Name: The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes (Video) | JoVE Journal
Uploaded: 2018-05-25T13:30:00.000+00:00
Duration: 9 min 47 s
Description: Learn to determine protease specificity in mouse lung tissue extracts using MALDI-TOF mass spectrometry. This protocol enables high-throughput analysis of multiple samples simultaneously for proteomics research.

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5 minutes

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/pubmed?term=((Role+of+proteolytic+enzymes+in+biological+regulation+%28a+review%29%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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/pubmed?term=((Determination+of+the+activity+of+proteolytic+enzymes+by+means+of+azocasein%5BTitle%5D)+AND+%22Vopr+Med+Khim%22%5BJournal%5D)">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/pubmed?term=((Enhanced+detection+of+casein+zymography+of+matrix+metalloproteinases%5BTitle%5D)+AND+%22Anal+Biochem%22%5BJournal%5D)">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/pubmed?term=((The+preparation+and+properties+of+two+new+chromogenic+substrates+of+trypsin%5BTitle%5D)+AND+%22Arch+Biochem+Biophys%22%5BJournal%5D)">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/pubmed?term=((Direct+photometric+or+fluorometric+assay+of+proteinases+using+substrates+containing+7-amino-4-trifluoromethylcoumarin%5BTitle%5D)+AND+%22Thromb+Res%22%5BJournal%5D)">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/pubmed?term=((Fluorescende+determination+of+carboxypeptidase+A+activity+based+on+electronic+energy+transfer%5BTitle%5D)+AND+%22Anal+Biochem%22%5BJournal%5D)">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/pubmed?term=((Profiling+constitutive+proteolytic+events+in+vivo%5BTitle%5D)+AND+%22Biochem+J%22%5BJournal%5D)">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/pubmed?term=((Identification+of+protease+specificity+by+combining+proteome-derived+peptide+libraries+and+quantitative+proteomics%5BTitle%5D)+AND+%22Mol+Cell+Proteomics%22%5BJournal%5D)">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/pubmed?term=((Identification+of+protease+specificity+using+biotin-labeled+substrates%5BTitle%5D)+AND+%22Open+Biochem+J%22%5BJournal%5D)">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/pubmed?term=((Iminobiotin+affinity+columns+and+their+application+to+retrieval+of+streptavidin%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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="https://pubs.acs.org/doi/abs/10.1021/ja00897a025">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/pubmed?term=((A+new+micro-test+for+the+detection+of+incomplete+coupling+reactions+in+solid-phase+peptide+synthesis+using+2%2C4%2C6-trinitrobenzenesulphonic+acid%5BTitle%5D)+AND+%22Anal.+Biochem%22%5BJournal%5D)">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/pubmed?term=((Recombinant+NeutraLite+avidin%3A+A+non-glycosylated%2C+acidic+mutant+of+chicken+avidin+that+exhibits+high+affinity+for+biotin+and+low+non-specific+binding+properties%5BTitle%5D)+AND+%22FEBS+Lett%22%5BJournal%5D)">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/pubmed?term=((Purification+of+human+liver+propionyl-CoA+carboxylase+by+carbon+tetrachloride+extraction+and+monomeric+avidin+affinity+chromatography%5BTitle%5D)+AND+%22Arch+Biochem+Biophys%22%5BJournal%5D)">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

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...

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

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

the-determination-protease-specificity-mouse-tissue-extracts-maldi

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Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

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.

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

Peptide arrays synthesized by the SPOT method can be used to analyze the substrate specificity of Protein lysine methyltransferases (PKMTs) and to define the substrate spectrum of PKMTs to understand their biological role. This protocol describes how to synthesize peptide arrays, methylate them with PKMTs, and analyze the results.

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

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.

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

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.

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

This workflow describes the performance of time- and cost-efficient enrichment of multiple protein post-translational modifications (PTMs) simultaneously for quantitative global proteomic analysis. The protocol utilizes peptide-level PTM enrichment with multiple conjugated antibodies, followed by data-independent acquisition mass spectrometry analysis to gain biological insights into PTM crosstalk.

Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization

This protocol covers a detailed analysis of peptidoglycan composition using liquid chromatography mass spectrometry coupled with advanced feature extraction and bioinformatic analysis software.

Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics

This protocol describes how to perform cell-type-specific protein labeling with azidonorleucine (ANL) using a mouse line expressing a mutant L274G-Methionine tRNA synthetase (MetRS*) and the necessary steps for labeled cell-type-specific proteins isolation. We outline two possible ANL administration routes in live mice by (1) drinking water and (2) intraperitoneal injections.

Cell-Type Specific Protein Purification and Identification from Complex Tissues Using a Mutant Methionine tRNA Synthetase Mouse Line

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.

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

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.

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

Learn to determine protease specificity in mouse lung tissue extracts using MALDI-TOF mass spectrometry. This protocol enables high-throughput analysis of multiple samples simultaneously for proteomics research.

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

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

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