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)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 ...

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

Universidad de Cartagena UDEC

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

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

We describe a simple protocol for identifying and quantifying the two components in binary mixtures of species possessing one or more similar proteins. Central to the method is the identification of 'corresponding proteins' in the species of interest, in other words proteins that are nominally the same but possess species-specific sequence differences. When subject to proteolysis, corresponding proteins will give rise to some peptides which are likewise similar but with species-specific variants. These are 'corresponding peptides'. Species-specific peptides can be used as markers for species determination, while pairs of corresponding peptides permit relative quantitation of two species in a mixture. The peptides are detected using multiple reaction monitoring (MRM) mass spectrometry, a highly specific technique that enables peptide-based species determination even in complex systems. In addition, the ratio of MRM peak areas deriving from corresponding peptides supports relative quantitation. Since corresponding proteins and peptides will, in the main, behave similarly in both processing and in experimental extraction and sample preparation, the relative quantitation should remain comparatively robust. In addition, this approach does not need the standards and calibrations required by absolute quantitation methods. The protocol is described in the context of red meats, which have convenient corresponding proteins in the form of their respective myoglobins. This application is relevant to food fraud detection: the method can detect 1% weight for weight of horse meat in beef. The corresponding protein, corresponding peptide (CPCP) relative quantitation using MRM peak area ratios gives good estimates of the weight for weight composition of a horse plus beef mixture.

We present a protocol for identifying and quantifying the components in mixtures of species possessing similar proteins. Mass spectrometry detects peptides for identification, and gives relative quantitation by ratios of peak areas. As a tool food for fraud detection, the method can detect 1% horse in beef.

种测定和定量混合物中使用肽应用的MRM质谱肉认证

We describe a simple protocol for identifying and quantifying the two components in binary mixtures of species possessing one or more similar proteins. ...

科研

生物化学

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

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

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

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 ...

Analysis of Histone Antibody Specificity with Peptide Microarrays

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The mass production and distribution of antibodies specific to histone PTMs has greatly facilitated research on these marks. As histone PTM antibodies are key reagents for many chromatin biochemistry applications, rigorous analysis of antibody specificity is necessary for accurate data interpretation and continued progress in the field. This protocol describes an integrated pipeline for the design, fabrication and use of peptide microarrays for profiling the specificity of histone antibodies. The design and analysis aspects of this procedure are facilitated by ArrayNinja, an open-source and interactive software package we recently developed to streamline the customization of microarray print formats. This pipeline has been used to screen a large number of commercially available and widely used histone PTM antibodies, and data generated from these experiments are freely available through an online and expanding Histone Antibody Specificity Database. Beyond histones, the general methodology described herein can be applied broadly to the analysis of PTM-specific antibodies.

This manuscript describes methods for applying peptide microarray technology to specificity profiling of antibodies that recognize histones and their post-translational modifications.

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 ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

RNA 结合区质量谱鉴定的样品制备

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

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

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of reactive oxygen species, and more. These fluctuations can lead to a widespread protein unfolding, aggregation, and cell death. Therefore, cells have evolved a dynamic and stress-specific network of molecular chaperones, which maintain a "healthy" proteome during stress conditions. ATP-independent chaperones constitute one major class of molecular chaperones, which serve as first-line defense molecules, protecting against protein aggregation in a stress-dependent manner. One feature these chaperones have in common is their ability to utilize structural plasticity for their stress-specific activation, recognition, and release of the misfolded client. 
In this paper, we focus on the functional and structural analysis of one such intrinsically disordered chaperone, the bacterial redox-regulated Hsp33, which protects proteins against aggregation during oxidative stress. Here, we present a toolbox of diverse techniques for studying redox-regulated chaperone activity, as well as for mapping conformational changes of the chaperone, underlying its activity. Specifically, we describe a workflow which includes the preparation of fully reduced and fully oxidized proteins, followed by an analysis of the chaperone anti-aggregation activity in vitro using light-scattering, focusing on the degree of the anti-aggregation activity and its kinetics. To overcome frequent outliers accumulated during aggregation assays, we describe the usage of Kfits, a novel graphical tool which allows easy processing of kinetic measurements. This tool can be easily applied to other types of kinetic measurements for removing outliers and fitting kinetic parameters. To correlate the function with the protein structure, we describe the setup and workflow of a structural mass spectrometry technique, hydrogen-deuterium exchange mass spectrometry, that allows the mapping of conformational changes on the chaperone and substrate during different stages of Hsp33 activity. The same methodology can be applied to other protein-protein and protein-ligand interactions.

One of the most challenging stress conditions that organisms encounter during their lifetime involves the accumulation of oxidants. During oxidative stress, cells heavily rely on molecular chaperones. Here, we present methods used to investigate the redox-regulated anti-aggregation activity, as well as to monitor structural changes governing the chaperone function using HDX-MS.

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

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have developed a simple non-labeled and non-radio-isotopic method using a matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis for a proofreading study. Here, a DNA polymerase [e.g., the Klenow fragment (KF) of Escherichia coli DNA polymerase I (pol I) in this study] in the presence of all four dideoxyribonucleotide triphosphates is used to process a mismatched primer-template duplex. The mismatched primer is then proofread/extended and subjected to MALDI-TOF MS. The products are distinguished by the mass change of the primer down to single nucleotide variations. Importantly, a proofreading can also be determined for internal single mismatches, albeit at different efficiencies. Mismatches located at 2-4-nucleotides (nt) from the 3' end were efficiently proofread by pol I, and a mismatch at 5 nt from the primer terminus showed only a partial correction. No proofreading occurred for internal mismatches located at 6 - 9 nt from the primer 3' end. This method can also be applied to DNA repair assays (e.g., assessing a base-lesion repair of substrates for the endo V repair pathway). Primers containing 3' penultimate deoxyinosine (dI) lesions could be corrected by pol I. Indeed, penultimate T-I, G-I, and A-I substrates had their last 2 dI-containing nucleotides excised by pol I before adding a correct ddN 5'-monophosphate (ddNMP) while penultimate C-I mismatches were tolerated by pol I, allowing the primer to be extended without repair, demonstrating the sensitivity and resolution of the MS assay to measure DNA repair.

A non-labeled, non-radio-isotopic method for DNA polymerase proofreading and a DNA repair assay was developed by using high-resolution MALDI-TOF mass spectrometry and a single nucleotide extension strategy. The assay proved to be very specific, simple, rapid, and easy to perform for proofreading and repair patches shorter than 9-nucleotides.

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

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 ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

血管紧张蛋白II图解的硝酸分析与鉴定

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

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.