Name: enrichment of bacterial lipoproteins and preparation of n-terminal lipopeptides for structural determination by mass spectrometry
Uploaded: 2018-05-21T12:30:00.000+00:00
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Description: Watch this Scientific Journal Video about Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry at JoVE.com

在梅瑞狄斯实验室的研究由埃伯利科学学院 (宾夕法尼亚州立大学) 提供的启动资金支持。我们感谢 Laremore 博士的专家技术咨询和获得设备在宾夕法尼亚州立大学的蛋白质组学和质谱的核心设施, 学院公园, PA, 在那里进行质谱分析。

1. 细胞生长和裂解
<ol><li>在15毫升胰蛋白酶大豆肉汤中生长细菌或类似的富媒体到晚期指数相 (OD600 1.0-1.5)。用离心法收割细胞, 用三缓冲盐水/EDTA (TBSE) 冲洗一次, 并继续使用协议或冷冻直至用途。 注: TBSE:20 毫米三盐酸盐 (HCl), pH 值 8.0, 130 毫米氯化钠 (氯化钠), 和5毫米乙二胺乙酸 (EDTA))。脂蛋白表达和修饰可能受生长条件 (例如酸度、盐度、生长介质) 和生长阶段15的影响。细胞生长可以按需要进行缩放, 但是建议用15毫升的细胞来单独制备脂蛋白, 因为过量的生物量可以降低脂蛋白的产量。一个15毫升的准备通常产生足够的样本, 以最佳负载2-4 车道上的标准 SDS 页迷你凝胶。</li>
	<li>并用重悬细胞在 800 ul TBSE 与1毫米苄磺酰氟 (PMSF) 和0.5 毫克/毫升溶菌酶。转移解决方案到一个2.0 毫升螺纹离心管与螺钉盖和 O 形环和孵化20分钟37摄氏度。 注意: 某些物种不容易溶菌酶溶解。应用适当的裂解酶代替以帮助裂解。</li>
	<li>将 800 ul 0.1 毫米氧化锆/二氧化硅微珠添加到管中, 在每一个周期之间的5个循环中, 以最大速度 (7000 rpm) 在均质机上摇晃, 以达到每一个循环之间的2分钟, 从而破坏细胞。</li>
	<li>离心样品在 3000 x g 5 分钟在4°c (对颗粒珠和未连续的细胞)。将上清液转移到新的2.0 毫升离心管上, 并保持冰。</li>
	<li>添加 200 ul TBSE 到剩余的颗粒, 并返回到均质机的一个额外的周期。离心机 (如上所述) 与上清液结合上清液 (应为 800-1000 ul 总容积)。</li>
</ol>2. TX-114 相分配法富集脂蛋白
<ol><li>用 TX-114 表面活性剂补充上清液到最终浓度为 2% (卷/量) 通过增加等量的 4% (音量/容量) 表面活性剂在冰冷 TBSE 和孵化在冰上1小时, 混合通过反转每 ~ 15 分钟。 注: 当冷却时, 上清和表面活性剂将被混溶。</li>
	<li>将管子转移到37摄氏度的水浴中, 孵化10分钟, 诱导相分离。在室温下, 将样品以 1万 x g 为10分钟离心, 以保持双相分离。</li>
	<li>轻轻地移开上水相并丢弃。在较低的表面活性剂阶段加入冰冷 TBSE, 将管子重新装入原体积, 并将其转化为混合。在冰上孵育10分钟。</li>
	<li>将管子转移到37摄氏度的水浴中, 孵化10分钟, 以诱导相分离, 然后离心机在 1万 x g 处室温下进行10分钟。</li>
	<li>重复步骤 2.3-2.4 次, 总共3个分色。移除上水相并丢弃。</li>
	<li>去除萃取过程中形成的沉淀蛋白颗粒 (可见于管底), 加入1的冰冷 TBSE 到表面活性剂相。离心机在4°c 在 1.6万 x g 为2分钟到颗粒不溶性蛋白质。 注: 样品应保持单一的阶段。如果相分离发生, 重新冷却样品和离心机。该颗粒一般由非脂蛋白污染物组成, 但可保留以供进一步分析。</li>
	<li>立即转移上清到一个新鲜的2.0 毫升离心管含有 1250 ul 100% 丙酮。吸管向上和向下彻底清洗样品从尖端, 因为它将是粘性的。混合通过反转和孵化隔夜在-20 °c 沉淀蛋白。</li>
	<li>离心样品在 1.6万 x g 在室温下为20分钟的颗粒脂蛋白, 注意管的方向。脂蛋白会沿着管子的外壁形成一层薄薄的白色薄膜。</li>
	<li>用100% 丙酮清洗颗粒两次。醒酒丙酮, 并允许样品风干。</li>
	<li>添加 20-40 ul 10 毫米三 HCl, pH 8.0 或标准 Laemmli SDS 页样本缓冲区16 , 并彻底并用重悬吹打上下对墙壁与沉淀脂蛋白。用吸管尖刮掉管子的一侧以去除脂蛋白。 注: 脂蛋白不会溶解在三个缓冲, 而是形成一个悬浮。<ol>
<li>储存脂蛋白在-20 °c, 直到使用。</li>
	</ol>
</li>
</ol>3. SDS 页、Electroblotting 和胭脂红的染色
<ol><li>使用标准方法16, 用 SDS 页分隔脂蛋白。 注: 适当的凝胶丙烯酰胺百分比将根据其预期用途和脂蛋白的大小而异。在这里,大肠杆菌脂蛋白被分离了10% 三甘氨酸凝胶, 而16.5% 三 tricine 凝胶用于小的大肠杆菌脂蛋白 Lpp17。</li>
	<li>使用标准的 electroblotting 程序将脂蛋白转移到硝化棉转移膜上。 注意: 使用 Bjerrum 谢弗-尼尔森缓冲器加 0.1% SDS 的半干转移是在 25 V, 1.3 mA 上执行15分钟18。交替地, 聚偏聚氟 (PVDF) 膜可以使用, 但由于它比硝化纤维素更疏水性, 它可能减少疏水性脂肽的有效洗脱从膜在以后的步骤。</li>
	<li>将硝化膜转移到容器中, 用胭脂红溶液 (0.2% (w/v) 胭脂红5% 乙酸) 覆盖。轻轻摇动5分钟, 或直到红色粉红色的条纹可见。</li>
	<li>倒入胭脂红溶液, 用 dH2O 小心冲洗硝化棉膜, 去除多余的污渍。 注: 胭脂红染色带将迅速 destain。如果乐队消失, 重复染色过程。</li>
	<li>用干净的刀片, 消费所需的带和转移到离心管。用1毫升 dH2O 洗涤三次, 完全 destain 带。 注意: 协议可以在这里暂停。将被切除的部分用水覆盖-20 摄氏度, 直到使用。</li>
	<li>将该部分转移到干净的表面, 用干净的剃刀刀片, 将硝化棉片切成大约1毫米 x 1 毫米的小块. 将这些碎片收集成一个0.5 毫升低蛋白结合离心管。</li>
	<li>用0.5 毫升新鲜制备的50毫米碳酸氢铵 (NH4HCO3), 用高效液相色谱 (HPLC) 级水中的 pH 值7.8 清洗两次工件。</li>
</ol>4. 胰蛋白酶消化, 从硝化棉膜中提取脂肽, 沉积到 MALDI 靶上, 并进行数据采集
<ol><li>并用重悬硝化棉片在 20 ul 20 微克/毫升溶液的胰蛋白酶在50毫米NH4HCO3, pH 7.8 在 HPLC 级水中。涡流混合, 然后简单地旋转, 以确保所有的部分完全覆盖的胰蛋白酶溶液。用石蜡膜覆盖管盖, 以防止蒸发和孵育消化过夜在37摄氏度。</li>
	<li>旋转样品在 1.6万 x g 三十年代和去除液体吹打。 注意: 这去除了 trypsinized 亲水性肽, 可以在以后被 MALDI 的 MS 用于蛋白质鉴定。</li>
	<li>在 HPLC 级水中加入 50 ul 0.5% 三氟乙酸酸 (TFA)。涡流混合, 然后简单地旋转样品, 以确保所有零件都被溶液覆盖。室温下孵育10分钟。用吹打除去液体。 警告:TFA 是有害的, 如果吸入。小心处理并在化学油烟机中使用。避免接触皮肤。</li>
	<li>重复步骤4.3 与 50 ul 10% 乙腈在 HPLC 级水。 警告:乙腈吸入有害。小心处理并在化学油烟机中使用。避免接触皮肤。</li>
	<li>重复步骤4.3 与 50 ul 20% 乙腈在 HPLC 级水。 注意: 这去除松散地束缚在硝化棉中的任何适度疏水肽。</li>
	<li>为洗脱紧密绑定的脂肽, 添加 15 ul 的新制成的10毫克/毫升α氰基-4-肉桂酸 (CHCA) 基质溶解在氯仿-甲醇 (2:1, v/v) 和孵化为10分钟在室温下, 间歇性涡流。<ol>
<li>交替地, 添加 15 ul 氯仿-甲醇洗脱脂肽, 并与基质混合在以后的时间。其他矩阵, 如 25-苯甲酸酸 (DHB), 应作为替代 CHCA, 如果获得不满意的结果, 为特定的脂肽组成。 警告:氯仿和甲醇都是有害的, 如果吸入。小心处理并在化学油烟机中使用。避免接触皮肤。</li>
		<li>
可选:为促进钠合物的形成, 用水碳酸氢钠 (NaHCO3) 补充氯仿-甲醇中 CHCA 的溶液, 最终浓度为1毫米。</li>
	</ol>
</li>
	<li>简单地旋转示例。用吸管, 小心地将液体转移到一个新的低蛋白结合离心管。此解决方案将包含 N 终端脂肽的大部分。 注: 硝化棉可部分或完全溶解在氯仿-甲醇溶液中。这不会对 MS 结果产生负面影响, 可以作为替代方法来增加脂肽的回报。PVDF 膜不会以同样的方式溶解。</li>
	<li>将洗脱脂肽的 1 ul 沉积在抛光钢 MALDI 靶上 CHCA。 注: 氯仿-甲醇将迅速蒸发, 留下结晶 CHCA 和脂肽。可选的第二 1 ul 整除可以放置到同一地点, 以增加样品浓度。氯仿-甲醇在沉积到靶上后可能会有很大的扩散, 因此应注意避免样品在靶上混合。建议将每个样品的多个斑点存入并拍摄。</li>
	<li>立即进行质谱分析。扫描现场的所有区域的脂肽信号, 特别是如果现场包含两层样本, 因为第二层可能推脂肽到现场的外缘。 注: 建议的启动激光强度为 25%, 但可能需要增加强度获取信号和总和多光谱 (20 或更多的扫描), 以达到足够的信噪比。在这里, 光谱是在一个 MALDI 的仪器上获得的 (参见材料表), 使用工厂配置的仪器方法对 700-3500 m/z范围的反射器进行正离子检测。该仪器用牛血清白蛋白 (BSA) 胰蛋白酶肽混合物进行校准。</li>
</ol>

<ol>
	<li>Babu, M. M., 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=A+database+of+bacterial+lipoproteins+(DOLOP)+with+functional+assignments+to+predicted+lipoproteins.">A database of bacterial lipoproteins (DOLOP) with functional assignments to predicted lipoproteins.</a> J Bacteriol. 188 (8), 2761-2773 (2006).</li><li>Narita, S., Tokuda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+lipoproteins;+biogenesis,+sorting+and+quality+control.">Bacterial lipoproteins; biogenesis, sorting and quality control.</a> Biochim Biophys Acta. 1862 (11), 1414-1423 (2016).</li><li>Kovacs-Simon, A., Titball, R. W., Michell, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipoproteins+of+bacterial+pathogens.">Lipoproteins of bacterial pathogens.</a> Infect Immun. 79 (2), 548-561 (2011).</li><li>Nguyen, M. T., G&#246;tz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipoproteins+of+Gram-Positive+Bacteria:+Key+Players+in+the+Immune+Response+and+Virulence.">Lipoproteins of Gram-Positive Bacteria: Key Players in the Immune Response and Virulence.</a> Microbiol Mol Biol Rev. 80 (3), 891-903 (2016).</li><li>Nakayama, H., Kurokawa, K., Lee, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipoproteins+in+bacteria:+structures+and+biosynthetic+pathways.">Lipoproteins in bacteria: structures and biosynthetic pathways.</a> FEBS J. 279 (23), 4247-4268 (2012).</li><li>Kurokawa, K., 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=Novel+bacterial+lipoprotein+structures+conserved+in+low-GC+content+Gram-positive+bacteria+are+recognized+by+Toll-like+receptor+2.">Novel bacterial lipoprotein structures conserved in low-GC content Gram-positive bacteria are recognized by Toll-like receptor 2.</a> J Biol Chem. 287 (16), 13170-13181 (2012).</li><li>Kurokawa, K., 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+Triacylated+ATP+Binding+Cluster+Transporter+Substrate-binding+Lipoprotein+of+Staphylococcus+aureus+Functions+as+a+Native+Ligand+for+Toll-like+Receptor+2.">The Triacylated ATP Binding Cluster Transporter Substrate-binding Lipoprotein of Staphylococcus aureus Functions as a Native Ligand for Toll-like Receptor 2.</a> J Biol Chem. 284 (13), 8406-8411 (2009).</li><li>Asanuma, M., 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=Structural+evidence+of+&#945;-aminoacylated+lipoproteins+of+Staphylococcus+aureus.">Structural evidence of &#945;-aminoacylated lipoproteins of Staphylococcus aureus.</a> FEBS J. 278 (5), 716-728 (2011).</li><li>Armbruster, K. M., Meredith, T. C. <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+the+Lyso-Form+N-Acyl+Intramolecular+Transferase+in+Low-GC+Firmicutes.">Identification of the Lyso-Form N-Acyl Intramolecular Transferase in Low-GC Firmicutes.</a> J Bacteriol. 199 (11), e00099-e00017 (2017).</li><li>Serebryakova, M. V., Demina, I. A., Galyamina, M. A., Kondratov, I. G., Ladygina, V. G., Govorun, V. 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+acylation+state+of+surface+lipoproteins+of+Mollicute+Acholeplasma+laidlawii.">The acylation state of surface lipoproteins of Mollicute Acholeplasma laidlawii.</a> J Biol Chem. 286 (26), 22769-22776 (2011).</li><li>Feng, S. H., Lo, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+mouse+spleen+B-cell+proliferation+and+secretion+of+immunoglobulin+by+lipid-associated+membrane+proteins+of+Mycoplasma+fermentans+incognitus+and+Mycoplasma+penetrans.">Induced mouse spleen B-cell proliferation and secretion of immunoglobulin by lipid-associated membrane proteins of Mycoplasma fermentans incognitus and Mycoplasma penetrans.</a> Infect Immun. 62 (9), 3916-3921 (1994).</li><li>Feng, S. H., Lo, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+extract+of+Mycoplasma+penetrans+proteinase+K-digested+lipid-associated+membrane+proteins+rapidly+activates+NF-kappaB+and+activator+protein+1.">Lipid extract of Mycoplasma penetrans proteinase K-digested lipid-associated membrane proteins rapidly activates NF-kappaB and activator protein 1.</a> Infect Immun. 67 (6), 2951-2956 (1999).</li><li>Luque-Garcia, J. L., Zhou, G., Spellman, D. S., Sun, T. -. T., Neubert, T. A. <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+electroblotted+proteins+by+mass+spectrometry:+protein+identification+after+Western+blotting.">Analysis of electroblotted proteins by mass spectrometry: protein identification after Western blotting.</a> Mol Cell Proteomics. 7 (2), 308-314 (2008).</li><li>Luque-Garcia, J. L., Zhou, G., Sun, T. -. T., Neubert, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Nitrocellulose+Membranes+for+Protein+Characterization+by+Matrix-Assisted+Laser+Desorption/Ionization+Mass+Spectrometry.">Use of Nitrocellulose Membranes for Protein Characterization by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.</a> Anal Chem. 78 (14), 5102-5108 (2006).</li><li>Kurokawa, K., 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=Environment-mediated+accumulation+of+diacyl+lipoproteins+over+their+triacyl+counterparts+in+Staphylococcus+aureus.">Environment-mediated accumulation of diacyl lipoproteins over their triacyl counterparts in Staphylococcus aureus.</a> J Bacteriol. 194 (13), 3299-3306 (2012).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227 (5259), 680-685 (1970).</li><li>Sch&#1235;gger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tricine-SDS-PAGE.">Tricine-SDS-PAGE.</a> Nat Protoc. 1, 16-22 (2006).</li><li>Gravel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Blotting+by+the+Semidry+Method.">Protein Blotting by the Semidry Method.</a> The Protein Protocols Handbook. , 321-334 (2002).</li><li>Webster, J., Oxley, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Identification+by+Peptide+Mass+Fingerprinting+using+MALDI-TOF+Mass+Spectrometry.">Protein Identification by Peptide Mass Fingerprinting using MALDI-TOF Mass Spectrometry.</a> The Protein Protocols Handbook. , 1117-1129 (2009).</li><li>Wilkins, M. 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=Detailed+peptide+characterization+using+PEPTIDEMASS+-+a+World-Wide-Web-accessible+tool.">Detailed peptide characterization using PEPTIDEMASS - a World-Wide-Web-accessible tool.</a> Electrophoresis. 18 (3-4), 403-408 (1997).</li><li>Gasteiger, E., 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=Protein+Identification+and+Analysis+Tools+on+the+ExPASy+Server.">Protein Identification and Analysis Tools on the ExPASy Server.</a> The Proteomics Protocols Handbook. , 571-607 (2005).</li><li>Perkins, D. N., Pappin, D. J. C., Creasy, D. M., Cottrell, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probability-based+protein+identification+by+searching+sequence+databases+using+mass+spectrometry+data.">Probability-based protein identification by searching sequence databases using mass spectrometry data.</a> Electrophoresis. 20 (18), 3551-3567 (1999).</li><li>Al-Saad, K. A., Zabrouskov, V., Siems, W. F., Knowles, N. R., Hannan, R. M., Hill, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-assisted+laser+desorption/ionization+time-of-flight+mass+spectrometry+of+lipids:+ionization+and+prompt+fragmentation+patterns.">Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of lipids: ionization and prompt fragmentation patterns.</a> Rapid Commun Mass Spectrom. 17 (1), 87-96 (2003).</li></ol>

作者没有什么可透露的。

本协议描述了两个不同阶段的脂蛋白表征: 富集 TX-114 相分配和结构确定的 MALDI MS。在 TX-114 萃取过程中, 额外的离心去除了在这个过程中沉淀的蛋白质, 其次是丙酮沉淀产生高浓度脂蛋白。通过将每个准备的规模限制为15毫升的细胞, 可以很容易地并行处理多个样本, 如果需要, 则在协议结束时将它们汇集在一起。
对于 MS 的结构分析, 将脂蛋白转移到硝化纤维素, 有利于胰蛋白酶...

细菌脂蛋白的特点是保守的 n-末端脂修饰半胱氨酸, 锚定球形蛋白领域的细胞膜表面。它们是普遍分布在细菌, 构成2到5% 的所有细胞基因在一个典型的基因组1。脂蛋白在多种细胞过程中起关键作用, 包括养分吸收、信号传导、蛋白质复合体的组装以及维持细胞包络结构完整性2。在致病性细菌中, 脂蛋白作为致病因子3,4。在感染过程中, 通过类似于收费受体 (TLR) 2 对 n-端脂肽的识别, 激发了先天免疫应答, 以清除入侵的病原体。根据 n-端酰化态, 脂蛋白通常由交替 TLR2 heterodimeric 配合物识别。TLR2-TLR1 承认 n-酰脂肽, 而 TLR2-TLR6 束缚自由脂肽α氨基终点。一旦绑定, 信号通路汇聚以诱导分泌促炎细胞因子3,4。
以前认为, 革兰氏阳性菌脂蛋白 diacylated, 革兰阴性菌 triacylated, 不同于不存在或存在酰胺相关脂肪酸的保守 n-端半胱氨酸残留。这种假设支持的是缺乏序列 orthologs 的革兰阳性基因组 Lnt, 革兰阴性 n-酰基转移酶, 形成 triacylated 脂蛋白5。然而, 最近的研究揭示了 Firmicutes 中缺乏lnt的脂蛋白 triacylation, 以及三种新的 n-端脂蛋白结构, 称为 peptidyl、溶血和n-乙酰形式67 ,8。这些发现引发了对可能尚未发现的脂蛋白形态的质疑, 还有关于这些新脂蛋白是如何制作的基本问题, 以及各种形式所传授的生理目的或优势。此外, 他们清楚地表明, 目前基因组无法预测脂蛋白结构。事实上, 我们最近发现了一种新的类脂蛋白 n-酰转移酶, 称为点燃, 从粪肠球菌和蜡样芽孢杆菌, 使溶血脂蛋白 9.这表明需要实验性地验证脂蛋白结构, 这可能是挑战, 由于其极其疏水性的性质和有限的方法, 以表征其分子结构。
为了促进对脂蛋白诱导宿主免疫应答以及 n 末端结构测定的研究, 我们已经修改了几种先前描述的协议, 以纯化细菌脂蛋白并制备 n-端胰蛋白酶。脂肽用于分析的 MALDI MS 6, 10, 11, 12.脂蛋白是丰富使用建立的海卫 X-114 (从今以后称为表面活性剂或 TX-114) 相分配方法, 优化去除污染的非脂蛋白和增加脂蛋白产量。这些脂蛋白适用于直接使用 TLR 化验或进一步纯化的 SDS 页。对于 MALDI MS, 脂蛋白转移到硝化棉膜提供了一个脚手架, 有效的原位胰蛋白酶消化, 洗涤, 并随后从膜表面洗脱, 导致高度纯化的 n-端脂肽。硝化棉已被证明可以促进样品处理和改善序列覆盖的高度疏水性肽从整体膜蛋白13,14, 以及脂蛋白9,10.该方法具有以极性为基础的分馏肽的额外优势, 在单实验中, 可以分析中间洗涤液与 n-末端结构测定同时进行高置信蛋白鉴定。.该协议独特的特点是有意的钠加成层, 以促进在 ms/毫秒内的父离子分裂为 dehydroalanyl 离子, 协助结构分配的N-酰化态。N 终点是与 TLR 识别脂蛋白有关的最大变量和关键特征。总之, 本议定书允许对脂蛋白进行深入和可重复性的研究, MALDI 的纯化和结构测定的各个阶段根据实验的总体目标容易适应。

<table><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.01 mm Zirconia/silica beads</td><td>BioSpec Products</td><td>110791012</td><td></td></tr><tr><td>Acetic acid</td><td>EMD</td><td>AX0073-9</td><td></td></tr><tr><td>Acetone</td><td>EMD</td><td>AX0116-6</td><td></td></tr><tr><td>Acetonitrile</td><td>EMD</td><td>AX0142-6</td><td></td></tr><tr><td>Ammonium bicarbonate</td><td>Fluka Analytical</td><td>09830</td><td></td></tr><tr><td>BioTrace NT Nitrocellulose</td><td>PALL Life Sciences</td><td>66485</td><td></td></tr><tr><td>Bovine serum albumin (BSA) digest standard</td><td>Protea</td><td>PS-204-1</td><td></td></tr><tr><td>Chloroform</td><td>Acros Organics</td><td>423550025</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Fisher Scientific</td><td>BP118</td><td></td></tr><tr><td>HPLC Grade water</td><td>EMD</td><td>WX0008-1</td><td></td></tr><tr><td>Lysozyme</td><td>Fisher Scientific</td><td>BP535-1</td><td></td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>34860</td><td></td></tr><tr><td>Phenylmethyl sulfonyl fluoride (PMSF)</td><td>Amresco</td><td>0754</td><td></td></tr><tr><td>Pierce trypsin protease</td><td>Thermo Scientific</td><td>90057</td><td></td></tr><tr><td>Ponceau S</td><td>Acros Organics</td><td>161470100</td><td></td></tr><tr><td>Protein LoBind Tube 0.5mL</td><td>Eppendorf</td><td>022431064</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>792519</td><td></td></tr><tr><td>Sodium chloride</td><td>Macron Fine Chemicals</td><td>7581-06</td><td></td></tr><tr><td>Trifluoroacetic acid (TFA)</td><td>Sigma-Aldrich</td><td>299537</td><td></td></tr><tr><td>Tris-hydrochloride (HCl)</td><td>Fisher Scientific</td><td>BP152</td><td></td></tr><tr><td>Triton X-114</td><td>Sigma-Aldrich</td><td>93422</td><td></td></tr><tr><td>Tryptic soy broth (TSB)</td><td>BD</td><td>211822</td><td></td></tr><tr><td>&amp;alpha;-cyano-4-hydroxycinnamic acid (MS Grade)</td><td>Sigma-Aldrich</td><td>C8982</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>MagNALyser</td><td>Roche</td><td></td><td></td></tr><tr><td>Trans-Blot Turbo Transfer System</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>MALDI-TOF target</td><td>Bruker Daltonics</td><td></td><td></td></tr><tr><td>Ultraflextreme MALDI-TOF-TOF</td><td>Bruker Daltonics</td><td></td><td>Equipped with a 355 nm frequency-tripled Nd:YAG smartbeam-II laser</td></tr></tbody></table>

enrichment of bacterial lipoproteins and preparation of n-terminal lipopeptides for structural determination by mass spectrometry

脂蛋白是哺乳动物先天免疫应答的细菌细胞包络和有效活化剂的重要成分。尽管它们对细胞生理学和免疫学都有重要意义, 但对于新的脂蛋白形态、合成方法以及各种形式对宿主免疫的影响, 仍有许多有待发现。为了对脂蛋白进行深入研究, 本协议描述了一种细菌脂蛋白的富集方法和 n-端胰蛋白酶脂肽的制备, 其结构由基质辅助激光解吸电离-飞行时间来确定。质谱 (MALDI)。在建立的海卫 X-114 相分治法中扩展了从细菌细胞膜中提取和富集的方法, 该协议包括去除非脂蛋白污染物的附加步骤, 增加脂蛋白产量和纯度。由于脂蛋白是常用的收费样受体 (TLR) 检测, 这是至关重要的第一个特点的 n-终端结构的 MALDI 的 MS. 本文提出了一种分离富集在 n-端的浓疏水性多肽的方法。脂肽适合直接分析的 MALDI 女士/ms. 经月桂酸钠分离的脂质体-丙烯酰胺凝胶电泳 (SDS 页) 被转移到硝化纤维膜上, 消化原位 胰蛋白酶, 依次洗涤去除极性胰蛋白酶肽, 最后洗脱与氯仿-甲醇。当与洗涤液中极性 trypsinized 肽的 MS 结合时, 这种方法提供了在单个实验中识别脂蛋白并表征其 n-终点的能力。故意的钠加成物也可以作为一种工具, 以促进更结构化的信息碎片谱。最终, 脂蛋白的丰富和对其 n-端结构的测定将允许对这种无处不在的细菌蛋白进行更广泛的研究。

协议的示意图在图 1中提供。图 2中显示了从粪肠球菌ATCC 19433 中提取的富含脂蛋白的分数 TX-114。为比较, 还显示了沉淀蛋白分数的带状模式。从这个分数的蛋白质证实了 MALDI-MS 是高度丰富的污染蛋白质以外的脂蛋白 (表 1)。在图 3中的质谱显示了 PnrA 的胰蛋白酶肽离子剖面, 该蛋白是在?...

Watch this Scientific Journal Video about Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry at JoVE.com

Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

质谱法富集细菌脂蛋白及 n-端脂肽的制备结构测定

用非离子表面活性剂相分配法对细菌脂蛋白的富集进行了描述, 直接用于 TLR 化验或其他应用。进一步详细的步骤, 以准备 n-终端胰蛋白酶脂肽的结构表征的质谱。

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their ...

enrichment-bacterial-lipoproteins-preparation-n-terminal-lipopeptides

Research

JoVE Journal

Immunology and Infection

8.6K 次观看.  Pennsylvania State University. 用非离子表面活性剂相分配法对细菌脂蛋白的富集进行了描述, 直接用于 TLR 化验或其他应用。进一步详细的步骤, 以准备 n-终端胰蛋白酶脂肽的结构表征的质谱。

视频: Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

隔离和脂质A的化学表征革兰氏阴性菌

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

科研

化学

Defining Substrate Specificities for Lipase and Phospholipase Candidates

Microorganisms produce a wide spectrum of (phospho)lipases that are secreted in order to make external substrates available for the organism. Alternatively, other (phospho)lipases may be physically associated with the producing organism causing a turnover of intrinsic lipids and frequently giving rise to a remodeling of the cellular membranes. Although potential (phospho)lipases can be predicted with a number of algorithms when the gene/protein sequence is available, experimental proof of the enzyme activities, substrate specificities, and potential physiological functions has frequently not been obtained. This manuscript describes the optimization of assay conditions for prospective (phospho)lipases with unknown substrate specificities and how to employ these optimized conditions in the search for the natural substrate of a respective (phospho)lipase. Using artificial chromogenic substrates, such as p-nitrophenyl derivatives, may help to detect a minor enzymatic activity for a predicted (phospho)lipase under standard conditions. Having encountered such a minor enzymatic activity, the distinct parameters of an enzyme assay can be varied in order to obtain a more efficient hydrolysis of the artificial substrate. After having determined the conditions under which an enzyme works well, a variety of potential natural substrates should be assayed for their degradation, a process that can be followed employing distinct chromatographic methods. The definition of substrate specificities for new enzymes, often provides hypotheses for a potential physiological role of these enzymes, which then can be tested experimentally. Following these guidelines, we were able to identify a phospholipase C (SMc00171) that degrades phosphatidylcholine to phosphocholine and diacylglycerol, in a crucial step for the remodeling of membranes in the bacterium Sinorhizobium meliloti upon phosphorus-limiting conditions of growth. For two predicted patatin-like phospholipases (SMc00930 and SMc01003) of the same organism, we could redefine their substrate specificities and clarify that SMc01003 is a diacylglycerol lipase.

Many predicted (phospho)lipases are poorly characterized with regard to their substrate specificities and physiological functions. Here we provide a protocol to optimize enzyme activities, search for natural substrates, and propose physiological functions for these enzymes.

定义底物特异性的脂肪酶和磷脂酶考生

Microorganisms produce a wide spectrum of (phospho)lipases that are secreted in order to make external substrates available for the organism. ...

生物化学

Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the face of turgor pressures several atmospheres in magnitude. Across the diverse shapes and sizes of the bacterial kingdom, the cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by short peptides. Peptidoglycan&#8217;s central importance to bacterial physiology underlies its use as an antibiotic target and has motivated genetic, structural, and cell biological studies of how it is robustly assembled during growth and division. Nonetheless, extensive investigations are still required to fully characterize the key enzymatic activities in peptidoglycan synthesis and the chemical composition of bacterial cell walls. High Performance Liquid Chromatography (HPLC) is a powerful analytical method for quantifying differences in the chemical composition of the walls of bacteria grown under a variety of environmental and genetic conditions, but its throughput is often limited. Here, we present a straightforward procedure for the isolation and preparation of bacterial cell walls for biological analyses of peptidoglycan via HPLC and Ultra Performance Liquid Chromatography (UPLC), an extension of HPLC that utilizes pumps to deliver ultra-high pressures of up to 15,000 psi, compared with 6,000 psi for HPLC. In combination with the preparation of bacterial cell walls presented here, the low-volume sample injectors, detectors with high sampling rates, smaller sample volumes, and shorter run times of UPLC will enable high resolution and throughput for novel discoveries of peptidoglycan composition and fundamental bacterial cell biology in most biological laboratories with access to an ultracentrifuge and UPLC.

The bacterial cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by peptides. Ultra Performance Liquid Chromatography provides high resolution and throughput for novel discoveries of peptidoglycan composition. We present a procedure for the isolation of cell walls (sacculi) and their subsequent preparation for analysis via UPLC.

通过超性能液相色谱对细菌细胞壁进行隔离和制备，进行组成分析

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the ...

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

脂滴隔离定量质谱分析

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the majority of exogenous fatty acids are present within host lipoprotein particles. Uncertainty remains as to the reservoirs of host fatty acids and the mechanisms by which bacteria extract fatty acids from the lipoprotein particles. In this work, we describe protocols for enrichment of low-density lipoprotein (LDL) particles from chicken egg yolk and determining whether LDLs serve as fatty acid reservoirs for S. aureus. This method exploits unbiased lipidomic analysis and chicken LDLs, an effective and economical model for the exploration of interactions between LDLs and bacteria. The analysis of S. aureus integration of exogenous fatty acids from LDLs is performed using high-resolution/accurate mass spectrometry and tandem mass spectrometry, enabling the characterization of the fatty acid composition of the bacterial membrane and unbiased identification of novel combinations of fatty acids that arise in bacterial membrane lipids upon exposure to LDLs. These advanced mass spectrometry techniques offer an unparalleled perspective of fatty acid incorporation by revealing the specific exogenous fatty acids incorporated into the phospholipids. The methods outlined here are adaptable to the study of other bacterial pathogens and alternative sources of complex fatty acids.

This method provides a framework for studying incorporation of exogenous fatty acids from complex host sources into bacterial membranes, particularly Staphylococcus aureus. To achieve this, protocols for the enrichment of lipoprotein particles from chicken egg yolk and subsequent fatty acid profiling of bacterial phospholipids utilizing mass spectrometry are described.

从鸡蛋黄中分离脂蛋白颗粒,研究细菌病原体脂肪酸并入膜磷脂

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the ...

Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral membrane enzymes, resulting in triacylated proteins. The first step in the lipoprotein modification pathway involves the transfer of a diacylglyceryl group from phosphatidylglycerol onto the prolipoprotein, resulting in diacylglyceryl prolipoprotein. In the second step, the signal peptide of prolipoprotein is cleaved, forming an apolipoprotein, which in turn is modified by a third fatty acid derived from a phospholipid. This last step is catalyzed by apolipoprotein N-acyltransferase (Lnt). The lipoprotein modification pathway is essential in most &#947;-proteobacteria, making it a potential target for the development of novel antibacterial agents. Described here is a sensitive assay for Lnt that is compatible with high-throughput screening of small inhibitory molecules. The enzyme and substrates are membrane-embedded molecules; therefore, the development of an in vitro test is not straightforward. This includes the purification of the active enzyme in the presence of detergent, the availability of alkyne-phospholipids and diacylglyceryl peptide substrates, and the reaction conditions in mixed micelles. Furthermore, in order to use the activity test in a high-throughput screening (HTS) setup, direct readout of the reaction product is preferred over coupled enzymatic reactions. In this fluorometric enzyme assay, the alkyne-triacylated peptide product is rendered fluorescent through a click-chemistry reaction and detected in a multiwell plate format. This method is applicable to other acyltransferases that use fatty acid-containing substrates, including phospholipids and acyl-CoA.

Presented here is a sensitive fluorescence assay to monitor apolipoprotein N-acyltransferase activity using diacylglyceryl peptide and alkyne-phospholipids as substrates with click-chemistry.

基于点击化学的载脂蛋白N-酰基转移酶荧光测定从酶表征到高通量筛选

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral ...

Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan structure are fairly conserved across all bacteria, there is also considerable variation between Gram-positives/negatives and between species. In addition, there are numerous known variations, modifications, or adaptations to the peptidoglycan that can occur within a bacterial species in response to growth phase and/or environmental stimuli. These variations produce a highly dynamic structure that is known to participate in many cellular functions, including growth/division, antibiotic resistance, and host defense avoidance. To understand the variation within peptidoglycan, the overall structure must be broken down into its constitutive parts (known as muropeptides) and assessed for overall cellular composition. Peptidoglycomics uses advanced mass spectrometry combined with high-powered bioinformatic data analysis to examine peptidoglycan composition in fine detail. The following protocol describes the purification of peptidoglycan from bacterial cultures, the acquisition of muropeptide intensity data through a liquid chromatograph&#8212;mass spectrometer, and the differential analysis of peptidoglycan composition using bioinformatics.

This protocol covers a detailed analysis of peptidoglycan composition using liquid chromatography mass spectrometry coupled with advanced feature extraction and bioinformatic analysis software.

肽聚糖的液相色谱质谱和生物信息学半定量分析

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan ...

Cell-Free Production of Proteoliposomes for Functional Analysis and Antibody Development Targeting Membrane Proteins

Membrane proteins play essential roles in a variety of cellular processes and perform vital functions. Membrane proteins are medically important in drug discovery because they are the targets of more than half of all drugs. An obstacle to conducting biochemical, biophysical, and structural studies of membrane proteins as well as antibody development has been the difficulty in producing large amounts of high-quality membrane protein with correct conformation and activity. Here we describe a &#8220;bilayer-dialysis method&#8221; using a wheat germ cell-free system, liposomes, and dialysis cups to efficiently synthesize membrane proteins and prepare purified proteoliposomes in a short time with a high success rate. Membrane proteins can be produced as much as in several milligrams, such as GPCRs, ion channels, transporters, and tetraspanins. This cell-free method contributes to reducing the time, cost and effort for preparing high-quality proteoliposomes, and provides suitable means for functional analysis of membrane proteins, drug targets screening, and antibody development.

This protocol describes an efficient cell-free method for production of high-quality proteoliposome by bilayer-dialysis method using wheat cell-free system and liposomes. This method provides suitable means for functional analysis of membrane proteins, drug targets screening, and antibody development.

无细胞生产蛋白脂质体，用于功能分析和靶向膜蛋白的抗体开发

Membrane proteins play essential roles in a variety of cellular processes and perform vital functions. Membrane proteins are medically important in drug ...

Sample Preparation using a Lipid Monolayer Method for Electron Crystallographic Studies

Electron crystallography is a powerful tool for high-resolution structure determination. Macromolecules such as soluble or membrane proteins can be grown into highly ordered two-dimensional (2D) crystals under favorable conditions. The quality of the grown 2D crystals is crucial to the resolution of the final reconstruction via 2D image processing. Over the years, lipid monolayers have been used as a supporting layer to foster the 2D crystallization of peripheral membrane proteins as well as soluble proteins. This method can also be applied to 2D crystallization of integral membrane proteins but requires more extensive empirical investigation to determine detergent and dialysis conditions to promote partitioning to the monolayer. A lipid monolayer forms at the air-water interface such that the polar lipid head groups remain hydrated in the aqueous phase and the non-polar, acyl chains, tails partition into the air, breaking the surface tension and flattening the water surface. The charged nature or distinctive chemical moieties of the head groups provide affinity for proteins in solution, promoting binding for 2D array formation. A newly formed monolayer with the 2D array can be readily transfer into an electron microscope (EM) on a carbon-coated copper grid used to lift and support the crystalline array. In this work, we describe a lipid monolayer methodology for cryogenic electron microscopic (cryo-EM) imaging.

Lipid monolayers have been used as a foundation for forming two-dimensional (2D) protein crystals for structural studies for decades. They are stable at the air-water interface and can serve as a thin supporting material for electron imaging. Here we present the proven steps on preparing lipid monolayers for biological studies.

使用脂质单层方法进行电子晶体学研究的样品制备

Electron crystallography is a powerful tool for high-resolution structure determination. Macromolecules such as soluble or membrane proteins can be grown ...

High-Resolution Studies of Proteins in Natural Membranes by Solid-State NMR

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy offers a unique access to probe the structure and dynamics of such proteins in biological membranes of increasing complexity. Here, we present modern solid-state NMR spectroscopy as a tool to study structure and dynamics of proteins in natural lipid membranes and at atomic scale. Such spectroscopic studies profit from the use of high-sensitivity ssNMR methods, i.e., proton-(1H)-detected ssNMR and DNP (Dynamic Nuclear Polarization) supported ssNMR. Using bacterial outer membrane beta-barrel protein BamA and the ion channel KcsA, we present methods to prepare isotope-labeled membrane proteins and to derive structural and motional information by ssNMR.

This work details robust basic routines on how to prepare isotope-labeled membrane protein samples and analyze them at high-resolution with modern solid-state NMR spectroscopy methods.

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy ...