Name: overlapping peptide library to map qa-1 epitopes in a protein
Uploaded: 2017-12-20T12:30:00.000+00:00
Duration: 8 min 4 s
Description: Watch this Scientific Journal Video about Overlapping Peptide Library to Map Qa-1 Epitopes in a Protein at JoVE.com

我们感谢佩内洛普. 加西亚的技术援助和这份手稿的准备工作。这项工作得到了一项研究创新补助金 (钻机) 从医学院在 PP1685 琳达大学 (681205-2967) 和试点补助金从国家多发性硬化症协会 () XT。

所有的实验都是遵照德克萨斯大学埃尔帕索分校和玛琳达大学的动物保育和使用委员会批准的机构动物保育和使用协议进行的。
1. OLP 库的生成覆盖了整个蛋白质的长度
<ol>
	<li>设计一个 OLP 库, 其中所有的多肽是15的长度, 和相邻的多肽重叠的11种氨基酸。 注: 在 MOG OLP 研究中, MOG 前体 [小家鼠] 的序列是从 NCBI 蛋白质数据库中检索到的, 遵循以下链接: https://www.ncbi.nlm.nih.gov/protein/NP_034944。MOG 前体 (247 种氨基酸), 其中包含信号和成熟的肽, 被使用, 因为大多数报告的 Qa-1 (HLA E) 表位是位于信号肽 (如 Qdm1)。从它的 N 端开始, 15-氨基肽的序列被辨认了, 因此二个毗邻肽重叠由11氨基酸 (图 1)。因此, 在247氨基酸 MOG 前体中, 准确地发现了59处。然而, 最后的 OLP 可以从12到15的范围, 取决于蛋白质的长度。此外, 我们选择 15-滨海图书馆, 因为我们和其他人已经表明, 15-滨海多肽, 当添加到树突状细胞 (DCs) 或巨噬, 可以有效地处理成表位, 以识别通过 CD8+ T 细胞14,15.</li>
	<li>在商业上购买每个单独的肽。5毫克每肽应该足够的筛选。处和截断的多肽 (步骤 6.1) 可盐水肽 (纯度约 50-70%)。最佳肽 (步骤 6.2) 的纯度将用于聚丙烯的生成和未来的生物分析应该和 #62; 90%。在无菌条件下重组多肽。</li>
	<li>在100% 亚砜中制作50毫克/毫升的单个肽类股。每多肽5毫克, 在每管中加入100μ l 的亚甲基亚砜, 混合, 并储存在-20 ° c 的多肽。这些股票将被用来做一个 OLP_pool 的股票, 生成的 CD8+ T 细胞系反应的 OLP_pool。此外, 这些股票还将用于10毫克/毫升个别肽类股, 以确定每个单独的肽的能力, 以刺激 Qa-1-restricted 反应在 OLP_pool 反应的 CD8+ T 细胞系。</li>
	<li>通过在新鲜的试管中加入等量的肽, 使 OLP_pool 的股票。这 OLP_pool 的股票含有100% 的亚甲基亚砜。在 MOG OLP 研究中, 有59处12。因此, OLP_pool 中各肽的浓度为847.46 微克/毫升 (50 毫克/毫升÷ 59)。</li>
	<li>通过稀释 (5x) 50 毫克/毫升在一个 V 底 96-井板中的不育的 H2O 中的储存量, 使10毫克/毫升的个体肽储存 (这股含有20% 亚砜)。使这些股票在一个96井板, 因为确定每一个单独的肽的 Qa-1-restricted 反应将在一个 96-井板使用8或12声道多通道吸管。 注: 所有肽, 包括处和截断多肽, 应稀释在一个 V 底96井板或96井样品机架充满1毫升管, 因为肽库的筛选是在96井板使用多通道吸管进行。如果是难以稀释的肽, 添加 1 N 氢氧化钠滴明智帮助解散肽。</li>
</ol>
2. 启动的 kb-/- db-/- CD8+ T 细胞与 OLP_pool 脉冲 Kb-/- b/-树突状细胞 (DCs)。
注意: 有两个主要的 Qa-1 等位基因: 一个是 Qa-1a, 另一个是 Qa-1b。由于通常用于学术研究的动物,例如C57BL/6 和 balb/c/c 小鼠, 携带 Qa-1b, 本协议描述了在蛋白质中映射 Qa-1b表位的过程。在本协议中使用的 CD8+ T 细胞从 Kb---Db-/小鼠 (C57BL/6 背景) 纯化, 其中 CD8+ t 细胞主要受经典 MHC-Ib 分子 (包括 Qa-1) 的限制。
<ol>
	<li>如前所述, 产生骨髓源性 DCs, 如12。
	<ol>
		<li>简言之, 培养骨髓单细胞悬浮液 (1 x 106细胞/毫升) 在 RPMI-10 培养基 (RPMI 1640 补充10% 胎牛血清, 5.5 x 10-5 M 2-基, 1 毫米钠丙酮酸盐, 和0.1 毫米不必要的氨基酸)包含 10 u/毫升 IL-4 和 100 u/毫升 GM-CSF 在一个6井板 (4 毫升/井) 在37° c, 5% CO2。</li>
		<li>两天后, 小心去除换药细胞, 添加新鲜培养基和细胞因子。</li>
		<li>在培养了两天的细胞后, 将含有新鲜培养基和细胞因子的换药细胞转移到一个新的6井板中。</li>
		<li>再培养两天的细胞, 并补充含有 LPS (0.1 µg/毫升) 的新鲜培养基和细胞因子, 以激活 DCs。</li>
		<li>24小时后, 收集 DCs 进行实验。</li>
	</ol>
	</li>
	<li>用3000拉德照射 DCs。
	<ol>
		<li>或者, 在 PBS 中, 在37° c 处将 DCs (5 x 107细胞/毫升) 与丝裂霉素 c-(50 微克/毫升) 进行处理, 20 分钟. 添加 RPMI-0 (RPMI 1640 无血清), 以填补管 (〜12毫升) 和旋转的细胞10分钟在一个表顶离心机在 300 x g. 丢弃清和重复再洗两次。这三洗是关键的, 因为任何痕量的丝裂霉素 C 可能抑制 CD8 的反应, 在 DC t 细胞共培养期间的 t 细胞.</li>
	</ol>
	</li>
	<li>在无血清培养基中将 DC 浓度调整为 5 x 106细胞/毫升 (目的-V 无血清培养基补充 5.5 x 10-5 M 2-基, 1 毫米丙酮酸钠, 和0.1 毫米不必要的氨基酸)。</li>
	<li>添加 OLP_pool 的股票解决方案, 其中包含100% 个亚甲基亚砜, 以使最终的亚砜浓度将是 0.5% (200x) 的 DCs。在 MOG OLP 研究中, OLP_pool 的每 OLP 浓度为847.46 微克/毫升;因此, 每个 OLP 在 DC 培养的最终浓度是4.2 微克/毫升 (847.46 微克/毫升÷ 200)。</li></ol>然而, 只要在细胞培养中的亚砜浓度小于 1%, 这种浓度就可以扩大到100微克/毫升。
	<li>室温下孵育 DCs 3 小时, 每15分钟轻轻摇动细胞。</li>
	<li>在这个潜伏期, 使用商用 CD8+ T 细胞纯化试剂盒, 从收获的脾脏或淋巴结中纯化 CD8 的+ T 细胞, 将细胞浓度调整到 10 x 106细胞/毫升无血清培养基含有 50 u/毫升白细胞介素 2 (IL-2) 和 100 u/毫升的 IL-7。</li>
	<li>现在, 将 CD8+ T 单元格添加到一个48井板中, 作为0.5 毫升/井 (在步骤2.8 中增加了0.5 毫升/OLP_pool 脉冲 DCs 后, CD8+ T 单元格的最终浓度将是 5 x 106单元格/mL)。 注意: 根据制造商提供的指示, 可以使用 CD8 阳性隔离试剂盒来纯化小鼠脾脏和淋巴结的 CD8+ T 细胞。获得的 CD8+ T 细胞没有珠子。</li>
	<li>旋下 OLP_pool 脉冲 DCs (300 x g, 10 分钟) 在 RT. 在无血清培养基中重建 OLP_pool 脉冲 DCs 至 2 x 106细胞/毫升, 并将0.5 毫升/井放入含有 CD8+ T 细胞的 48-井板中 (OLP 的最终浓度_pool 脉冲 DCs 是 1 x 106细胞/毫升, 最终浓度的 CD8+ T 细胞是 5 x 106细胞/毫升, 最终浓度的 IL-2 是 25 u/毫升, 最终浓度的 IL-7 是 50 u/毫升)。培养细胞在37° c 和 5% CO2。</li>
	<li>在4天, 删除和丢弃约400μ l 培养基, 并添加500μ l 的新鲜无血清培养基含有 100 u/毫升 IL-2 和 100 u/毫升的 IL-7 每井。孵育细胞在37° c 和 5% CO2。</li>
	<li>在7或8天, re-stimulate OLP_pool 的 CD8+ T 细胞与 OLP_pool。</li>

3. Restimulation 的 CD8+ T 细胞的 OLP_pool
<ol>
	<li>四天前 re-stimulation 的 OLP_pool 引物 CD8 的+ T 细胞, 准备 2% (v/v) 聚丙烯酰胺珠解决方案: 洗涤2克聚丙烯酰胺珠两次在20毫升内毒素无 H2O 或 PBS。将聚丙烯酰胺微珠离心 (400 x g) 用于5分钟和重100毫升的 PBS。15磅/米的高压釜在室温下20分钟贮存。</li>
	<li>注射小鼠腹腔与1毫升/小鼠的无菌2% 聚丙烯酰胺珠溶液, 以吸引迁移的单核/巨噬细胞进入腹膜腔16。</li>
	<li>四天以后, 牺牲动物由 CO2药剂过量。
	<ol>
		<li>在无菌条件下, 在腹部中心切开一个小开口, 这样开口就足以传递5毫升的转移吸管。用 RPMI-0 填充转移吸管, 并通过开口将吸管插入腹腔。</li>
		<li>用移冲洗腹腔。从腹腔内取出尽可能多的液体到无菌管 (这些是腹膜巨噬细胞)。重复冲洗步骤 4-5 次。</li>
	</ol>
	</li>
	<li>用3000拉德照射腹膜巨噬细胞。
	<ol>
		<li>或者, 用丝裂霉素 C 治疗腹膜巨噬细胞 (遵循步骤2.2 中描述的步骤)。</li>
	</ol>
	</li>
	<li>在无血清培养基中调节腹腔巨噬细胞的浓度为 5 x 106 。添加 OLP_pool 的股票 (最后的亚砜浓度小于 1%) 和 M CSF (最终 concentration=100 U/毫升)。 注: 在本 MOG OLP 研究中, 我们使用了0.5% 的亚甲基亚砜。因此, MOG OLP_pool 的股票被稀释 200x (例如1 μ l 的 MOG OLP_pool 的股票被添加到199μ l 的腹膜巨噬细胞)。因此, 每个 OLP 的最终浓度是4.2 微克/毫升)。</li>
	<li>在48良好的组织培养板中添加200μ l/井 (1 x 106细胞/井), 并在37° c 处孵育该板块 4 h。</li>
	<li>用200μ l/5ml RPMI-0 的温和洗涤去除换药细胞和聚丙烯酰胺珠。</li>
	<li>收集并汇集步骤2.10 中的 OLP_pool 引物 CD8+ T 单元格。在含有 25 u/毫升 IL-2 和 50 u/毫升 IL-7 的无血清培养基中, 将 OLP_pool 引物 CD8+ T 细胞浓度调整为 1 x 106细胞/毫升。增加1毫升/井到 48-井板, 其中包含 OLP_pool 脉冲腹膜巨噬细胞。</li>
	<li>四天后, 在48井板中补充培养基。在48井培养皿中去除并丢弃大约400μ l 培养基。在每个井中加入500μ l 的新鲜无血清培养基, 含有100的 u/毫升 IL-2 和100毫升的 IL-7。培养细胞在37° c 和 5% CO2。</li>
	<li>三或四天后, 检查 OLP_pool-restimulated CD8+ T 细胞的 OLP_pool 特异, Qa-1-restricted 反应酶联 immunospot 检测 (ELISPOT)。在这一点之后, re-stimulate CD8 的+ T 单元格每 7-10 天。 注: 巨噬细胞是首选的 re-stimulation OLP_pool 引物的 CD8+ T 细胞。我们注意到, CD8 的+ T 细胞之后的增长比 DCs体外更好。</li>
</ol>
4. OLP_pool-restimulated CD8+ T 细胞系中的 OLP_pool 特异性、Qa-1-restricted 反应的测定
注: OLP_pool OLP_pool restimulated CD8+ T 细胞系中的特异性、Qa-1-restricted 反应是由 IFNγ在 OLP_pool 或 C1R 出现后由 C1R 分泌物所决定的。Qa-1b使用 IFNγ ELISPOT 检测的单元格。C1R 细胞可以在商业上获得。C1RQa-1b单元格可以通过换 C1R 单元格的 Qa-1 慢向量生成。
<ol>
	<li>添加100μ l/井的捕获抗干扰素γ抗体稀释在涂层缓冲 (PBS) 到 ELISPOT 板的井。</li></ol>在4° c 的夜间密封和孵化盘。
	<li>第二天, 丢弃含有捕获抗干扰素抗体的涂层缓冲液, 并添加200μ l/well-blocking 溶液 (无血清培养基), 并在室温下孵育2小时的板。</li>
	<li>照射 C1R 和 C1R。Qa-1b单元格, 具有9600拉德。
	<ol>
		<li>或者, 对待 C1R 和 C1R。Qa-1b细胞与丝裂霉素 C 如步骤2.2 所述, 除了治疗时间将是30分钟。</li>
	</ol>
	</li>
	<li>调整 C1R 和 C1R。Qa-1 在无血清培养基中的b细胞为 4 x 106细胞/毫升。</li>
	<li>丢弃板中的阻挡液, 添加 C1R 和 C1R。Qa-1b单元格位于50μ l/井 (20万细胞/井) 和混合。</li>
	<li>添加50μ l/100x 稀释 OLP_pool 库存 (在无血清培养基中), 并适当混合。在室温下孵育 2-3 小时。</li>
	<li>将 OLP_pool-restimulated CD8+ T 单元格调整为 1-2 x 106单元格/ml, 在含有 150 U/毫升 IL-7 的无血清培养基中, 并将50μ l/井 (5万-10万细胞/井) 添加到盘中。离心板 (58 x g) 为5分钟。</li>
	<li>在37° c 和 5% CO2的夜间孵育盘。</li>
	<li>吸气细胞悬浮。用去离子水 (250 μ l/井) 洗涤2次井。每洗一步, 允许水井浸泡5分钟。</li>
	<li>洗井3次与洗涤缓冲 I (PBS 包含 0.05% Tween20)。每洗一步, 允许水井浸泡1分钟。丢弃洗涤缓冲器。</li>
	<li>在稀释缓冲液中添加100μ l 的检测抗体 (PBS 含有10% 胎牛血清 (FBS))。</li>
	<li>在室温下孵育2小时。</li>
	<li>丢弃检测抗体溶液。洗涤井3次与250µL/井洗涤缓冲 i. 允许井在每个洗涤步骤浸泡1分钟。</li>
	<li>在稀释缓冲液中加入100μ l/好的酶共轭 (亲和-HRP) 稀释1:100 稀释。</li>
	<li>在室温下孵育1小时的板材。</li>
	<li>放弃酶共轭溶液。洗涤井4次与250µL/井洗涤缓冲 i. 允许井在每个洗涤步骤浸泡1分钟。</li>
	<li>洗井2次与250µL/洗井缓冲 II (PBS)。</li>
	<li>添加100µL 的衬底溶液 (混合 1 drop=20 µL 的原与1毫升的共体基底), 每井。监视5到60分钟的现场开发。</li>
	<li>当期望的结果开始出现时, 通过用蒸馏水冲洗水井来停止基底反应。</li>
	<li>在室温下晾干2小时或一夜, 直到完全干燥。去除板下的塑料托盘将有利于干燥。将盘子放在一个密封的塑料袋里, 直到被分析。</li>
	<li>通过在解剖显微镜下检查或使用 ELISPOT 板读取器来手动枚举斑点。请参见图 2中的代表结果。</li>

5. 在 OLP_pool 特异的 CD8+ T 细胞系中, 测定 OLP_pool 中的单个肽, 刺激 Qa-1 限制的干扰素-γ分泌
<ol>
	<li>使用上述的干扰素-γ ELISPOT 检测方法, 确定刺激 OLP_pool 特异的、Qa-1-restrictred 的干扰素分泌物的单个肽, 在 OLP_pool 特定的 CD8 的 T 细胞系中 (每个单独的肽的最终浓度为 ELISOPT 试验是10微克/毫升)。请参见图 3中的代表性结果。 注意: OLP 特定的、Qa-1-restircted 的响应被定义为响应, 它在 C1R 的存在中至少是响应的三倍。Qa-1b单元格, 但没有 OLP。在 MOG OLP 研究中, OLP68、OLP96 和 OLP105 都达到了这个标准。因此, 所有三处可能包含 Qa-1 表位12 (图 3)。然而, OLP105 始终给予最强烈的回应;因此, 我们对 OLP105 (图 4和图 5)12进行了详细的分析。</li>
</ol>
6. 在一个15度的 OLP 中确定最佳的 Qa-1 表位, 它在 OLP_pool 特定的 CD8+ T 细胞系中激发特定的、Qa-1-restricted 的反应
<ol>
	<li>合成了一个 15 OLP 的 C 和 N-末期截断肽, 如图 4所示。</li>
	<li>在 OLP 特定的 CD8+ t 细胞系中, 通过刺激 CD8 的+ t 细胞与每个被截断的肽, 以及在 C1R 或 C1R 的存在下的父母15的 OLP。Qa-1b使用以上描述的干扰素 ELISPOT 检测细胞。每个单独的肽的最终浓度用于 ELISPOT 测定是10微克/毫升。肽被定义为最佳的 Qa-1 表位, 如果肽: 1) 一致地给予相似或更强的干扰素-γ反应, 与原始的 15-在 C1R 的存在相比。Qa-1b单元格;2) 介于 8-10-滨海 (9-滨海肽的首选) 之间的肽长度绑定到 MHC I 分子15,17。请参见图 5中的代表性结果。</li>
</ol>

<ol>
	<li>Aldrich, C. J., 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+a+Tap-dependent+leader+peptide+recognized+by+alloreactive+T+cells+specific+for+a+class+Ib+antigen.">Identification of a Tap-dependent leader peptide recognized by alloreactive T cells specific for a class Ib antigen.</a> Cell. 79 (4), 649-658 (1994).</li><li>Vance, R. E., Kraft, J. R., Altman, J. D., Jensen, P. E., Raulet, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+CD94/NKG2A+is+a+natural+killer+cell+receptor+for+the+nonclassical+major+histocompatibility+complex+(MHC)+class+I+molecule+Qa-1(b).">Mouse CD94/NKG2A is a natural killer cell receptor for the nonclassical major histocompatibility complex (MHC) class I molecule Qa-1(b).</a> J Exp Med. 188 (10), 1841-1848 (1998).</li><li>Oliveira, C. 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=The+nonpolymorphic+MHC+Qa-1+mediates+CD8++T+cell+surveillance+of+antigen-processing+defects.">The nonpolymorphic MHC Qa-1 mediates CD8+ T cell surveillance of antigen-processing defects.</a> J Exp Med. 207 (1), 207-221 (2010).</li><li>Nagarajan, N. A., Gonzalez, F., Shastri, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonclassical+MHC+class+Ib-restricted+cytotoxic+T+cells+monitor+antigen+processing+in+the+endoplasmic+reticulum.">Nonclassical MHC class Ib-restricted cytotoxic T cells monitor antigen processing in the endoplasmic reticulum.</a> Nat Immunol. 13 (6), 579-586 (2012).</li><li>Hu, D., 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=Analysis+of+regulatory+CD8+T+cells+in+Qa-1-deficient+mice.">Analysis of regulatory CD8 T cells in Qa-1-deficient mice.</a> Nat Immunol. 5 (5), 516-523 (2004).</li><li>Tang, X., 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=Regulation+of+immunity+by+a+novel+population+of+Qa-1-restricted+CD8alphaalpha+TCRalphabeta++T+cells.">Regulation of immunity by a novel population of Qa-1-restricted CD8alphaalpha+TCRalphabeta+ T cells.</a> J Immunol. 177 (11), 7645-7655 (2006).</li><li>Leavenworth, J. W., Tang, X., Kim, H. J., Wang, X., Cantor, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amelioration+of+arthritis+through+mobilization+of+peptide-specific+CD8++regulatory+T+cells.">Amelioration of arthritis through mobilization of peptide-specific CD8+ regulatory T cells.</a> J Clin Invest. 123 (3), 1382-1389 (2013).</li><li>Wu, Y., Zheng, Z., Jiang, Y., Chess, L., Jiang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+specificity+of+T+cell+regulation+that+enables+self-nonself+discrimination+in+the+periphery.">The specificity of T cell regulation that enables self-nonself discrimination in the periphery.</a> Proc Natl Acad Sci U S A. 106 (2), 534-539 (2009).</li><li>Panoutsakopoulou, V., 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=Suppression+of+autoimmune+disease+after+vaccination+with+autoreactive+T+cells+that+express+Qa-1+peptide+complexes.">Suppression of autoimmune disease after vaccination with autoreactive T cells that express Qa-1 peptide complexes.</a> J Clin Invest. 113 (8), 1218-1224 (2004).</li><li>Tang, X., Maricic, I., Kumar, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-TCR+antibody+treatment+activates+a+novel+population+of+nonintestinal+CD8+alpha+alpha++TCR+alpha+beta++regulatory+T+cells+and+prevents+experimental+autoimmune+encephalomyelitis.">Anti-TCR antibody treatment activates a novel population of nonintestinal CD8 alpha alpha+ TCR alpha beta+ regulatory T cells and prevents experimental autoimmune encephalomyelitis.</a> J Immunol. 178 (10), 6043-6050 (2007).</li><li>Holderried, T. A., Lang, P. A., Kim, H. J., Cantor, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+disruption+of+CD8++Treg+activity+enhances+the+immune+response+to+viral+infection.">Genetic disruption of CD8+ Treg activity enhances the immune response to viral infection.</a> Proc Natl Acad Sci U S A. 110 (52), 21089-21094 (2013).</li><li>Wang, X., 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=Targeting+Non-classical+Myelin+Epitopes+to+Treat+Experimental+Autoimmune+Encephalomyelitis.">Targeting Non-classical Myelin Epitopes to Treat Experimental Autoimmune Encephalomyelitis.</a> Sci Rep. 6, 36064 (2016).</li><li>Lo, W. F., Dunn, C. D., Ong, H., Metcalf, E. S., Soloski, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+and+host+factors+involved+in+the+major+histocompatibility+complex+class+Ib-restricted+presentation+of+Salmonella+Hsp+60:+novel+pathway.">Bacterial and host factors involved in the major histocompatibility complex class Ib-restricted presentation of Salmonella Hsp 60: novel pathway.</a> Infect Immun. 72 (5), 2843-2849 (2004).</li><li>Xu, W., 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=The+nucleocapsid+protein+of+Rift+Valley+fever+virus+is+a+potent+human+CD8++T+cell+antigen+and+elicits+memory+responses.">The nucleocapsid protein of Rift Valley fever virus is a potent human CD8+ T cell antigen and elicits memory responses.</a> PLoS One. 8 (3), e59210 (2013).</li><li>Schumacher, T. N., 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=Peptide+selection+by+MHC+class+I+molecules.">Peptide selection by MHC class I molecules.</a> Nature. 350 (6320), 703-706 (1991).</li><li>Zhang, X., Goncalves, R., Mosser, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+and+characterization+of+murine+macrophages.">The isolation and characterization of murine macrophages.</a> Curr Protoc Immunol. , (2008).</li><li>Guo, H. 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=Different+length+peptides+bind+to+HLA-Aw68+similarly+at+their+ends+but+bulge+out+in+the+middle.">Different length peptides bind to HLA-Aw68 similarly at their ends but bulge out in the middle.</a> Nature. 360 (6402), 364-366 (1992).</li><li>Soloski, M. J., Metcalf, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+involvement+of+class+Ib+molecules+in+the+host+response+to+infection+with+Salmonella+and+its+relevance+to+autoimmunity.">The involvement of class Ib molecules in the host response to infection with Salmonella and its relevance to autoimmunity.</a> Microbes Infect. 3 (14-15), 1249-1259 (2001).</li><li>Smith, T. R., 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=Dendritic+cells+use+endocytic+pathway+for+cross-priming+class+Ib+MHC-restricted+CD8alphaalpha+TCRalphabeta++T+cells+with+regulatory+properties.">Dendritic cells use endocytic pathway for cross-priming class Ib MHC-restricted CD8alphaalpha+TCRalphabeta+ T cells with regulatory properties.</a> J Immunol. 182 (11), 6959-6968 (2009).</li><li>Kalra, A., Mukherjee, P., Chauhan, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+fine+specificity+of+the+immune+response+to+a+Plasmodium+falciparum+rhoptry+neck+protein,+PfAARP.">Characterization of fine specificity of the immune response to a Plasmodium falciparum rhoptry neck protein, PfAARP.</a> Malar J. 15, 457 (2016).</li><li>Kambayashi, 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=The+nonclassical+MHC+class+I+molecule+Qa-1+forms+unstable+peptide+complexes.">The nonclassical MHC class I molecule Qa-1 forms unstable peptide complexes.</a> J Immunol. 172 (3), 1661-1669 (2004).</li></ol>

作者声明没有利益冲突。

在这里, 我们描述了一个用于分析蛋白质中 Qa-1 表位的协议。关于该议定书, 以前还报告了其他一些战略。首先, 异基因 CD8+ T 单元格线和克隆用于标识 Qdm1。其次, 一个假定的 Qa-1-binding 主题从分析 Qdm 用于识别 HSP60p216-224 和 TCRBV8.1 表位9,18。第三, 从蛋白质中单独重叠的肽被用于免疫动物。随后, 从免疫动物中分离出来的 CD8+<...

Qa-1 属于一组经典主要组织相容性复合体 1b (MHC-Ib) 分子的小鼠。它的人类同源是 HLA E。以前的证据表明 Qa-1 分子具有重要的生物学功能。首先, Qa-1 分子在测量细胞结构和功能完整性方面起着重要作用。在这方面, Qa-1 分子已经进化了一些策略来监测细胞的正常功能。一个这样的策略使 Qa-1 分子能够形成与加工的领导肽 (表位),即Qa-1 行列式修饰 (Qdm), 从经典的 MHC Ia 分子在内质网1处理的复合物。这些 Qa-1/Qdm 配合物随后在细胞表面显示, 并结合 nk 细胞抑制性 NKG2A 受体, 抑制 nk 杀伤活性2。如果 MHC-Ia 分子的表达丢失, 细胞 (如恶性细胞) 就会对 NK 的杀伤2敏感。另一种策略使 Qa-1 分子能够在细胞表面形成新的 Qa-1/epitope 配合物 (与抗原处理相关的运输载体)3和/或 ERAAP (内质网肽相关抗原处理)4 (这两种缺陷经常发生在恶性细胞中)。表达这些新的 Qa-1/epitope 复合体的细胞可以通过表位特异的 Qa-1-restricted CD8+ T 细胞识别和消除。其次, Qa-1 分子诱导免疫调节5。在这方面, Qa-1/epitope 配合物已被证明刺激 CD8+调节 T (t) 细胞, 这是重要的预防免疫介导的损害 self-tissues67,8 ,9,10。第三, Qa-1-restricted CD8+ t 细胞已被证明可以限制免疫应答病毒感染11。
因此, 具体增加表位特定的 Qa-1-restrictred CD8+ T 细胞是一个潜在的可行的策略, 以消除异常细胞, 增强免疫调节, 并为控制的大小病毒诱导的免疫应答。虽然尚未确定是否增加表位特定的 Qa-1-restricted CD8+ T 细胞可以增强免疫监测和限制病毒诱导的免疫应答, 但我们的实验室和其他人已经清楚地表明免疫 Qa-1 表位可增强 Qa-1-restricted CD8 t 细胞特异性自身免疫 CD4 + t 细胞的功能, 从而有效控制 CD4+ t 细胞介导的自身免疫性疾病实验性反应性脑脊髓炎动物模型的多样性 ( 人多发性硬化动物模型 )6,10, 胶原诱导关节炎 ( 人类类风湿关节炎动物模型 )7, 以及肥胖糖尿病 (人类1型糖尿病动物模型)8。此外, 我们发现, 免疫与组织特定的 Qa-1 表位导致特定的控制免疫介导的炎症, 在该组织通过增强 CD8+ t 细胞12。上述临床前研究的成功表明, 需要对 Qa-1 表位免疫进行全面评估, 以治疗组织特异性免疫介导的疾病, 并有可能治疗与自来水缺乏症相关的其他疾病, 并ERAAP
因此, 有一个技术的需求, 可以可靠和快速地分析 Qa-1 表位的蛋白质。在这方面, 描述了数量有限的生物重要的 Qa-1 表位。大多数这些 Qa-1 表位被确定偶然在研究 CD8+ T 细胞对细菌的反应13, 细胞缺乏在 TAP3, 细胞缺陷在 ERAAP4, 和细胞, 导致 EAE6, 9。因此, 一个高吞吐量的技术是可取的, 以确定生物重要的 Qa-1 表位在一个定义的蛋白质。在下面, 我们描述一个重叠的肽 (OLP) 图书馆策略, 映射功能的 Qa-1 表位在一个蛋白质使用 Qa-1-resrticted CD8+ T 细胞系特定的 OLP 池 (OLP_pool) 的蛋白质。

<table><tbody><tr><td>The protein to be analyzed</td><td>N/A</td><td>N/A</td><td>Sequence of the protein can be obtained from NCBI</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>Cat#: D2650 SIGMA</td><td>DMSO should be sterile and cell culture tested.</td></tr><tr><td>K&lt;sup&gt;b-/-&lt;/sup&gt;D&lt;sup&gt;b-/-&lt;/sup&gt; mice</td><td>The Lackson Laboratory</td><td>Stock#: 019995</td><td>We used Taconic H2-K&lt;sup&gt;b&lt;/sup&gt;H2-D&lt;sup&gt;b&lt;/sup&gt; doube knockout mice (Cat#: 4215-F and 4215-M) which however are not available anymore.</td></tr><tr><td>AIM V Serum Free Medium</td><td>ThermoFisher Scientific</td><td>Cat#: 12055091</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>ThermoFisher Scientific</td><td>Cat#: 21985023</td><td></td></tr><tr><td>Sodium pyruvate</td><td>ThermoFisher Scientific</td><td>Cat#: 11360070</td><td></td></tr><tr><td>Nonessential Amino Acids</td><td>ThermoFisher Scientific</td><td>Cat#: 11140076</td><td></td></tr><tr><td>Dynabeads CD8 Positive Isolation Kit</td><td>ThermoFisher Scientific</td><td>Cat#: 11333D</td><td></td></tr><tr><td>Bio-Gel P-100</td><td>Bio-Rad</td><td>Cat#: 150-4171</td><td></td></tr><tr><td>Phoshate Balanced Solution (PBS)</td><td>ThermoFisher Scientific</td><td>Cat#: 20012027</td><td></td></tr><tr><td>Trasfer pipette</td><td>Globe Scientific</td><td>Mfg#: 137238</td><td></td></tr><tr><td>Murine M-CSF</td><td>PeproTech</td><td>Cat#: 315-02</td><td></td></tr><tr><td>48-well tissue culture plates</td><td>USA Scientific</td><td>Cat#: CC7682-7548</td><td></td></tr><tr><td>Corning Costar TC-treated Multiple well Plates, 96-well, V-shaped bottom</td><td>Sigma-Aldrich</td><td>Cat#: Z372129 Sigma</td><td></td></tr><tr><td>1ml deep 06-well PP plate, sterile</td><td>USA Scientific</td><td>Item#: 1896-1110</td><td></td></tr><tr><td>Recombinant murine IL-2</td><td>PeproTech</td><td>Cat#: 212-12</td><td></td></tr><tr><td>Recombinant murine IL-7</td><td>PeproTech</td><td>Cat#L: 217-17</td><td></td></tr><tr><td>Capture anti-IFN-&amp;gamma; antibody</td><td>BD Biosciences</td><td>Cat#: 551881</td><td></td></tr><tr><td>ELISPOT plate</td><td>Sigma-Aldrich</td><td>Cat#: S2EM004M99</td><td></td></tr><tr><td>C1R</td><td>ATCC</td><td>Cat#: ATCC CRL-1993</td><td></td></tr><tr><td>C1R.Qa-1&lt;sup&gt;b&lt;/sup&gt;</td><td></td><td></td><td>Custom made (GenBank access#: NM_010398.3)</td></tr><tr><td>Qa-1 lentiviral vector</td><td>GeneCopoeia</td><td>Product#: Mm02955</td><td></td></tr><tr><td>Detection anti-IFN-&amp;gamma; antibody</td><td>BD Biosciences</td><td>Cat#: 551881</td><td></td></tr><tr><td>Tween20</td><td>Sigma-Aldrich</td><td>Cat#: P9416</td><td></td></tr><tr><td>Streptavidin-HRP</td><td>BD Biosciences</td><td>Cat#: BD557630</td><td></td></tr><tr><td>AEC substrate</td><td>BD Biosciences</td><td>Cat#: 551951</td><td></td></tr><tr><td>ImmunoSpot Analyzer</td><td>ImmunoSpot</td><td></td><td>Any immunoSpot analyer should work for this purpose.</td></tr></tbody></table>

overlapping peptide library to map qa-1 epitopes in a protein

Qa-1 (HLA E 在人) 属于一组经典主要组织相容性复合体 1b (MHC-Ib) 分子。最近的数据表明, Qa-1 分子在测量细胞的结构和功能完整性, 诱导免疫调节, 并限制免疫应答病毒感染的重要作用。此外, 通过表位免疫功能增强 Qa-1-restricted CD8 的+ T 细胞, 在几种自身免疫性疾病动物模型中显示了治疗效果,例如: 实验过敏脑脊髓炎,胶原诱导的关节炎, 肥胖糖尿病。因此, 迫切需要一种方法, 可以有效和快速地确定功能 Qa-1 表位的蛋白质。在这里, 我们描述一个协议, 利用 Qa-1-restricted CD8+ T 细胞系特定的重叠肽 (OLP) 库, 以确定 Qa-1 表位的蛋白质。这个 OLP 库包含15的重叠多肽, 覆盖了整个蛋白质的长度, 相邻的多肽与11种氨基酸重叠。使用该协议, 我们最近确定了一个 9 Qa-1 表位的髓鞘胶质糖蛋白 (MOG)。这一新映射的 MOG Qa-1 表位被证明是为了诱导特定的抗原, Qa-1-restricted CD8+ T 细胞增强髓鞘特异的免疫调节。因此, 该协议对于将来研究 Qa-1-restricted CD8+ T 单元格的新目标和功能是有用的。

一种覆盖整个蛋白质长度的 OLP 库的设计
从蛋白质的 N 末端开始, 每个肽是15氨基酸 (15)。因此, 第一肽跨越位置1到位置15。第二肽的 N 末端与第一个肽的 C 末端重叠由11氨基酸。因此, 第二肽跨越位置5到位置19。将其余的肽设计到蛋白质的 C 端末端 (图 1)。我们选择了 15-滨海图书馆, 因为我们和其他人已经表?...

Watch this Scientific Journal Video about Overlapping Peptide Library to Map Qa-1 Epitopes in a Protein at JoVE.com

Overlapping Peptide Library to Map Qa-1 Epitopes in a Protein

重叠肽库在蛋白质 Qa-1 表位中的映射

Qa-1 (HLA) 属于一组经典主要组织相容性复杂的1b 分子。免疫 Qa-1-binding 表位已表明, 以加强组织特异性免疫调节和改善一些自身免疫性疾病。在此, 我们描述一个重叠的肽库策略, 以确定 Qa-1 表位的蛋白质。

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b (MHC-Ib) molecules. Recent data suggest that Qa-1 molecules ...

overlapping-peptide-library-to-map-qa-1-epitopes-in-a-protein

Research

JoVE Journal

Immunology and Infection

6.8K 次观看.  Loma Linda University. Qa-1 (HLA) 属于一组经典主要组织相容性复杂的1b 分子。免疫 Qa-1-binding 表位已表明, 以加强组织特异性免疫调节和改善一些自身免疫性疾病。在此, 我们描述一个重叠的肽库策略, 以确定 Qa-1 表位的蛋白质。

视频: Overlapping Peptide Library to Map Qa-1 Epitopes in a Protein

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

Biopanning bacterial display libraries is a proven technique for discovery of peptide affinity reagents, a robust alternative to antibodies. The semi-automated sorting method herein has streamlined biopanning to decrease the occurrence of false positives. Here we illustrate the thought process and techniques applied in evaluating candidates and minimizing downstream analysis.

细菌显示文库的半自动化筛选肽亲和试剂的发现与分离结果分析

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

科研

生物化学

Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against human leukocyte antigens (HLA). Histocompatibility guidelines are based on laboratory tests of anti-HLA immunity, which presents either as pre-existing or de novo generated HLA antibodies that constitute a major transplantation barrier. Current tests are built on a single-antigen beads (SAB) platform using a fixed set of ~100 preselected recombinant HLA antigens to probe transplant sera. However, in humans there exist a far greater variety of HLA types, with no two individuals other than identical twins who can share the same combination of HLA sequences. While advanced technologies for HLA typing and direct sequencing can precisely capture any mismatches in DNA sequence between a donor's and recipient's HLA, the SAB assay, due to its limited variety in sequence representation, is unable to precisely detect alloantibodies specifically against the donor HLA mismatches. We sought to develop a complementary method using a different technology to detect and characterize anti-donor HLA antibodies on a personalized basis. The screening tool is a custom peptide array of donor HLA-derived sequences for probing post-transplant sera of the organ recipient to assess the risk for antibody-mediated rejection. On a single array for one donor-recipient pair, up to 600 unique peptides are made based on the donor's HLA protein sequences, each peptide carrying at least one mismatched residue in a 15-amino acid sequence. In our pilot experiments to compare antigen patterns for pre- and post-transplant sera on these arrays, we were able to detect anti-HLA signals with the resolution that also allowed us to pinpoint the immune epitopes involved. These personalized antigen arrays allow high-resolution detection of donor-specific HLA epitopes in organ transplantation.

Mismatches in human leukocyte antigen (HLA) sequences between organ donor and recipient pairs are the major cause of antibody-mediated rejection in organ transplantation. Here we present the use of custom antigen arrays that are based on individual donors' HLA sequences to probe anti-donor HLA alloantibodies in organ recipients.

个性化肽阵列在器官移植 HLA 抗体检测中的研究

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against ...

A Fast and Quantitative Method for Post-translational Modification and Variant Enabled Mapping of Peptides to Genomes

Cross-talk between genes, transcripts, and proteins is the key to cellular responses; hence, analysis of molecular levels as distinct entities is slowly being extended to integrative studies to enhance the understanding of molecular dynamics within cells. Current tools for the visualization and integration of proteomics with other omics datasets are inadequate for large-scale studies. Furthermore, they only capture basic sequence identify, discarding post-translational modifications and quantitation. To address these issues, we developed PoGo to map peptides with associated post-translational modifications and quantification to reference genome annotation. In addition, the tool was developed to enable the mapping of peptides identified from customized sequence databases incorporating single amino acid variants. While PoGo is a command line tool, the graphical interface PoGoGUI enables non-bioinformatics researchers to easily map peptides to 25 species supported by Ensembl genome annotation. The generated output borrows file formats from the genomics field and, therefore, visualization is supported in most genome browsers. For large-scale studies, PoGo is supported by TrackHubGenerator to create web-accessible repositories of data mapped to genomes that also enable an easy sharing of proteogenomics data. With little effort, this tool can map millions of peptides to reference genomes within only a few minutes, outperforming other available sequence-identity based tools. This protocol demonstrates the best approaches for proteogenomics mapping through PoGo with publicly available datasets of quantitative and phosphoproteomics, as well as large-scale studies.

Here we present the proteogenomic tool PoGo and protocols for fast, quantitative, post-translational modification and variant enabled mapping of peptides identified through mass spectrometry onto reference genomes. This tool is of use to integrate and visualize proteogenomic and personal proteomic studies interfacing with orthogonal genomics data.

一种快速、定量的后向基因组转化和变异的多肽映射方法

Cross-talk between genes, transcripts, and proteins is the key to cellular responses; hence, analysis of molecular levels as distinct entities is slowly ...

遗传学

Immunopeptidomics: Isolation of Mouse and Human MHC Class I- and II-Associated Peptides for Mass Spectrometry Analysis

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science of investigating the composition of peptides presented by major histocompatibility complex (MHC) class I and class II molecules using mass spectrometry (MS) technology platforms. Among all the steps in an MS-based immunopeptidomics workflow, sample preparation is critically important for capturing high-quality data of therapeutic relevance. Here, step-by-step instructions are described to isolate MHC class I and II-associated peptides by immunoaffinity purification from quality control samples, from mouse (EL4 and A20), and human (JY) cell lines more specifically. The various reagents and specific antibodies are thoroughly described to isolate MHC-associated peptides from these cell lines, including the steps to verify the beads-binding efficiency of the antibody and the elution efficiency of the MHC-peptide complexes from the beads. The protocol can be used to establish and standardize an immunopeptidomics workflow, as well as to benchmark new protocols. Moreover, the protocol represents a great starting point for any non-experts in addition to foster the intra- and inter-laboratory reproducibility of the sample preparation procedure in immunopeptidomics.

Here, we present a protocol for the purification of MHC class I and class II peptide complexes from mouse and human cell lines providing high-quality immunopeptidomics data. The protocol focuses on sample preparation using commercially available antibodies.

免疫肽组学:分离小鼠和人MHC I类和II类相关肽用于质谱分析

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science ...

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that may facilitate the development of vaccines and peptide drugs. Peptide scanning is a simple and efficient method that straightforwardly maps the linear epitope recognized by a monoclonal antibody (mAb). Here, the authors present an epitope determination methodology involving serially truncated recombinant proteins, synthetic peptide design, and dot-blot hybridization for the antigenic recognition of nervous necrosis virus coat protein using a neutralizing mAb. This technique relies on the dot-blot hybridization of synthetic peptides and mAbs on a polyvinylidene fluoride (PVDF) membrane. The minimum antigenic region of a viral coat protein recognized by the RG-M56 mAb can be narrowed down by step-by-step trimmed peptide mapping onto a 6-mer peptide epitope. In addition, alanine scanning mutagenesis and residue substitution can be performed to characterize the binding significance of each amino acid residue making up the epitope. The residues flanking the epitope site were found to play critical roles in peptide conformation regulation. The identified epitope peptide may be used to form crystals of epitope peptide-antibody complexes for an x-ray diffraction study and functional competition, or for therapeutics.

Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

肽扫描辅助一个单克隆抗体识别的线性B细胞表位的鉴定

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that ...

免疫与感染

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.

加上质谱肽免疫亲和富集的蛋白质定量

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

生物学

Biosensor-based High Throughput Biopanning and Bioinformatics Analysis Strategy for the Global Validation of Drug-protein Interactions

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation of the drug-protein interactions is highly desirable for not only understanding the molecular mechanisms underlying therapeutic efficacy but also for assessing drug characteristics, such as adsorption, distribution, metabolism, excretion, and toxicity (ADMET) for clinical use. Here, we present a biosensor-based high throughput strategy for the biopanning of T7 phage-displayed short peptides that can be easily displayed on the phage capsid. Subsequent analysis of the amino acid sequences of peptides containing short segments, as &#34;broken relics&#34;, of the drug-binding sites using bioinformatics programs in receptor ligand contact (RELIC) suite, is also shown. By applying this method to two clinically approved drugs, an anti-tumor irinotecan, and an anti-flu oseltamivir, the detailed process for collecting the drug-recognizing peptide sequences and highlighting the drug-binding sites of the target proteins are explained in this paper. The strategy described herein can be applied for any small molecules of interest.

This study aimed to present a strategy for identifying drug-peptide interactions. The strategy involves the biopanning of drug-recognizing short peptides based on a quartz-crystal microbalance (QCM) biosensor, followed by bioinformatics analysis for quantitatively assessing the information obtained for the drug recognition and annotation of the drug-binding sites on proteins.

基于生物传感器的高通量生物跨越和生物信息学分析战略，用于全球药物-蛋白质相互作用验证

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation ...

生物工程

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

高通量MHC II类分子结合分析抗原表位肽的定量分析

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

鉴定蛋白质相互作用的网站使用多肽阵列

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

A Peptide Microarray Technology for Specificity Profiling of Antibodies

Source: Cornett, E. M., et al. <a href="https://app.jove.com/v/55912">Analysis of Histone Antibody Specificity with Peptide Microarrays.</a> J. Vis. Exp. (2017).
This video demonstrates a technique that applies peptide microarray technology to profile the specificity of antibodies recognizing post-translational modifications (PTMs) on histone peptides. A microarray slide containing immobilized fluorophore-conjugated histone peptides with known PTM combinations is treated with target PTM-specific antibodies. This process helps identify the antibody&#39;s specificity for the target and its potential cross-reactivity with non-target PTMs.

用于抗体特异性分析的肽微阵列技术

1. Designing the Array Slide and Source Plate Layout

	Under the &#39;Plan a slide layout&#39; heading on the ArrayNinja interface, click on the link that ...