the dorsomedial, striatum of animals with severe dyskinesia involving facial, trunk and orolingual muscles (Fig. 5). Furthermore, MALDI IMS revealed a correlation between dyskinesia severity and levels of des-tyrosine alpha-neoendorphin, representing a previously unknown mechanism of functional inactivation of dynorphins in the striatum as the removal of N-terminal tyrosine reduces the dynorphin's opioid-receptor binding capacity9. This is the first study on neuropeptide characterization in LID using MALDI IMS and the results highlight the potential of the technique for application in all fields of biomedical research.

Dopamine replacement pharmacotherapy using L-DOPA is the most commonly used symptomatic treatment of Parkinson&#x2019;s disease, but is accompanied by side effects including involuntary abnormal movements, termed dyskinesia 1. Here, a protocol for MALDI imaging mass spectrometry is presented that detects changes in rat brain neuropeptide levels related to dyskinesia.

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 ...

Medicine

Detection of Protease Activity by Fluorescent Peptide Zymography

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using electrophoresis through a resolving gel embedded with a degradable substrate. This method differs from traditional gel zymography in that a quenched fluorogenic peptide is covalently incorporated into the resolving gel instead of full length proteins, such as gelatin or casein. Use of the fluorogenic peptides enables direct detection of proteolytic activity without additional staining steps. Enzymes within the biological samples cleave the quenched fluorogenic peptide, resulting in an increase in fluorescence. The fluorescent signal in the gels is then imaged with a standard fluorescent gel scanner and quantified using densitometry. The use of peptides as the degradable substrate greatly expands the possible proteases detectable with zymographic techniques.

Here, we present a detailed protocol for a modified zymographic technique in which fluorescent peptides are used as the degradable substrate in place of native proteins. Electrophoresis of biological samples in fluorescent peptide zymograms enables detection of a wider range of proteases than previous zymographic techniques.

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using ...

Cancer Research

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis&#8212;in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before&#8212;and provide some representative results.

Here we present a protocol to perform polysome profiling on the isolated perfused mouse heart. We describe methods for heart perfusion, polysome profiling, and analysis of the polysome fractions with respect to mRNAs, miRNAs, and the polysome proteome.

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

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 ...

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, including RNA processing, signal transduction, DNA damage response, miRNA biogenesis, and translation.
In recent years, thanks to biochemical and analytical developments, mass spectrometry (MS)-based proteomics has emerged as the most effective strategy to characterize the cellular methyl-proteome with single-site resolution. However, identifying and profiling in vivo protein R-methylation by MS remains challenging and error-prone, mainly due to the substoichiometric nature of this modification and the presence of various amino acid substitutions and chemical methyl-esterification of acidic residues that are isobaric to methylation. Thus, enrichment methods to enhance the identification of R-methyl-peptides and orthogonal validation strategies to reduce False Discovery Rates (FDR) in methyl-proteomics studies are required.
Here, a protocol specifically designed for high-confidence R-methyl-peptides identification and quantitation from cellular samples is described, which couples metabolic labeling of cells with heavy isotope-encoded Methionine (hmSILAC) and dual protease in-solution digestion of whole cell extract, followed by off-line High-pH Reversed Phase (HpH-RP) chromatography fractionation and affinity enrichment of R-methyl-peptides using anti-pan-R-methyl antibodies. Upon high-resolution MS analysis, raw data are first processed with the MaxQuant software package and the results are then analyzed by hmSEEKER, a software designed for the in-depth search of MS peak pairs corresponding to light and heavy methyl-peptide within the MaxQuant output files.

Protein Arginine (R)-methylation is a wide-spread post-translational modification regulating multiple biological pathways. Mass spectrometry is the best technology to globally profile the R-methyl-proteome, when coupled to biochemical approaches for modified peptide enrichment. The workflow designed for the high confidence identification of global R-methylation in human cells is described here.

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, ...

Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis

This protocol identifies the immunity proteins of the bactericidal enzymes: colicin E3 and bacteriocin, produced by a pathogenic Escherichia coli strain using antibiotic induction, and identified by MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. The immunity protein of colicin E3 (Im3) and the immunity protein of bacteriocin (Im-Bac) were identified from prominent b- and/or y-type fragment ions generated by the polypeptide backbone cleavage (PBC) on the C-terminal side of aspartic acid, glutamic acid, and asparagine residues by the aspartic acid effect fragmentation mechanism. The software rapidly scans in silico protein sequences derived from the whole genome sequencing of the bacterial strain. The software also iteratively removes amino acid residues of a protein sequence in the event that the mature protein sequence is truncated. A single protein sequence possessed mass and fragment ions consistent with those detected for each immunity protein. The candidate sequence was then manually inspected to confirm that all detected fragment ions could be assigned. The N-terminal methionine of Im3 was post-translationally removed, whereas Im-Bac had the complete sequence. In addition, we found that only two or three non-complementary fragment ions formed by PBC are necessary to identify the correct protein sequence. Finally, a promoter (SOS box) was identified upstream of the antibacterial and immunity genes in a plasmid genome of the bacterial strain.

Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis

Here we present a protocol for the rapid identification of proteins produced by genomically sequenced pathogenic bacteria using MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. Metastable protein ions fragment because of the aspartic acid effect and this specificity is exploited for protein identification.

This protocol identifies the immunity proteins of the bactericidal enzymes: colicin E3 and bacteriocin, produced by a pathogenic Escherichia coli strain ...

A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors

Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein phosphatases (PPPs) coordinate over 90% of all cellular serine/threonine dephosphorylation. Deregulation of protein phosphorylation has been implicated in the pathophysiology of various diseases, including cancer and neurodegeneration. Despite their widespread activity, the molecular mechanisms controlling PPPs and those controlled by PPPs are poorly characterized. Here, a proteomic approach termed phosphatase inhibitor beads and mass spectrometry (PIB-MS) is described to identify and quantify PPPs, their posttranslational modifications, and their interactors in as little as 12 h using any cell line or tissue. PIB-MS utilizes a non-selective PPP inhibitor, microcystin-LR (MCLR), immobilized on sepharose beads to capture and enrich endogenous PPPs and their associated proteins (termed the PPPome). This method does not require the exogenous expression of tagged versions of PPPs or the use of specific antibodies. PIB-MS offers an innovative way to study the evolutionarily conserved PPPs and expand our current understanding of dephosphorylation signaling.

Here, we present a protocol for the enrichment of endogenous phosphoprotein phosphatases and their interacting proteins from cells and tissues and their identification and quantification by mass spectrometry-based proteomics.

Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein ...

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic mechanisms of proteolytic enzymes1-3. With the advent of mass spectrometry-based proteomics, the enzymatically-catalyzed incorporation of 18O-atoms from stable isotopically enriched water has become a popular method to quantitatively compare protein expression levels (reviewed by Fenselau and Yao4, Miyagi and Rao5 and Ye et al.6). 18O-labeling constitutes a simple and low-cost alternative to chemical (e.g. iTRAQ, ICAT) and metabolic (e.g. SILAC) labeling techniques7. Depending on the protease utilized, 18O-labeling can result in the incorporation of up to two 18O-atoms in the C-terminal carboxyl group of the cleavage product3. The labeling reaction can be subdivided into two independent processes, the peptide bond cleavage and the carboxyl oxygen exchange reaction8. In our PALeO (protease-assisted labeling employing 18O-enriched water) adaptation of enzymatic 18O-labeling, we utilized 50% 18O-enriched water to yield distinctive isotope signatures. In combination with high-resolution matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), the characteristic isotope envelopes can be used to identify cleavage products with a high level of specificity. We previously have used the PALeO-methodology to detect and characterize endogenous proteases9 and monitor proteolytic reactions10-11. Since PALeO encodes the very essence of the proteolytic cleavage reaction, the experimental setup is simple and biochemical enrichment steps of cleavage products can be circumvented. The PALeO-method can easily be extended to (i) time course experiments that monitor the dynamics of proteolytic cleavage reactions and (ii) the analysis of proteolysis in complex biological samples that represent physiological conditions. PALeO-TimeCourse experiments help identifying rate-limiting processing steps and reaction intermediates in complex proteolytic pathway reactions. Furthermore, the PALeO-reaction allows us to identify proteolytic enzymes such as the serine protease trypsin that is capable to rebind its cleavage products and catalyze the incorporation of a second 18O-atom. Such "double-labeling" enzymes can be used for postdigestion 18O-labeling, in which peptides are exclusively labeled by the carboxyl oxygen exchange reaction. Our third strategy extends labeling employing 18O-enriched water beyond enzymes and uses acidic pH conditions to introduce 18O-stable isotope signatures into peptides.

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Stable isotope labeling workflows employing 18O-enriched water (LeO-workflows) are versatile tools for quantitative and qualitative proteomics studies. In protease-assisted (PALeO) workflows, 18O-atoms are introduced by proteolytic cleavage and carboxyl oxygen exchange reactions mediated by proteases. In the acid-catalyzed (ALeO) workflow, 18O-atoms are introduced by carboxyl oxygen exchange at low pH.

Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic ...

Biology

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

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

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Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

Detection of Protease Activity by Fluorescent Peptide Zymography

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis

A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors

Protease- and Acid-catalyzed Labeling Workflows Employing ¹⁸O-enriched Water

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins