We are grateful to Hannah Kleinschmidt for excellent technical support. This paper was funded by the Open Access support program of the Deutsche Forschungsgemeinschaft and the publication fund of the Georg August Universita&#776;t Go&#776;ttingen.

1.考虑生物安全条件准备一个安全的工作环境<ol><li>熟悉实验室和安全法规的相关性与微生物工作。大多数人类病原微生物必须在生物安全水平进行处理2的条件，但一些，如伤寒沙门菌，需要生物安全3级上处理每一病原体可在www.cdc.gov/biosafety访问级别的信息。 </li><li>不管具体微生物的生物危害的分类，把中随附传染性废物传染性病原体必须在处置前进行高压灭菌接触的所有材料。尊重有害物质和生物物质的区域安全指导。确保立即和妥善处置可能受污染的材料（生物危害）的那个合适的容器可用。 </li><li>确保无菌器械（接种环路），解决方案和文化传媒（琼脂平板）的细菌开始培养前可用。 </li><li>处理感染性微生物后，立即用抗菌肥皂和温水手。 </li></ol> 2.选择参考与收藏株<ol><li>选择并获得一个标准的基因组测序的参考隔离随着编码蛋白质的序列，最好是在FASTA格式。如果有更多的基因组测序的菌株是可用的，包括这些在分析中。 注意:此分离物/这些菌株稍后将被用来预测在质谱中观察到的质量峰的标识（见第7）。 </li><li>选择并在它们覆盖的种类或感兴趣的亚种的系统发育这样的方式获得各种不同的潜在菌株。 注意:这些菌株稍后将被用来证明在人群中的生物标志物的变化（见第8节）。 </li><li>确保整个收集和参考株（S）是亲20 -佩尔利由各自的黄金标准这个特殊的有机体18类型。 注意:这可能包括各种（分）-typing方法，但是很可能会采取MLST，这仍然是为了阐明最微生物物种的遗传多样性的标准方法。 </li><li>推断集合中的发展史，从打字数据， 例如计算系统发生，使用与MEGA6软件MLST数据21算术平均（UPGMA）的加权对群法。对于MLST数据，也请教MLST数据库和分配序列类型和各自的克隆物22。 注意:这将稍后被用来分析MSPP的一致性与早期金标准打字方法（见第9节）。 </li></ol> 3.准备一个MALDI靶板小心:TFA为强酸。使用不当TFA负有严重的皮肤灼伤，眼部损害和严重的刺激邻风险F中的上呼吸道吸入。因此，严格的安全措施必须得到尊重和适当的个人防护装备（PPE），包括护目镜，面罩，适当的手套，靴子，甚至是一个完整的防护服是必要的，在处理TFA。可能接触TFA必须具有有效的排风系统处理下足够的通风物质进行控制。在通风不良的情况下，必须使用经批准的过滤器的呼吸器。此外，TFA是有害的水生生物具有长期持续影响。必须避免TFA的废水中的任何释放到环境中。 注:斑点样到MALDI目标之前，彻底清洁目标板，如果板使用过。 <ol><li>制备，用30毫升去离子水和70ml纯100ml乙醇70％含水乙醇溶液中。 </li><li>通过混合50制备250μl的80％的含水三氟乙酸（TFA）溶液的去离子水和200微升100％TFA的反应管和涡旋管1分钟微升。 </li><li>通过将其放入一个玻璃盘和浸没它在70％含水乙醇中约5分钟，在室温下清洗MALDI靶。 </li><li>冲洗下热水目标。 </li><li>用纸巾，擦拭深入靶板用70％的乙醇水溶液，除去所有以前的样本和其他潜在的杂物。 </li><li>如果需要进一步清洁，在热水冲洗一边用纸巾擦拭。 </li><li>除去残留的和潜在的隐形，污染物，通过覆盖目标表面用80％的薄层TFA水溶液（〜每96点100微升）和擦拭干净的纸巾所有目标位置。 </li><li>最后，冲洗目标除去酸，擦干用纸巾，并使其保持至少15分钟，在室温下蒸发残留液体。 </li></ol> 4.一个制备45;氰基-4-羟基 - 肉桂酸矩阵解法含有内校准物<ol><li>通过在1ml的50％乙腈，47.5％水和2.5％TFA的混合物中溶解10毫克α氰基-4-羟基肉桂酸（HCCA）制备的饱和基质溶液。残留未溶解的HCCA仍将如果溶液是饱和的。 </li><li>加入重组人胰岛素作为内部校准。对于这一点，在-20℃用于进一步使用制备于50％乙腈水溶液，等分试样并储存原液至10微克/微升的最终浓度。 </li></ol> 5. MALDI-TOF质谱注意:特异于感兴趣的生物的培养条件必须被使用。对于MALDI-TOF MS的样品可以通过涂抹标本制作或萃取来制备，这取决于有机体（见8.4.1）。而乙醇甲酸提取方法提供的病原体足够失活，涂抹标本制作具有下SUFF要执行icient生物安全条件的要求（见第1节）。通常，有基体的涂布后没有感染的危险性，但对于具体病原体所需的特定灭活协议。因此，例如诺卡物种的MALDI-TOF MS需要在沸水中，通过蛋白23的乙醇沉淀后的细菌的先前裂解。 EIKhéchine 等 。开发了一种程序分枝杆菌的失活，在含有水和0.5％吐温20 24螺旋盖试管在95℃下加热该细菌菌落1小时。 <ol><li>涂片标本<ol><li>散布细菌菌落的针头大小的量直接在MALDI靶板位置（&#39;发现&#39;）。 </li><li>覆盖每个点用1微升含有内部校准HCCA常规基质或基质的，并留下在室温下crystalize。为确定校准峰的精确质量，覆盖控制SPOT 1微升试验标准和1微升含内部校准HCCA基质。 注意:在此，作为试验标准大肠杆菌 DH5阿尔法的提取物被用于演示在MALDI-TOF MS的特性的蛋白质的指纹。它已与延伸的可检测质量范围的上限两种蛋白质掺入它。 </li></ol></li><li>提取方法<ol><li>收获来自琼脂板培养大约五菌落接种环，并在300微升完全暂停双蒸水在1.5ml反应管中。加入900微升无水乙醇，直到细菌菌落完全暂停反复吹打调匀。 注意:在这个步骤是可能的，并且很好地建立到样品存储在-20℃。此外，病原体灭活可以通过划线1-10微升提取物的上下面在最佳生长条件孵育合适的琼脂平板进行测试。成功激活是由没有微生物生长的指示。 </li><li>离心样品以13,000rpm xg离心1分钟，弃去上清液，并干燥在室温下沉淀10分钟。吹打在50μl的70％甲酸向上和向下彻底重悬沉淀。 </li><li>加入50微升乙腈混匀。离心13,000 XG 2分钟取出碎片。转移1μl的上清液到上的MALDI靶盘一个样本位置和离开干燥，在室温下5分钟。 </li><li>覆盖每个点用1微升含有内部校准HCCA矩阵的和离开在室温下crystalize。 </li></ol></li><li>质谱记录 注意:峰采集从质谱是使用标准程序建议进行（质心算法; S / N比:2;相对强度阈:2％;峰宽3米/ Z，基线减法:顶帽） <ol><li>根据制造商的protoc校准仪器醇。 </li><li>对于每一个点，聚集在100射击步600谱。 <ol><li>进入质谱仪的组态软件的"AutoXecute"选项卡。打开"方法"通过左键点击到"方法"按钮，选择方法如 "MBT_AutoX"从下拉菜单。 </li><li>左键单击"方法"菜单的"编辑"按钮右侧，打开"AutoXecute法编辑器"。转到"积累"选项卡。将"总结"的值改为"600"和_x_"的拍摄步骤"的值改为"100"，"令人满意枪"。 </li></ol></li></ol></li><li>内置频谱校准程序 注意:一分钟的测量误差是固有的质谱。根据间歇仪器的使用，仪器的温度和重新校准，所获得的测量值可以实验之间变化。继前测量仪器校准和<html>测量后的频谱校准内部校准是确保频谱之间的可比性最精确的方法。 <ol><li>执行每次校准峰值列表以下程序: <ol><li>启动频谱浏览器（ 例如 ，flexAnalysis和开放频谱:菜单"文件"→"打开..."） </li><li>与校准峰创建质量控制列表:菜单中的"方法"→"打开..."。 </li><li>选择方法:MBT_Standard.FAMSMethod→"打开"。 </li><li>编辑质量控制列表:取消所有校准物。 </li><li>在底部新增校准物峰:峰标签:"Insulin_HIStag [M + H] + _魅力"; m_z:"5808.29";公差[PPM]:"50";检查校准物复选框。 </li><li>另存为， 例如 ，"MSPP校准表"。 </li></ol></li><li>对于每一个光谱，从列表中选择的校准高峰，单击"自动分配"，然后按"确定"。 </li></ol></li></ol>ove_title"&gt; 6。检查内部校准程序<ol><li>实验确定校准峰的精确质量。 <ol><li>准备两个点与1微升测试标准（每步5.1.1）。叠加先用1微升定期HCCA矩阵，第二次用1微升校准-飙升矩阵。 </li><li>获得各点（5.3节）质谱，并在内部校准的测试标准峰（第5.4节）。 </li><li>通过与频谱浏览器中打开它们（ 例如，flexAnalysis和开放频谱:菜单"文件"→"打开..."）叠加两种光谱的预期质量，并找到峰（胰岛素m / z = 5,808.29），这应该是目前在校准物-掺入谱，但不与常规的矩阵得到的频谱。 </li></ol></li><li>检查校准物山顶不是由利益的有机体的任何其他生物标志物遮蔽。 <ol><li>与基准应变和奥雅纳准备两个点（5.1节）rlay先用1微升正规矩阵，第二次用1微升校准-飙升矩阵。 </li><li>收购点都质谱（第5.3节），并通过与频谱浏览器中打开它们叠加所产生的光谱（ 例如，flexAnalysis和开放频谱:菜单"文件"→"打开..."）。确保校准峰是清晰可见与尖刺矩阵获得，而不是由另一相邻信号遮蔽的频谱。如果不是这种情况下，选择另一校准了特定生物体。 注意:使用尖刺内部校准显著提高精度，以确定生物标志物群众的变化。采用这种方法，下至1大质量的差异可以被检测出来。或者，从生物体始发也不变群众可以用作校准物。然而，根据定义，所有的生物衍生群众必须考虑潜在变量，除非证明并非如此。 </li></ol></li></ol><ol><li>测量参考应变的质谱，使用矩阵尖刺与内部校准物。 <ol><li>散布细菌菌落（第5.1节）或1微升细菌蛋白提取物（第5.2节）的一个针头大小的量直接在MALDI靶板位置（&#39;发现&#39;）。 </li><li>覆盖用1微升含有内部校准HCCA矩阵的各点，并保持目标盘在室温（5.1.2节/ 5.2.4）至crystalize。 </li><li>参考株的记录质谱（第5.3节）。 </li></ol></li><li>内部校准参考频谱的校准质量（这里:在m / z = 5,806.29胰岛素），并随后通过基线减法（高顶礼帽）和平滑（参数:SavitzkyGolay;宽度:2 M / Z，10个循环）前处理。 <ol><li>启动频谱浏览器（ 例如，flexAnalysis和开放频谱:菜单"文件"→"打开...;"）。 </li><li>选择方法:下拉菜单"方法"→"打开..."，左键单击选择的方法， 例如，"MBT_Standard.FAMSMethod"→"打开"。 </li><li>从下拉菜单中选择"校准"选择"内部......"校准光谱。打开一个窗口，列出校准峰（S）（第5.4节）。左键单击校准峰（胰岛素）→左键单击"确定"。从"过程"下拉菜单中选择"过程谱"。 </li><li>为基线减法激活在右侧在光谱列表中的光谱。从"过程"下拉菜单中选择"减质谱基线"。 </li><li>平滑频谱，激活在光谱列表中的光谱在右侧。从"过程"下拉菜单中选择"平稳质谱基线"。 </li></ol></li><li>从参考菌株的基因组测序数据，计算出theoretica升单同位素通过使用序列比对编辑翻译的DNA序列插入相应的氨基酸序列的分子量每个编码的蛋白质的。复制粘贴在ExPASy生物信息资源门户网站（http://web.expasy.org/compute_pi/）这种蛋白质序列在输入框中。按"点击这里"来计算的pI / MW。在C的情况下杆菌 SSP。doylei计算的14检测的生物标志物的质量。 </li><li>将结果复制到电子表格中，随着含有该基因标识符的一列和下一个的分子量。排序方式计算分子量行，方便群众更容易查找。注:其他栏目都是可选的;功能注释以后可能会进行解释特别有用。 </li><li>插入第二列到电子表格的脱methioninated形式的分子量，从单同位素分子量减去135道尔顿。注:这是因为一些蛋白质经过交通过蛋白水解除去N-末端蛋氨酸的翻译后修饰。 </li><li>通过查找从上述（ 表1）中制备的基因组表中的测量的质量的每个主要测量生物标志物的质量分配给从基准应变算出的质量。 </li><li>如果生物标记离子不能被分配到预测的基因产物，可以考虑其他的翻译后修饰（甲基化，乙酰化，异戊烯等 ;参见http://www.abrf.org/delta-mass进行修改的汇编和相关的质量变化）。对于在感兴趣的生物体中经常观察到任何其它已知的翻译后修饰的另一列添加到表并重新计算分子量与对于去methioninated形式的过程。 </li><li>设置其他电子表格选项卡，并记录每个生物标记离子在一个单独的表列中的质量和标识符。 </li></ol> 8.评估变异生物标志物人口<ol><li>从校准获得收集质谱分离，作为参照株（S）（5.4.2节）来完成。 </li><li>确定在质谱变体的生物标志物。变体的质量的特征在于不存在从参考光谱和新型质量在基准不存在的外观已知质量的。的质量差必须符合的单个氨基酸交换（ 表2），或它们的组合。 </li><li>在每行一个分离物，记录每个生物标记，并在各自的表列中的预测的同种型的测量的质量。 注意:很少，一些质量偏移可能归因于几个不同的氨基酸交换， 例如，两个，N-交换用D和Q用E交换，并且反之亦然，导致0.985达（ 表2）的质量转变。这种内在的问题不能由质谱单独解决。因此，具有相同质量的蛋白质序列水平不同亚型必须被视为单个MSPP类型。在特定生物标记基因离子并行Sanger测序证实，虽然MSPP，键入C.没有发生问题杆菌 SSP。doylei隔离在该研究中使用的集合。 </li><li>确认新颖MSPP类型通过PCR放大和测序的各个生物标志物的基因。反过来，这也可作为该生物标记的身份已被正确地分配的确认。 <ol><li>培养细菌的最佳生长条件下，分离物。 C.文化杆菌 SSP。doylei在37℃下微需氧条件下（5％O 2，10％的CO 2，85％N 2）根据补充有5％绵羊血的哥伦比亚琼脂菌株。孵育约48小时。使用单独的琼脂平板上的每个隔离，避免交叉污染。 </li><li>提取根据制造商的说明使用适当的DNA提取试剂盒/自动化机械的细菌分离株的基因组DNA。 </li> &lt;利&gt;扩增使用表3中所列的引物的各自的生物标志物的基因进行下列的条件下所有的PCR反应:在94℃变性30秒;退火在55℃持续30秒;延伸在72℃30秒。 </li><li>确定使用的基因组DNA的适当量由Sanger测序每个扩增子的DNA序列（通常600-700纳克的DNA是在约100毫微克/微升的浓度足够）和扩增引物之一（通常这是通过使用的服务提供者的）。 </li></ol></li><li>在一个单独的表（ 见表4），通过使用适当的翻译工具（ 例如 ，Transseq:http://www.ebi.ac.uk/Tools/st/）记录每个新颖亚型的推导蛋白质序列25。 </li></ol> 9.计算基于MSPP，系统发育和比较黄金标准<ol><li>串联的特定生物标记的氨基酸序列属于到t他MSPP型菌株成一个连续序列使用序列比对编辑器，如BIOEDIT（http://www.mbio.ncsu.edu/bioedit/bioedit.html）26或生物标志物的电子表格。 </li><li>作为金标准分型数据， 例如 ，UPGMA聚类21完成由集群计算系统发育。 </li><li>基于MSPP，系统发育比较与黄金标准4，13所获得的之一。 </li></ol>

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	<li>Seng, P., 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=Ongoing+revolution+in+bacteriology:+routine+identification+of+bacteria+by+matrix-assisted+laser+desorption+ionization+time-of-flight+mass+spectrometry.">Ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.</a> Clin Infect Dis. 49 (4), 543-551 (2009).</li><li>Bader, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI-TOF-MS-based+species+identification+and+typing+approaches+in+medical+mycology.">MALDI-TOF-MS-based species identification and typing approaches in medical mycology.</a> Proteomics. 13 (5), 788-799 (2013).</li><li>Sandrin, T. R., Goldstein, J. E., Schumaker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI+TOF+MS+profiling+of+bacteria+at+the+strain+level:+a+review.">MALDI TOF MS profiling of bacteria at the strain level: a review.</a> Mass Spectrom Rev. 32 (3), 188-217 (2013).</li><li>Zautner, A. 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=Discrimination+of+multilocus+sequence+typing-based+Campylobacter+jejuni+subgroups+by+MALDI-TOF+mass+spectrometry.">Discrimination of multilocus sequence typing-based Campylobacter jejuni subgroups by MALDI-TOF mass spectrometry.</a> BMC Microbiol. 13, 247 (2013).</li><li>Reil, 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=Recognition+of+Clostridium+difficile+PCR-ribotypes+001,+027+and+126/078+using+an+extended+MALDI-TOF+MS+system.">Recognition of Clostridium difficile PCR-ribotypes 001, 027 and 126/078 using an extended MALDI-TOF MS system.</a> Eur J Clin Microbiol Infect Dis. 30 (11), 1431-1436 (2011).</li><li>Kuhns, M., Zautner, A. 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=Rapid+discrimination+of+Salmonella+enterica+serovar+Typhi+from+other+serovars+by+MALDI-TOF+mass+spectrometry.">Rapid discrimination of Salmonella enterica serovar Typhi from other serovars by MALDI-TOF mass spectrometry.</a> PLoS One. 7 (6), e40004 (2012).</li><li>Wolters, 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=MALDI-TOF+MS+fingerprinting+allows+for+discrimination+of+major+methicillin-resistant+Staphylococcus+aureus+lineages.">MALDI-TOF MS fingerprinting allows for discrimination of major methicillin-resistant Staphylococcus aureus lineages.</a> Int J Med Microbiol. 301 (1), 64-68 (2011).</li><li>Josten, 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=Analysis+of+the+matrix-assisted+laser+desorption+ionization-time+of+flight+mass+spectrum+of+Staphylococcus+aureus+identifies+mutations+that+allow+differentiation+of+the+main+clonal+lineages.">Analysis of the matrix-assisted laser desorption ionization-time of flight mass spectrum of Staphylococcus aureus identifies mutations that allow differentiation of the main clonal lineages.</a> J Clin Microbiol. 51 (6), 1809-1817 (2013).</li><li>Lu, J. J., Tsai, F. J., Ho, C. M., Liu, Y. C., Chen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+biomarker+discovery+for+identification+of+methicillin-resistant+and+vancomycin-intermediate+Staphylococcus+aureus+strains+by+MALDI-TOF.">Peptide biomarker discovery for identification of methicillin-resistant and vancomycin-intermediate Staphylococcus aureus strains by MALDI-TOF.</a> Anal Chem. 84 (13), 5685-5692 (2012).</li><li>Novais, A., 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=MALDI-TOF+mass+spectrometry+as+a+tool+for+the+discrimination+of+high-risk+Escherichia+coli+clones+from+phylogenetic+groups+B2+(ST131)+and+D+(ST69,+ST405,+ST393).">MALDI-TOF mass spectrometry as a tool for the discrimination of high-risk Escherichia coli clones from phylogenetic groups B2 (ST131) and D (ST69, ST405, ST393).</a> Eur J Clin Microbiol Infect Dis. , (2014).</li><li>Matsumura, Y., 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=Detection+of+extended-spectrum-beta-lactamase-producing+Escherichia+coli+ST131+and+ST405+clonal+groups+by+matrix-assisted+laser+desorption+ionization-time+of+flight+mass+spectrometry.">Detection of extended-spectrum-beta-lactamase-producing Escherichia coli ST131 and ST405 clonal groups by matrix-assisted laser desorption ionization-time of flight mass spectrometry.</a> J Clin Microbiol. 52 (4), 1034-1040 (2014).</li><li>Christner, 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=Rapid+MALDI-TOF+Mass+Spectrometry+Strain+Typing+during+a+Large+Outbreak+of+Shiga-Toxigenic+Escherichia+coli.">Rapid MALDI-TOF Mass Spectrometry Strain Typing during a Large Outbreak of Shiga-Toxigenic Escherichia coli.</a> PLoS One. 9 (7), e101924 (2014).</li><li>Zautner, A. E., Masanta, W. O., Weig, M., Gro&#223;, U., Bader, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+Spectrometry-based+PhyloProteomics+(MSPP):+A+novel+microbial+typing+Method.">Mass Spectrometry-based PhyloProteomics (MSPP): A novel microbial typing Method.</a> Scientific Reports. 5, (2015).</li><li>Dasti, J. I., Tareen, A. M., Lugert, R., Zautner, A. E., Gross, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Campylobacter+jejuni:+a+brief+overview+on+pathogenicity-associated+factors+and+disease-mediating+mechanisms.">Campylobacter jejuni: a brief overview on pathogenicity-associated factors and disease-mediating mechanisms.</a> Int J Med Microbiol. 300 (4), 205-211 (2010).</li><li>Zautner, A. 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=Seroprevalence+of+campylobacteriosis+and+relevant+post-infectious+sequelae.">Seroprevalence of campylobacteriosis and relevant post-infectious sequelae.</a> Eur J Clin Microbiol Infect Dis. 33 (6), 1019-1027 (2014).</li><li>Zautner, A. E., Herrmann, S., Gro&#223;, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Campylobacter+jejuni+-+The+Search+for+virulence-associated+factors.">Campylobacter jejuni - The Search for virulence-associated factors.</a> Archiv Fur Lebensmittelhygiene. 61 (3), 91-101 (2010).</li><li>Dingle, K. 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=Multilocus+sequence+typing+system+for+Campylobacter+jejuni.">Multilocus sequence typing system for Campylobacter jejuni.</a> J Clin Microbiol. 39 (1), 14-23 (2001).</li><li>Dingle, K. 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=Molecular+characterization+of+Campylobacter+jejuni+clones:+a+basis+for+epidemiologic+investigation.">Molecular characterization of Campylobacter jejuni clones: a basis for epidemiologic investigation.</a> Emerg Infect Dis. 8 (9), 949-955 (2002).</li><li>Cody, A. 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=Real-time+genomic+epidemiological+evaluation+of+human+Campylobacter+isolates+by+use+of+whole-genome+multilocus+sequence+typing.">Real-time genomic epidemiological evaluation of human Campylobacter isolates by use of whole-genome multilocus sequence typing.</a> J Clin Microbiol. 51 (8), 2526-2534 (2013).</li><li>Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MEGA6:+Molecular+Evolutionary+Genetics+Analysis+version+6.0.">MEGA6: Molecular Evolutionary Genetics Analysis version 6.0.</a> Mol Biol Evol. 30 (12), 2725-2729 (2013).</li><li>Jolley, K. A., Chan, M. S., Maiden, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mlstdbNet+-+distributed+multi-locus+sequence+typing+(MLST)+databases.">mlstdbNet - distributed multi-locus sequence typing (MLST) databases.</a> BMC Bioinformatics. 5, 86 (2004).</li><li>Verroken, A., 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=Evaluation+of+Matrix-Assisted+Laser+Desorption+Ionization-Time+of+Flight+Mass+Spectrometry+for+Identification+of+Nocardia+Species.">Evaluation of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Nocardia Species.</a> J Clinl Microbiol. 48 (11), 4015-4021 (2010).</li><li>El Kh&#233;chine, A., Couderc, C., Flaudrops, C., Raoult, D., Drancourt, M. <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+Identification+of+Mycobacteria+in+Routine+Clinical+Practice.">Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Identification of Mycobacteria in Routine Clinical Practice.</a> PLoS ONE. 6 (9), e24720 (2011).</li><li>Goujon, 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+new+bioinformatics+analysis+tools+framework+at+EMBL-EBI.">A new bioinformatics analysis tools framework at EMBL-EBI.</a> Nucleic Acids Research. 38, 695-699 (2010).</li><li>Hall, 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=BioEdit:+a+user-friendly+biological+sequence+alignment+editor+and+analysis+program+for+Windows+95/98/NT.">BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT.</a> Nucleic Acids Symposium Series. 41, 95-98 (1999).</li><li>Jolley, K. A., 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=Ribosomal+multilocus+sequence+typing:+universal+characterization+of+bacteria+from+domain+to+strain.">Ribosomal multilocus sequence typing: universal characterization of bacteria from domain to strain.</a> Microbiology. 158, 1005-1015 (2012).</li><li>Suarez, S., 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=Ribosomal+proteins+as+biomarkers+for+bacterial+identification+by+mass+spectrometry+in+the+clinical+microbiology+laboratory.">Ribosomal proteins as biomarkers for bacterial identification by mass spectrometry in the clinical microbiology laboratory.</a> J Microbiol Methods. 94 (3), 390-396 (2013).</li><li>Teramoto, 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=Phylogenetic+classification+of+Pseudomonas+putida+strains+by+MALDI-MS+using+ribosomal+subunit+proteins+as+biomarkers.">Phylogenetic classification of Pseudomonas putida strains by MALDI-MS using ribosomal subunit proteins as biomarkers.</a> Anal Chem. 79 (22), 8712-8719 (2007).</li><li>Teramoto, K., Kitagawa, W., Sato, H., Torimura, M., Tamura, T., Tao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+analysis+of+Rhodococcus+erythropolis+based+on+the+variation+of+ribosomal+proteins+as+observed+by+matrix-assisted+laser+desorption+ionization-mass+spectrometry+without+using+genome+information.">Phylogenetic analysis of Rhodococcus erythropolis based on the variation of ribosomal proteins as observed by matrix-assisted laser desorption ionization-mass spectrometry without using genome information.</a> J Biosci Bioeng. 108 (4), 348-353 (2009).</li><li>Bernhard, M., Weig, M., Zautner, A. E., Gross, U., Bader, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+on-target+lysis+(YOTL),+a+procedure+for+making+auxiliary+mass+spectrum+data+sets+for+clinical+routine+identification+of+yeasts.">Yeast on-target lysis (YOTL), a procedure for making auxiliary mass spectrum data sets for clinical routine identification of yeasts.</a> J Clin Microbiol. 52 (12), 4163-4167 (2014).</li><li>Stark, 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=Mass+spectrometric+profiling+of+Bacillus+cereus+strains+and+quantitation+of+the+emetic+toxin+cereulide+by+means+of+stable+isotope+dilution+analysis+and+HEp-2+bioassay.">Mass spectrometric profiling of Bacillus cereus strains and quantitation of the emetic toxin cereulide by means of stable isotope dilution analysis and HEp-2 bioassay.</a> Anal Bioanal Chem. 405 (1), 191-201 (2012).</li></ol>

The authors have nothing to disclose.

在建立MSPP计划的最关键的一步是生物标志物离子身份的明确的遗传决定。如果它是不可能无疑鉴定的生物标记，那么它应该被排除在方案13。 C.在菌 SSP。doylei方案包括14种不同的生物标志物离子。这些都是5少比C.在检测到C之间杆菌 SSP。 杆菌 MSPP方案13。最显著差异菌 SSP。 菌和C.杆菌 SSP。...

在过去十年中，时间飞行基质辅助激光解吸电离质谱（MALDI-TOF MS）已经发展到在临床微生物学1,2微生物属和种鉴定高度评价标准方法。物种鉴定是基于完整细胞或细胞裂解物的小蛋白指纹记录。在常规的临床微生物学中使用的质谱仪的典型质量范围是2-20 kDa的。此外，所得的光谱可以被用于鉴别在下面种菌株和低于亚种级别3。创业初期的研究已经确定了具体的生物标志物离子来株的空肠弯曲菌 4特定亚型， 难辨梭状芽孢杆菌 5， 沙门氏菌 SSP。 伤寒 沙门菌 6， 金黄色葡萄球菌 7 - 9，和Escherichia大肠杆菌10 - 12。 对应等位基因亚型几个变量的生物标记物群众相结合，提供了更深层次的子类型的选项。此前，我们成功地实施大规模型材这些变化转换成一个叫做C.质谱法基于phyloproteomics（MSPP）有意义的和可重复的phyloproteomic关系的方法菌 SSP。 杆菌菌株收集13。 MSPP可以使用质谱等同于基于DNA序列分型技术如多位点序列分型（MLST）。 弯曲杆菌是细菌性胃肠炎的全球领先的原因14，15。作为弯曲菌病感染后后遗症的结果，即，格林-巴利综合症，反应性关节炎和炎症性肠病可能出现16。感染的主要来源是从鸡，火鸡，猪，牛，羊，鸭，牛奶及地表水15,17污染畜肉。因此，在食品安全的背景下定期流行病学监测研究是必要的。 MLST是"黄金标准"的分子分型弯曲杆菌种18。因为基于MLST方法桑格测序是劳动密集，耗时且相对昂贵的，MLST打字被限制为相对小的分离同伙。因此，有必要更便宜和更快的子类型的方法。这种需要可以通过质谱方法，如MSPP得到满足。 本文介绍用空肠弯曲菌 SSP的集合MSPP打字了详细的方案。doylei株及其与MLST潜力比较。

<table border="0" cellpadding="0" cellspacing="0" width="1348">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">acetonitrile</td>
			<td style="width:663px;">Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">34967</td>
			<td style="width:337px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Autoflex III TOF/TOF 200 system</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td style="width:119px;">GT02554 G201</td>
			<td>Mass spectrometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">bacterial test standard BTS</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td style="width:119px;">604537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">BioTools 3.2 SR1</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td>263564</td>
			<td>Software Package</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">Bruker IVD Bakterial Test Standard</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td style="width:119px;">8290190</td>
			<td>5 tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG8843</td>
			<td>ATCC 49349;IMVS 1141;NCTC 11951;strain 093</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG9143</td>
			<td>Goossens Z90</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG7790</td>
			<td>ATCC 49350;CCUG 18265;Kasper 71;LMG 8219;NCTC 11847</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG9243</td>
			<td>Goossens N130</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG8871</td>
			<td>NCTC A603/87</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG9255</td>
			<td>Goossens B538</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG8870</td>
			<td>NCTC A613/87</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Columbia agar base&#160;</td>
			<td>Merck, Darmstadt, Germany</td>
			<td style="width:119px;">1.10455 .0500</td>
			<td>500 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Compass for FlexSeries 1.2 SR1</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td>251419</td>
			<td>Software Package</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">defibrinated sheep blood&#160;</td>
			<td>Oxoid Deutschland GmbH, Wesel, Germany</td>
			<td>SR0051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">ethanol</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">02854 Fluka</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">formic acid</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">F0507</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">HCCA matrix</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td style="width:119px;">604531</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Kimwipes paper tissue</td>
			<td style="width:663px;">Kimtech Science via Sigma-Aldrich, Taufkirchen, Germany</td>
			<td>Z188956</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">MALDI Biotyper 2.0</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td>259935</td>
			<td>Software Package</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Mast Cryobank vials</td>
			<td>Mast Diagnostica, Reinfeld, Germany</td>
			<td style="width:119px;">CRYO/B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">MSP 96 polished steel target</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td>224989</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAamp DNA Mini Kit&#160;</td>
			<td style="width:663px;">Qiagen, Hilden, Germany</td>
			<td style="width:119px;">51304</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">recombinant human insulin</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">I2643</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">trifluoroacetic acid</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">T6508</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">water, molecular biology-grade</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">W4502</td>
			<td></td>
		</tr>
	</tbody>
</table>

subtyping of campylobacter jejuni ssp. doylei isolates using mass spectrometry-based phyloproteomics (mspp)

MALDI-TOF MS提供不仅在种和亚种水平，但甚至低于，在应变水平区分某些细菌的可能性。可检测生物标志物离子的等位基因亚型产生具体的分离质量的变化。质谱为基础的phyloproteomics（MSPP）是一种新技术，它结合了质谱检测生物标志物的群众一个方案，允许从比较基因组分离特定质量偏移phyloproteomic关系扣除测序参考株。然后将推断的氨基酸序列被用于计算基于MSPP-系统树图。 在这里，我们通过键入空肠弯曲杆菌 SSP描述MSPP的工作流程。doylei的7株分离的集合。这七株人类起源和多位点序列分（MLST）的展示了其遗传多样性。 MSPP打字造成了七种不同的MSPP序列类型，充分反映他们的PHYlogenetic关系。 C.在菌 SSP。doylei MSPP方案包括14个不同的生物标志物离子，主要是核糖体蛋白在2至11 kDa的质量范围。 MSPP可以在原则上，可以适用于其他质谱平台具有延伸的质量范围。因此，这种技术有可能成为应变水平的微生物类型的有用工具的潜力。

此前，我们成功地建立了一个C. MSPP方案菌 SSP。 空肠弯曲菌 13。在这里，我们的目的是同级亚种C.延长方法菌 SSP。doylei。在这个特定的设置，七C.菌 SSP。doylei株微生物微生物学根特大学BCCM / LMG比利时根特/实验室比利时集合收购。用于我们的分析所有7株均来源于人。基因组测序菌株ATCC 49349（LMG 8843），最初是?...

Watch this Scientific Journal Video about Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics MSPP at JoVE.com

Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics MSPP

基于质谱的phyloproteomics（MSPP）用于键入空肠弯曲杆菌过磷酸钙的集合。doylei隔离在相比于多位点序列分型（MLST）的应变水平。

器的子类型<em&gt;空肠弯曲菌</em&gt; SSP。<em&gt; doylei</em&gt;分离株使用基于光谱质谱PhyloProteomics（MSPP）

MALDI-TOF MS offers the possibility to differentiate some bacteria not only at the species and subspecies level but even below, at the strain level. ...

subtyping-campylobacter-jejuni-ssp-doylei-isolates-using-mass

Research

JoVE Journal

Genetics

Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics (MSPP)

8.5K 次观看.  Universitätsmedizin Göttingen. 基于质谱的phyloproteomics（MSPP）用于键入空肠弯曲杆菌过磷酸钙的集合。doylei隔离在相比于多位点序列分型（MLST）的应变水平。 

视频: Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics MSPP

Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) is described. DNA extraction is carried out using a silica membrane-based DNA extraction kit. After the PCR amplification using a detergent-free PCR buffer, restriction enzymes are added to the solution without purifying the reaction solution. A reverse-phase silica monolith column and a Fourier transform high resolution mass spectrometer having a modified Kingdon trap analyzer are employed for separation and detection, respectively. The mobile phase, consisting of 400 mM 1,1,1,3,3,3-hexafluoro-2-propanol, 15 mM triethylamine (pH 7.9) and methanol, is delivered at a flow rate of 0.4 ml/min. The cycle time for LC/ESI-MS analysis is 8 min including equilibration of the column. Deconvolution software having an isotope distribution model of the oligonucleotide is used to calculate the corresponding monoisotopic mass from the mass spectrum. For analysis of oligonucleotides (range 26-79 nucleotides), mass accuracy was 0.62 &#177; 0.74 ppm (n = 280) and excellent accuracy and precision were sustained for 180 hr without use of a lock mass standard.

An improved polymerase chain reaction-restriction fragment length polymorphism method for genotyping pufferfish species by liquid chromatography/mass spectrometry is described. A reverse-phase silica monolith column is employed for separating digested amplicons. This method can elucidate the monoisotopic masses of oligonucleotides, which is useful for identifying base composition.

河豚有毒改进的聚合酶链反应 - 限制性片段长度多态性分型液相色谱/质谱法

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by ...

科研

遗传学

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) refers to the experimental situation in which evolution is observed using living organisms under controlled conditions and stressors; organisms are thereby artificially forced to make evolutionary changes. Microorganisms are subject to a variety of stressors in the environment and are capable of regulating certain stress-inducible proteins to increase their chances of survival. Naturally occurring spontaneous mutations bring about changes in a microorganism's genome that affect its chances of survival. Long-term exposure to chemostat culture provokes an accumulation of spontaneous mutations and renders the most adaptable strain dominant. Compared to the colony transfer and serial transfer methods, chemostat culture entails the highest number of cell divisions and, therefore, the highest number of diverse populations. Although chemostat culture for ALE requires more complicated culture devices, it is less labor intensive once the operation begins. Comparative genomic and transcriptome analyses of the adapted strain provide evolutionary clues as to how the stressors contribute to mutations that overcome the stress. The goal of the current paper is to bring about accelerated evolution of microorganisms under controlled laboratory conditions.

Here, we present a protocol to obtain adaptive laboratory evolution of microorganisms under conditions using chemostat culture. Also, genomic analysis of the evolved strain is discussed.

程序的微生物实验室自适应演化的恒化

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) ...

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

单细胞测序拷贝数改变的检测

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been investigated in various organs and tissues in rat. However, standard methods for the purification of miRNAs and detection of their expression in rat peritoneal membrane have not been well established. We have developed an effective and reliable method to purify and quantify miRNAs using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) in rat peritoneal membrane. This protocol consists of four steps: 1) purification of peritoneal membrane sample; 2) purification of total RNA including miRNA from peritoneal membrane sample; 3) reverse transcription of miRNA to produce cDNA; and 4) qRT-PCR to detect miRNA expression. Using this protocol, we successfully determined that the expression of six miRNAs (miRNA-142-3p, miRNA-21-5p, miRNA-221-3p, miRNA-223-3p, miRNA-327, and miRNA-34a-5p) increased significantly in the peritoneal membrane of a rat peritoneal fibrosis model compared with those in control groups. This protocol can be used to study the profile of miRNA expression in the peritoneal membrane of rats in many pathological conditions.

Here we present a protocol for the detection of microRNA expression in rat peritoneal membrane using quantitative real-time reverse-transcription polymerase chain reaction. This method is suitable for studying the microRNA expression profile in rat peritoneal membrane in several pathological conditions.

使用定量实时PCR检测大鼠腹膜中的微小RNA表达

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been ...

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. Therefore, DNA-protein interactions regulate multiple physiological, pathophysiological, and biological functions, such as cell differentiation, cell proliferation, cell cycle control, chromosome stability, epigenetic gene regulation, and cell transformation. In eukaryotic cells, the DNA interacts with histone and nonhistone proteins and is condensed into chromatin. Several technical tools can be used to analyze DNA-protein interactions, such as the Electrophoresis (gel) Mobility Shift Assay (EMSA) and DNase I footprinting. However, these techniques analyze the protein-DNA interaction in vitro, not within the cellular context. Chromatin immunoprecipitation (ChIP) is a technique that captures proteins at their specific DNA binding sites, thereby allowing for the identification of DNA-protein interactions within their chromatin context. It is done by fixation&#160;of the DNA-protein interaction, followed by&#160;immunoprecipitation of the protein of interest. Subsequently, the genomic site that the protein was bound to is characterized. Here, we describe and discuss ChIP and demonstrate its analytical value for the identification of the Transforming Growth Factor-&#946; (TGF-&#946;)-induced binding of the transcription factor SMAD2 to SMAD Binding Elements (SBE) within the promoter region of the tyrosine-protein kinase Kit (c-KIT) receptor ligand Stem Cell Factor (SCF).

DNA-protein interactions are essential for multiple biological processes. During the evaluation of cellular functions, the analysis of DNA-protein interactions is indispensable for understanding gene regulation. Chromatin immunoprecipitation (ChIP) is a powerful tool to analyze such interactions in vivo.

使用染色质免疫沉淀分析c-KIT配体启动子

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for protein synthesis by translating codons in mRNA into amino acid sequences. tRNAs were long considered as house-keeping molecules that lacked regulatory functions. However, a growing body of evidence indicates that cellular tRNA levels fluctuate in correspondence to varying conditions such as cell type, environment, and stress. The fluctuation of tRNA expression directly influences gene translation, favoring or repressing the expression of particular proteins. Ultimately comprehending the dynamic of protein synthesis requires the development of methods able to deliver high-quality tRNA profiles. The method that we present here is named SPOt, which stands for Streamlined Platform for Observing tRNA. SPOt consists of three steps starting with metabolic labeling of cell cultures with radioactive orthophosphate, followed by guanidinium thiocyanate-phenol-chloroform extraction of radioactive total RNAs and finally hybridization on in-house printed macroarrays. tRNA levels are estimated by quantifying the radioactivity intensities at each probe spot. In the protocol presented here we profile tRNAs in Mycobacterium smegmatis mc2155, a nonpathogenic bacterium often used as a model organism to study tuberculosis.

We describe an original transfer RNA analysis platform named SPOt (Streamlined Platform for Observing tRNA). SPOt simultaneously measures cellular levels of all tRNAs in biological samples, in only three steps, and in less than 24 hours.

使用 Macroarrays 的转移 rna 的代谢标记和剖面分析

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for ...

Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation

When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in production or degradation of RNA. This protocol describes a method for measurement of RNA production, using 5-Bromouridine labelling of RNA followed by immunoprecipitation, which enables investigation of RNA synthesized within a short timeframe (e.g., 1 h). The advantage of 5-Bromouridine-labelling and immunoprecipitation over the use of toxic transcriptional inhibitors, such as &#945;-amanitin and actinomycin D, is that there are no or very low effects on cell viability during short-term use. However, because 5-Bromouridine-immunoprecipitation only captures RNA produced within the short labelling time, slowly produced as well as rapidly degraded RNA can be difficult to measure by this method. The 5-Bromouridine-labelled RNA captured by 5-Bromouridine-immunoprecipitation can be analyzed by reverse transcription, quantitative polymerase chain reaction, and next generation sequencing. All types of RNA can be investigated, and the method is not limited to measuring mRNA as is presented in this example.

This method can be used to measure RNA synthesis. 5-Bromouridine is added to cells and incorporated into synthesized RNA. RNA synthesis is measured by RNA extraction immediately after labelling, followed by 5-Bromouridine-targeted immunoprecipitation of labelled RNA and analysis by reverse transcription and quantitative polymerase chain reaction.

用 5-Bromouridine 标记和免疫沉淀 RNA 合成的研究

When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in ...

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective tag single nucleotide polymorphism (SNP) approach using a multiplexed genotyping assay with mass spectrometry, to investigate gene pathway associations in clinical cohorts. We investigate the food allergy candidate locus Interleukin13 (IL13) as an example. This method efficiently maximizes the coverage by taking advantage of shared linkage disequilibrium (LD) within a region. Selected LD SNPs are then designed into a multiplexed assay, enabling up to 40 different SNPs to be analyzed simultaneously, boosting cost-effectiveness. Polymerase chain reaction (PCR) is used to amplify the target loci, followed by single nucleotide extension, and the amplicons are then measured using matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF) mass spectrometry. The raw output is analyzed with the genotype calling software, using stringent quality control definitions and cut-offs, and high probability genotypes are determined and output for data analysis.

Identification of genetic variants contributing to complex human disease allows us to identify novel mechanisms. Here, we demonstrate a multiplex genotyping approach to candidate genes or gene pathway analysis that maximizes the coverage at low cost and is amenable to cohort-based studies.

应用多重基因分型和质谱法进行临床队列研究的候选基因检测

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...

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 ...

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, and was deployed to West Africa during the recent Ebola virus epidemic, highlighting this possibility. In addition to its practical advantages (low cost, ease of equipment transport and use), this technology also provides fundamental advantages over second-generation sequencing approaches, particularly the very long read length, ability to directly sequence RNA, and real-time availability of data. Raw read accuracy is lower than with other sequencing platforms, which represents the main limitation of this technology; however, this can be partially mitigated by the high read depth generated. Here, we present a field-compatible protocol for sequencing of the mRNAs encoding for Niemann-Pick C1, which is the cellular receptor for ebolaviruses. This protocol encompasses extraction of RNA from animal blood samples, followed by RT-PCR for target enrichment, barcoding, library preparation, and the sequencing run itself, and can be easily adapted for use with other DNA or RNA targets.

Nanopore sequencing is a novel technology that allows cost-effective sequencing in remote locations and resource-poor settings. Here, we present a protocol for sequencing of mRNAs from whole blood that is compatible with such conditions.

纳米测序全血 mRNA 测序

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...