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
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

这项工作部分是由日本制药大学的日本制药大学研究补助金 (2016) 和下个 KAKENHI 赠款号 JP17854179 资助的。

日本制药大学伦理委员会批准了动物实验协议, 并按照日本制药大学实验室动物的护理和使用指导方针进行。该协议已被调整的组织提取物从代表的小鼠。小鼠是从日本的公司购买的。
1. Iminobiotin 标记衬底的制备
基板采用 9-fluorenylmethyloxycarbonyl 基团 (芴甲氧羰基) 保护的氨基酸进行固相合成, 如下11所述。使用表 2确定所用的芴...

<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>

本协议采用 MALDI 质谱法对组织提取物中的粗样品中的蛋白酶特异性进行识别, 并可方便地进行多样本加工。特别是, 我们操纵 pH 值导致基材特异性的改变。
亲和生物素复合物 (ABC) 方法在生物化学中得到了广泛应用, 其结合特异性, 在我们的协议中得到了应用。由于 ABC 的强烈亲和力, 亲和对生物素的约束几乎是不可逆转的。因此, 对 abc 的亲和纯化效率极低。因此, 我们报告说, ...

蛋白酶通过裂解蛋白质调节生理过程, 并且在特定时间和地点启动蛋白酶活性的调控1。因此, 确定局部调节组织的表达和活性是很重要的, 并开发一种新的检测蛋白酶的方法。本研究提出的方法旨在鉴别蛋白酶的特异性, 同时检测蛋白酶。
有几种检测蛋白酶特异性的方法。azocasein 方法在检测蛋白酶2中是众所周知的, 但在获取进一步信息的能力方面是有限的。酶谱法是另一种综合的蛋白酶检测方法, 可用于测定蛋白酶的分子量3, 但它不能用于检查基底特异性。蛋白酶特异性可以通过光谱分析, 使用 nitroanilide4的基质, 和荧光化验, 使用香豆素基底5或荧光共振能量转移 (烦恼) 化验6。这些方法使单个井中所含基底的单特异性检测得以实现。本研究在不同条件下考察组织提取物的蛋白酶特异性, 因为这些萃取物含有多种蛋白酶。蛋白酶活性受多种因素的制约, 包括 pH 值、辅、假体组和离子强度。近年来, 蛋白质组学方法已被用于鉴定蛋白酶特异性;例如, 由 Biniossek et al报告的方法。7使用蛋白质组来确定基材的特异性, 即使是在粗提取物中, 也可以精确地测定识别多个氨基酸序列8的蛋白酶的裂解活性。但是, 这种方法不适合对大量样品进行分析。相比之下, 我们的方法可以同时处理多个样本, 利用基质辅助激光解吸/电离飞行时间 (MALDI) 质谱进行样品分析, 便于快速、简便地检测出基质。
本文概述了用 MALDI 质谱法测定蛋白酶特异性的方法, 以测量独特基质的分子量。在图 1和表 1中显示了被切割基底的分子形式以及它们的理论分子权重。以前的研究使用了含有 polyglycine 间隔和生物素9的基质;然而, 这些基质是有缺陷的, 因为 polyglycine 序列可能会由识别甘氨酸序列的酶所切割。此外, 亲和和生物素之间的高度亲和性可能导致回收率低。为了改善这些缺点, 我们在本研究中合成了一种独特的基质, 它由聚乙二醇 (PEG)、iminobiotin 和氨基酸组成, 它可以识别出裂解部位 (图 1)。为了区分相似分子量的氨基酸, 在 PEG 间隔与氨基酸在劈裂部位添加 d-丝氨酸。
该基板的 N 端被标记为 iminobiotin, 这使得从粗样品中进行亲和纯化。使用 iminobiotin 而不是生物素是至关重要的;生物素与亲和有很强的亲和性, 这导致亲和树脂的生物素标记基质的回收率较低, 而 iminobiotin 对亲和的亲和性可以通过 pH 值来改变。在 ph 值为9的条件下, Iminobiotin 标记的基板将绑定到亲和, 而亲和在 4 ph 值以下的条件下释放 Iminobiotin 标记的基底。因此, iminobiotin 用于亲和纯化10。总之, 我们描述了一个详细的协议, 以检测蛋白酶特异性使用独特的基质。

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

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

蛋白酶具有多种生物学功能, 包括蛋白质活化/失活和食物消化。鉴定蛋白酶特异性对揭示蛋白酶功能具有重要意义。本研究提出的方法通过基质辅助激光解吸/电离飞行时间 (MALDI) 质谱法测定独特基质的分子量来确定蛋白酶的特异性。基体中含有 iminobiotin, 而劈裂部位由氨基酸组成, 间隔由聚乙二醇组成。被劈裂的基底将产生一个独特的分子量, 使用裂解氨基酸。该方法的优点之一是可以用粗样品在一锅中进行, 也适用于多种样品的评估。在本文中, 我们描述了一个简单的实验方法优化, 从小鼠肺组织提取的样本, 包括组织抽取, 消化基质的放置到样品, 消化基质的纯化在不同的 pH 条件下, 用 MALDI 质谱法测定基质的分子量。总之, 这项技术可以识别的蛋白酶特异性从组织提取物中获得的 MALDI 的质谱, 可以很容易地扩大到多样本处理。

模型蛋白酶鉴定蛋白酶特异性方法的评价
利用 TPCK 胰蛋白酶和 V8 蛋白酶对该系统的效率进行了评价, 得到了基体的特异性。TPCK-胰蛋白酶和 V8 蛋白酶被估计为在赖氨酸和精氨酸残留物的 C 末端, 以及天门冬氨酸和谷氨酸残留物的羧基端, 分别对氨基酸序列进行劈裂。表 1显示了基板的理论分子量和由特定蛋白酶劈裂的...

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

MALDI 质谱法测定小鼠组织提取物蛋白酶特异性: 操作 PH 值导致特异性改变

在这里, 我们提出了一个协议, 以确定蛋白酶特异性的粗鼠组织提取物使用 MALDI 的光谱。

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

Research

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

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

解决由蓝变性PAGE和蛋白质相关剖析亲和纯化的蛋白复合物

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

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用肽微阵列分析组蛋白抗体特异性

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

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

用氢-氘交换质谱法定义 Hsp33's 氧化还原调节陪护活动和 Hsp33 映射构象变化

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

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

单核苷酸延长和 MALDI 质谱分析校对及 DNA 修复方法

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

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

通过肽浓缩和质谱检测蛋白质泛化位点检测

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

Using the Open-Source MALDI TOF-MS IDBac Pipeline for Analysis of Microbial Protein and Specialized Metabolite Data

使用开源 MALDI TOF-MS IDBac 管道分析微生物蛋白和专用代谢物数据

In order to visualize the relationship between bacterial phylogeny and specialized metabolite production of bacterial colonies growing on nutrient agar, ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

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

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

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