Name: the determination of protease specificity in mouse tissue extracts by maldi-tof mass spectrometry: manipulating ph to cause specificity changes
Uploaded: 2018-05-25T13:30:00.000+00:00
Duration: 9 min 47 s
Description: 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

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...

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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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6.8K 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

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 ...

TMT Sample Preparation for Proteomics Facility Submission and Subsequent Data Analysis

Proteomic technologies are powerful methodologies that can aid our understanding of mechanisms of action in biological systems by providing a global view of the impact of a disease, treatment, or other condition on the proteome as a whole. This report provides a detailed protocol for the extraction, quantification, precipitation, digestion, labeling, and subsequent data analysis of protein samples. Our optimized TMT labeling protocol requires a lower tag-label concentration and achieves consistently reliable data. We have used this protocol to evaluate protein expression profiles in a variety of mouse tissues (i.e., heart, skeletal muscle, and brain) as well as cells cultured in vitro. In addition, we demonstrate how to evaluate thousands of proteins from the resulting dataset.

We present an optimized tandem mass tag (TMT) labeling protocol that includes detailed information for each of the following steps: protein extraction, quantification, precipitation, digestion, labeling, submission to a proteomics facility, and data analyses.

Proteomic technologies are powerful methodologies that can aid our understanding of mechanisms of action in biological systems by providing a global view ...

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, ...

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 ...

Enrichment of Host Cell Proteins in Monoclonal Antibody Drug Products

Host Cell Protein Analysis using Enrichment Beads Coupled with Limited Digestion

Host cell proteins (HCPs) are impurities that can adversely affect therapeutic proteins, even in small quantities. To evaluate the potential risks associated with drug products, methods have been developed to identify low-abundance HCPs. A crucial approach for developing a sensitive HCP detection method involves enriching HCPs while simultaneously removing monoclonal antibodies (mAbs) before analysis, utilizing liquid chromatography-mass spectrometry (LC-MS).
This protocol offers detailed instructions for enriching host cell proteins using commercially available proteome enrichment beads. These beads contain a diverse library of hexapeptide ligands with specific affinities for different proteins. The protocol also incorporates limited digestion and subsequent peptide detection using nano LC-MS/MS. By employing these techniques, HCPs with low abundance can be enriched over 7000-fold, resulting in an impressive detection limit as low as 0.002 ppm. Significantly, this protocol enables the detection of 850 HCPs with a high level of confidence using a NIST mAb. Moreover, it is designed to be user-friendly and includes a video demonstration to assist with its implementation. By following these steps, researchers can effectively enrich and detect HCPs, enhancing the sensitivity and accuracy of risk assessment for drug products.

Author Spotlight: Detecting Low-Abundant Host Cell Proteins in Drug Products Using Enrichment Beads and Limited Digestion 

A protocol is presented for enriching host cell proteins (HCPs) from drug products (DP) and detecting peptides using proteome enrichment beads. The method is demonstrated using an in-house manufactured monoclonal antibody (mAb) drug substance (DS), which is a well-characterized reference material for evaluating and comparing different methods in terms of performance.

Host cell proteins (HCPs) are impurities that can adversely affect therapeutic proteins, even in small quantities. To evaluate the potential risks ...

Chemistry

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Biology

Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that transduce the mechanical energy of sound into an electro-chemical energy processed by the peripheral and central nervous systems. Several proteomic techniques have been developed to study the cochlear inner ear, such as two-dimensional difference gel electrophoresis (2D-DIGE), antibody microarray, and mass spectrometry (MS). MS is the most comprehensive and versatile tool in proteomics and in conjunction with separation methods can provide an in-depth proteome of biological samples. Separation methods combined with MS has the ability to enrich protein samples, detect low molecular weight and hydrophobic proteins, and identify low abundant proteins by reducing the proteome dynamic range. Different digestion strategies can be applied to whole lysate or to fractionated protein lysate to enhance peptide and protein sequence coverage. Utilization of different separation techniques, including strong cation exchange (SCX), reversed-phase (RP), and gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) can be applied to reduce sample complexity prior to MS analysis for protein identification.

Proteome analysis of the cochlear sensory epithelium can be challenging due to its small size and because membrane proteins are difficult to isolate and identify. Both membrane and soluble proteins can be identified by combining multiple preparative methods and separation techniques along with high-resolution mass spectrometry.

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that ...

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.

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease (AD), amyloid-beta peptides form protofibrils in the extracellular space, which act as seeds that gradually grow and mature into large amyloid plaques. Despite this basic understanding, current knowledge of the amyloid fibril structure, composition, and deposition patterns in the brain is limited. One major barrier has been the inability to isolate highly purified amyloid fibrils from brain extracts. Affinity purification and laser capture microdissection-based approaches have been previously used to isolate amyloids but are limited by the small quantity of material that can be recovered. This novel, robust protocol describes the biochemical purification of amyloid plaque cores using sodium dodecyl sulfate (SDS) solubilization with sucrose density gradient ultracentrifugation and ultrasonication and yields highly pure fibrils from AD patients and AD model brain tissues. Mass spectrometry (MS)-based bottom-up proteomic analysis of the purified material represents a robust strategy to identify nearly all the primary protein components of amyloid fibrils. Previous proteomic studies of proteins in the amyloid coronae have revealed an unexpectedly large and functionally diverse collection of proteins. Notably, after refining the purification strategy, the number of co-purifying proteins was reduced by more than 10-fold, indicating the high purity of the isolated SDS insoluble material. Negative staining and immuno-gold electron microscopy allowed confirmation of the purity of these preparations. Further studies are required to understand the spatial and biological attributes that contribute to the deposition of these proteins into amyloid inclusions. Taken together, this analytical strategy is well-positioned to increase the understanding of amyloid biology.

This biochemical purification method with mass spectrometry-based proteomic analysis facilitates the robust characterization of amyloid fibril cores, which may accelerate the identification of targets for preventing Alzheimer's disease.

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease ...