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

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确定所用的芴甲氧羰基化合物, 这取决于所需的氨基酸和合成阶段。
<ol><li>将0.2 克芴甲氧羰基-NH-萨尔树脂与10毫升 n-, n-甲基酰胺 (DMF) 在合成柱上。</li>
	<li>在 DMF 中加入5毫升30% 哌啶。岩石的解决方案为10分钟, 以切割 n-芴甲氧羰基基团。</li>
	<li>
检查 n-芴甲氧羰基裂解与硝基苯酸 (TNBS) 测试12: 添加10µL 1% TNBS 在 DMF 和10µL (diisopropylethylamine) 到 10 x 75 毫米2玻璃管, 然后转移到玻璃管的少量树脂。n-芴甲氧羰基裂解树脂应发展为橙色的颜色。
	<ol>
<li>如果此结果为负值, 请重复步骤 1.2-1.3。</li>
	</ol>
</li>
	<li>用5毫升的 DMF 洗涤树脂三倍。</li>
	<li>
添加5毫升的 DMF, 其中含有0.3 毫摩尔的芴甲氧羰基化合物, 列于表 2 (从阶段1中使用的化合物开始), 0.3 毫摩尔 (6-氯-1 h benzotriazol-1 基)-n, n, n ', n-'-tetramethyluronium 六氟磷酸盐 (HCTU), 0.3 毫摩尔 1-Hydroxy-1H-benzotriazole, 一水合物 (对 hobt) 和0.6 毫摩尔 diisopropylethylamine (DIPEA)。岩石的解决方案为30分钟与树脂夫妇。
	<ol>
<li>使用 TNBS 测试检查此步骤。如果结果是肯定的, 这意味着反应没有足够的进展, 这一步必须重复。</li>
	</ol>
</li>
	<li>用5毫升的 DMF 洗涤树脂三倍。</li>
	<li>重复步骤 1.2-1.6, 改变在步骤1.5 中使用的芴甲氧羰基化合物, 根据表 2, 重覆伸长反应。</li>
	<li>在三氟乙酸酸中加入1毫升2.5% 水, 1% triisopropylsilane (TIS) 和 2.5% ethanedithiol (EDT), 以从树脂和保护基团中切割多肽。岩石解决方案为60分钟。</li>
	<li>过滤, 并收集滤液在一个50毫升管。</li>
	<li>加入40毫升的冷二乙醇, 并在-20 摄氏度隔夜贮存。</li>
	<li>离心机 4000 x g 在-20 °c 为30分钟收集颗粒。</li>
	<li>用干燥 3 h 去除二乙基醚。</li>
	<li>加入1毫升50% 乙腈水, 溶解粗肽。Lyophilize 去除三氟乙酸酸的溶液。重复此步骤几次。</li>
	<li>
使用 C18 墨盒柱纯化多肽。
	<ol>
<li>使用3毫升乙腈 (ACN) 激活 C18 墨盒柱上的树脂。</li>
		<li>平衡与5毫升 5% ACN, 0.1% TFA 在 H2O。</li>
		<li>将粗合成肽装入 C18 墨盒柱中。</li>
		<li>用10毫升 5% ACN, 0.1% TFA 在 H2O 洗涤。</li>
		<li>洗脱3毫升 60% ACN, 0.1% TFA 在 H 2 O 中的样品.</li>
	</ol>
</li>
	<li>
用 MALDI 质谱法测定其分子量, 检查肽的含量。
	<ol>
<li>在靶板上用吸管1µL 淋洗。在50% 乙腈中立即加入饱和α氰基-4-肉桂酸 (CHCA) 溶液, 0.1% 三氟乙酸酸</li>
		<li>空气干燥使混合物结晶。</li>
		<li>根据仪器制造商的协议获取样品的质谱。<ol>
<li>手动将 MALDI 质谱仪设置为正反射模式。</li>
			<li>用 CHCA、缓激肽片段 1-7 (monoisotopic 分子量 = 756.3997 Da) 和血管紧张素 II (monoisotopic 分子量 = 1045.5423 Da) 校准质谱仪。</li>
			<li>获得合成肽的质谱, 然后通过与理论分子量的比较进行评价。</li>
		</ol>
</li>
	</ol>
</li>
</ol>2. 组织提取物的制备
<ol><li>麻醉在异氟醚吸入腔内的雄性小鼠, 并通过脚趾捏检查麻醉深度。弄死颈椎脱位对小鼠进行了实验。</li>
	<li>使用剪刀, 使中线腹部切口通过皮肤延伸到剑突过程。</li>
	<li>切开隔膜, 收集所有肺组织。把纸巾切成小块。</li>
	<li>用冰冷的50毫米三缓冲盐水冲洗组织三次 (pH 7.4)。</li>
	<li>重量的组织和转移约100毫克的组织到 12 x 75 毫米2玻璃管。</li>
	<li>加入0.3 毫升的萃取缓冲器 (50 毫米三缓冲器, pH 7.4, 含0.1% 聚 (oxyethylene) 对辛醚)。</li>
	<li>
用混合均质器融汇肺组织样品。
	<ol>
<li>把样品放在冰上冷却。</li>
		<li>融汇的样品在 2万 rpm 三十年代在搅拌机。重复此步骤, 直到样品完全均匀。 注: 不要连续融汇超过1分钟, 以避免温度升高。</li>
	</ol>
</li>
	<li>将匀浆1000µL 到1.5 毫升管和离心机5分钟, 在 1万 x g 处4摄氏度。</li>
	<li>将上清的500µL 转移到一个新的1.5 毫升塑料管上。</li>
	<li>使用10µL 的样品, 以确定蛋白质浓度, 使用的方法, 如二辛可宁酸 () 或考马斯灿烂的蓝色 (CBB) 方法。</li>
	<li>为防止冷冻, 在样品中加入80% 甘油, 最终浓度为50% 甘油。</li>
	<li>将样品保存在-80 摄氏度, 直到进一步使用。</li>
</ol>3. 蛋白酶和组织提取物中基质的消化
<ol><li>
添加10µL 0.1 µg/µL 的 n-甲苯磺酰基-l-苯丙氨酸氯甲基酮 (TPCK)-胰蛋白酶, 0.1 µg/µL 金黄色葡萄球菌 V8 蛋白酶, 或10µg/µL 的组织提取成890µL 的消化缓冲。
	<ol>
<li>为了澄清蛋白酶特异性的差异取决于 ph 值, 使用缓冲调整到 ph 值 3, 5, 7 和9。消化缓冲的组成在表 3中显示。</li>
		<li>用非活性蛋白酶在组织提取物中进行阴性对照, 在沸水中孵化 (或加热100摄氏度) 进行10分钟。</li>
	</ol>
</li>
	<li>添加10µL 的 iminobiotin 标记基底 (溶解在二甲基亚砜 (亚砜), 最终浓度 = 100 pmol/10 µL)。</li>
	<li>孵育3小时在37°c 消化 iminobiotin 标记的底物。</li>
	<li>孵化后, 用沸水停止底物的消化 (或以100摄氏度加热) 10 分钟。</li>
	<li>在4摄氏度的 1万 x g 处离心消化基质5分钟。</li>
	<li>将上清液转移到新的1.5 毫升管中。</li>
</ol>4. Iminobiotin 标记衬底的纯化
<ol><li>
添加50µL 50% 链亲和素琼脂糖珠在1米三缓冲 (pH 9) 包含0.1% 聚 (oxyethylene) 对辛醚。
	<ol>
<li>使用 ph 值测试纸确保溶液在 ph 值9左右。如果溶液的 ph 值较低, 则通过加入1米的三缓冲 (ph 9) (含0.1% 聚 (oxyethylene) 对辛醚), 将其调整到大约 ph 值9。这一步很重要, 因为它涉及到将 iminobiotin 标记的基板绑定到链亲和素。</li>
	</ol>
</li>
	<li>在4°c 一夜之间孵化, 在旋转轮上连续的温和混合, 将 iminobiotin 标记的基板绑定到链亲和素。在孵化过程中, 珠子应该保持悬浮状态。</li>
	<li>离心机为1分钟在 1万 x g 在4°c。移除上清。</li>
	<li>
洗涤珠五倍如下:
	<ol>
<li>添加1000µL 的洗涤缓冲器 (50 毫米的三缓冲器, pH 9) 的珠子。</li>
		<li>清洗10分钟在4°c 与连续温和的混合在一个转子车轮上。在孵化过程中, 珠子应该保持悬浮状态。</li>
		<li>离心机为1分钟在 1万 x g 在4°c 和去除上清。</li>
	</ol>
</li>
	<li>
添加100µL 0.2% 三氟乙酸酸的珠子。
	<ol>
<li>使用 ph 值测试纸确保溶液低于 pH 值2。如果溶液的 ph 值高, 则通过加入1% 三氟乙酸酸将 ph 值调整到低于2。</li>
	</ol>
</li>
	<li>在室温下孵育30分钟, 在旋转轮上连续温和搅拌。</li>
	<li>离心机为1分钟在 1万 x g 在4°c, 然后转移上清到一个新的1.5 毫升管子。</li>
</ol>5. 样品制备和基质辅助激光解吸/电离飞行时间质谱 (MALDI 质谱)
注: 以下步骤中的所有解决方案均应采用高效液相色谱 (HPLC) 级或氨基酸序级水来制备。
<ol><li>将乙腈500µL 到 C18 树脂上活化。取出二手乙腈。重复此步骤三次。</li>
	<li>平衡 C18 树脂, 100 µL 0.1% 三氟乙酸酸。删除淋洗。重复此步骤三次。</li>
	<li>要装载样品, 用吸管将样品与树脂粘合。</li>
	<li>用20µL 0.1% 三氟乙酸酸清洗树脂。重复此步骤五次。</li>
	<li>洗脱样品与3µL 60% 乙腈在0.1% 三氟乙酸酸。将淋洗在靶板上进行 MALDI 质谱测定。立即加入饱和α氰基-4-肉桂酸 (CHCA) 溶液在50% 乙腈0.1% 三氟乙酸酸。</li>
	<li>空气干燥使混合物结晶。</li>
	<li>
根据仪器制造商的协议获取样品的质量谱。
	<ol>
<li>手动将 MALDI 质谱仪设置为正反射模式。</li>
		<li>用 CHCA、缓激肽片段 1-7 (monoisotopic 分子量 = 756.3997 Da) 和血管紧张素 II (monoisotopic 分子量 = 1045.5423 Da) 校准质谱仪。</li>
		<li>获取样品的质谱, 密切关注质谱从700到 900 Da 为生物素标记基质的劈裂形式。</li>
	</ol>
</li>
	<li>使用表 1标识检测到的峰值。适当分子量的检测表明, 在相关氨基酸的 C 终点处有裂解。</li>
</ol>

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作者声明没有利益冲突。

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)&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

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 ...

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

Research

JoVE Journal

Biochemistry

6.7K 次观看.  Nihon Pharmaceutical University. 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。

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

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

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

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

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

基质辅助飞行的完整蛋白质（MALDI-TOF）质谱分析激光解吸/电离飞行时间大于100 kDa的

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

科研

化学

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

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

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

MALDI成像帕金森病的神经肽质谱

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

医学

Detection of Protease Activity by Fluorescent Peptide Zymography

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

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

荧光肽酶谱法检测蛋白酶活性

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

癌症研究

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.

Langendorff 灌注后离体小鼠心脏 Polysomes 的动态蛋白质组学及 miRNA 分析

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

生物化学

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.

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 ...

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.

基于质谱的蛋白质组学方法用于全局和高置信度的蛋白质R-甲基化分析

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

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

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

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

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

使用MALDI-TOF-TOF-MS/MS和自上而下的蛋白质组学分析鉴定 大肠杆菌 中的抗菌免疫蛋白

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

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

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

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

一种基于质谱的磷酸化蛋白磷酸酶及其相互作用物鉴定方法

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

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

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

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

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

蛋白酶和酸催化的贴标工作流程，用人<sup&gt; 18</sup&gt; O-富氧水

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

生物学

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.

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

摘要

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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

摘要

探索更多视频

此视频中的章节

基质辅助飞行的完整蛋白质（MALDI-TOF）质谱分析激光解吸/电离飞行时间大于100 kDa的

MALDI成像帕金森病的神经肽质谱

荧光肽酶谱法检测蛋白酶活性

Langendorff 灌注后离体小鼠心脏 Polysomes 的动态蛋白质组学及 miRNA 分析

通过肽浓缩和质谱检测蛋白质泛化位点检测

基于质谱的蛋白质组学方法用于全局和高置信度的蛋白质R-甲基化分析

使用MALDI-TOF-TOF-MS/MS和自上而下的蛋白质组学分析鉴定 大肠杆菌 中的抗菌免疫蛋白

一种基于质谱的磷酸化蛋白磷酸酶及其相互作用物鉴定方法

蛋白酶和酸催化的贴标工作流程，用人

捕获复合质谱 - 一个强大的工具来确定新型C-二GMP效应蛋白

使用MALDI-TOF-TOF-MS/MS和自上而下的蛋白质组学分析鉴定大肠杆菌中的抗菌免疫蛋白