我们感谢克里斯托弗Masselon博士（iRTV，东航，格勒诺布尔）和病毒感染的成员和癌症组在肠易激综合征，其关键手稿的评价和有益的讨论。我们感谢西里尔殿的蛋白质CRM1的一种恩赐。这种科学工作在格勒诺布尔指导中心（; UMS 3518 CNRS-CEA-UJF-EMBL ISBG）的质谱设施发生了。它是财政由法国基础设施一体化结构生物学计划（FRISBI，ANR-10-INSB-05-02）的支持，GRAL（ANR-10的LabX-49-01）[格勒诺布尔伙伴关系中的结构生物学]和法国国家科学研究中心（CNRS）。

1。蛋白样品制备:缓冲交易所（可选） 5-25微升微摩尔浓度的蛋白（1〜20微米）的是必要的。缓冲交换可以采用离心超滤设备（ 如的Vivaspin，赛多利斯）或离心凝胶过滤柱（ 如微型生物分拆6层析柱，Bio-Rad公司）29,30进行。缓冲液交换步骤可重复2〜3倍。缓冲交换的详细描述过往呈29,30。例如，我们利用20mM的三（羟甲基）氨基甲烷（ 即三）pH为8的最终缓冲液中。 注意:该步骤可以在许多情况下可以省略。它应进行时的蛋白质样品中含有的分子或缓冲剂，可以强烈地与MS检测干涉[ 例如甘油; 4 - （2 -羟基乙基）-1 -哌嗪乙磺酸，HEPES]。它也是有用改善曲先进而精湛的谱。 2。矩阵中提高自己的纯度（可选）令再结晶<ol><li>倒10毫升40％乙醇（乙醇）进入一个耐热烧瓶中。 </li><li>添加600毫克的矩阵。 </li><li>使用水浴和一个电阻加热器，温基质溶液，并搅拌，直到该矩阵用玻璃棒或磁力搅拌器的完全溶解。 </li><li>直到你有一个饱和溶液重复步骤2。 </li></ol> 注意:使用过大体积的溶剂或溶解远低于溶液沸点的矩阵将导致结晶或无结晶的差的产率都没有。 <ol start="5"><li>让溶液慢慢冷却。你可以把它在室温下进行几个小时，然后在4℃下过夜。注意:如果结晶都没有形成，结晶可以通过使用玻璃搅拌棒刮擦烧杯（正好在溶液表面的下方）的内部进行诱导。 </li><li>通过过滤收集矩阵晶体。 </li><li>漂洗结晶，用冰冷的溶剂的最小量和滤液它们。 </li><li>使结晶干燥。可以使用真空此步骤。 </li></ol> 3。 MALDI不锈钢目标的清洁<ol><li>冲洗MALDI板用甲醇（甲醇），用清洁纸巾实验室应用（ 如的Kimwipe）特制轻轻擦拭。 </li><li>冲洗目标与H 2 O和清洁纸巾擦拭。 </li><li>插入MALDI板在600毫升的烧杯中，并用50％乙醇覆盖。 </li><li>超声处理的对象中用于在超声浴中10分钟的EtOH溶液。 </li><li>如果有剩余的上盘残留物，重复再次1-4步骤。 </li><li>最后，用甲醇目标（或水，见下面的注释），倾斜于所有的液体被收集在一个清洁的组织方式。让靶干燥，在室温或使用氮气气流。 </li></ol> 注意:类似的过程，先前描述的15。 注意:用于目标的最后漂洗溶剂（甲醇或水）确定一些目标特性。如果目标是用MeOH冲洗，样品涂在目标使用水时，远远不止。 4。 α-CHCA薄层液的制备及其沉积在MALDI靶<ol><li>溶解的α-CHCA，在丙酮中，以使饱和溶液。 </li><li>通过手（没有任何吸管）蘸10微升末端（ 例如 GELoader提示，Eppendorf）中的α-CHCA饱和溶液:少量的溶液将在尖端流（由于毛细现象）。 </li><li>触摸非常迅速（ 即 1秒）的MALDI靶用移液管尖和沉积在MALDI靶的α-CHCA丙酮溶液。这将构成基质的薄层，其中所述蛋白质样品将被存入。尝试生成一薄层斑点尽可能小。 </li></ol> 注意:当使用SA为基体，我们准备利用SA的丙酮饱和溶液薄薄的一层。 5。 SA，α-CHCA，DHB和CHCA_DHB混合25:虎斑溶液的制备<ol><li>制备20毫克/毫升的SA溶液的ACN，0.1％TFA（70:30，体积/体积）。 </li><li>制备在乙腈20毫克/毫升α-CHCA溶液和5％甲酸（70:30，体积/体积）命名为"α-CHCA溶液"]。 </li><li>制备在乙腈20毫克/毫升DHB溶液和0.1％三氟乙酸（TFA）（70:30，体积/体积）命名为"DHB溶液"]。 </li><li>混合"的α-CHCA解决方案"和"DHB解决方案"在1:1的比例（体积/体积），获得"CHCA_DHB解决方案"。 </li></ol> 注意:一个可以混合"的α-CHCA解决方案"和"DHB解决方案"在不同的比例，与质量小于100 kDa的蛋白质进行分析时。对于EXA投影机的简易40:60α-CHCA和DHB（体积/体积）之间的比值得到一个更好的分辨率，但不敏感（见讨论）。 6。在MALDI靶沉积样品<ol><li>存款0.5微升先前准备的α-CHCA薄层上的蛋白质样品。紧接在此之后，加入0.5微升的基质溶液（ 即 SA溶液或α-CHCA溶液或「CHCA_DHB混合物"）的。这意味着在目标样品与基质混合。 </li></ol> 注意:通常1的蛋白质样品与基质以1:1的比例混合。这个比率是用于MALDI谱的良好质量的关键。如果你想测试不同的样品浓度，可以使用ACN_formic酸溶液（如上所述），以稀释样品。 注意:如果你愿意，你可以混合样品和基质在试管中，然后存入混"sample_matrix解决方案化"的薄层上。 注意:我们建议您测试不同的样品浓度，因为稀释样品可让您减少 ​​污染物的干扰。稀释样品，你可以使用ACN_formic酸溶液（如上所述），以稀释样品。 7。校准物沉积<ol><li>存款0.5微升的校准标准（ 如 "蛋白质标准二"，布鲁克道尔顿，不来梅），再加入0.5μL基质溶液中。 </li></ol> 注:装入旁边的蛋白质样品点进行MALDI板（见讨论）的校准。 8。电离光谱采集校准物和蛋白质样品一旦样品和校准物干燥，你可以观察在显微镜下"斑"。这个观察是可选的，可能主要感兴趣的新手。为了获得样品的光谱，插入第ê目标在MALDI-TOF仪器，并选择合适的仪器参数，这些参数为不同的每种类型的设备。为了充分利用相应的设定应该遵循制造商的建议。作为一般的考虑分析完整蛋白质时，应该使用该仪器的线性模式（而不是在反射模式，这是适当的肽分析）。通常情况下，我们利用我们的仪器在正离子模式。此外，一个关键的参数是"脉冲离子提取"，这提高了仪器的分辨率31。在简单术语中，"脉冲离子提取"可以被定义为样品离子的产生，当离子被引向检测器加速的时间之间的停顿。在分析完整蛋白质，应使用一个"脉冲离子提取"（ 例如 500纳秒）观察肽时，大于（ 例如 80纳秒）。 选择适当的m / z范围，获取光谱ØF中的校准和校准仪器。当您获得您的样品的光谱，使用适当的激光强度（见讨论）。

<ol>
	<li>Rupp, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biomolecular Crystallography : Principles, Practice, and Application to Structural Biology. , (2010).</li><li>Cohen, S. L., Chait, B. T. <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+as+a+tool+for+protein+crystallography.">Mass spectrometry as a tool for protein crystallography.</a> Annual review of biophysics and biomolecular structure. 30, 67-85 (2001).</li><li>Chait, B. T. <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--a+useful+tool+for+the+protein+X-ray+crystallographer+and+NMR+spectroscopist.">Mass spectrometry--a useful tool for the protein X-ray crystallographer and NMR spectroscopist.</a> Structure. 2, 465-467 (1994).</li><li>Gans, 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=Stereospecific+isotopic+labeling+of+methyl+groups+for+NMR+spectroscopic+studies+of+high-molecular-weight+proteins.">Stereospecific isotopic labeling of methyl groups for NMR spectroscopic studies of high-molecular-weight proteins.</a> Angew Chem Int Ed Engl. 49, 1958-1962 (2010).</li><li>Wilm, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+electrospray+ionization.">Principles of electrospray ionization.</a> Molecular &amp; cellular proteomics : MCP. 10, M111 009407 (2011).</li><li>Urban, P. L., Amantonico, A., Zenobi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lab-on-a-plate:+extending+the+functionality+of+MALDI-MS+and+LDI-MS+targets.">Lab-on-a-plate: extending the functionality of MALDI-MS and LDI-MS targets.</a> Mass spectrometry reviews. 30, 435-478 (2011).</li><li>De Hoffmann, E., Stroobant, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mass Spectrometry: Principles and Applications. , (2007).</li><li>Karas, M., Hillenkamp, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+desorption+ionization+of+proteins+with+molecular+masses+exceeding+10,000+daltons.">Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons.</a> Analytical chemistry. 60, 2299-2301 (1988).</li><li>Spengler, B., Cotter, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultraviolet+laser+desorption/ionization+mass+spectrometry+of+proteins+above+100,000+daltons+by+pulsed+ion+extraction+time-of-flight+analysis.">Ultraviolet laser desorption/ionization mass spectrometry of proteins above 100,000 daltons by pulsed ion extraction time-of-flight analysis.</a> Analytical chemistry. 62, 793-796 (1990).</li><li>Beavis, R. C., Chait, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-accuracy+molecular+mass+determination+of+proteins+using+matrix-assisted+laser+desorption+mass+spectrometry.">High-accuracy molecular mass determination of proteins using matrix-assisted laser desorption mass spectrometry.</a> Analytical chemistry. 62, 1836-1840 (1990).</li><li>Hillenkamp, F., Karas, M., Beavis, R. C., Chait, B. T. <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+mass+spectrometry+of+biopolymers.">Matrix-assisted laser desorption/ionization mass spectrometry of biopolymers.</a> Analytical chemistry. 63, 1193A-1203A (1991).</li><li>Beavis, R. C., Chait, B. T. <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+mass-spectrometry+of+proteins.">Matrix-assisted laser desorption ionization mass-spectrometry of proteins.</a> Methods in enzymology. 270, 519-551 (1996).</li><li>Xiang, F., Beavis, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+to+increase+contaminant+tolerance+in+protein+matrix-assisted+laser+desorption/ionization+by+the+fabrication+of+thin+protein-doped+polycrystalline+films.">A method to increase contaminant tolerance in protein matrix-assisted laser desorption/ionization by the fabrication of thin protein-doped polycrystalline films.</a> Rapid Communications in Mass Spectrometry. 8, 199-204 (1994).</li><li>Cadene, M., Chait, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robust,+detergent-friendly+method+for+mass+spectrometric+analysis+of+integral+membrane+proteins.">A robust, detergent-friendly method for mass spectrometric analysis of integral membrane proteins.</a> Analytical chemistry. 72, 5655-5658 (2000).</li><li>Fenyo, 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=MALDI+sample+preparation:+the+ultra+thin+layer+method.">MALDI sample preparation: the ultra thin layer method.</a> J. Vis. Exp.. (3), e192 (2007).</li><li>Gabant, G., Cadene, M. <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+of+full-length+integral+membrane+proteins+to+define+functionally+relevant+structural+features.">Mass spectrometry of full-length integral membrane proteins to define functionally relevant structural features.</a> Methods. 46, 54-61 (2008).</li><li>Hook, 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=Long+range+allosteric+control+of+cytoplasmic+dynein+ATPase+activity+by+the+stalk+and+C-terminal+domains.">Long range allosteric control of cytoplasmic dynein ATPase activity by the stalk and C-terminal domains.</a> The Journal of biological chemistry. 280, 33045-33054 (2005).</li><li>Vorm, O., Roepstorff, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+sequence+information+derived+by+partial+acid+hydrolysis+and+matrix-assisted+laser+desorption/ionization+mass+spectrometry.">Peptide sequence information derived by partial acid hydrolysis and matrix-assisted laser desorption/ionization mass spectrometry.</a> Biological mass spectrometry. 23, 734-740 (1994).</li><li>Beavis, R. C., Chait, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cinnamic+acid+derivatives+as+matrices+for+ultraviolet+laser+desorption+mass+spectrometry+of+proteins.">Cinnamic acid derivatives as matrices for ultraviolet laser desorption mass spectrometry of proteins.</a> Rapid Communications in Mass Spectrometry. 3, 432-435 (1989).</li><li>Madler, S., Boeri Erba, ., E, R., Zenobi, <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+for+Studying+Noncovalent+Complexes+of+Biomolecules.">MALDI-ToF Mass Spectrometry for Studying Noncovalent Complexes of Biomolecules.</a> Topics in current chemistry. , (2012).</li><li>Wortmann, A., Pimenova, T., Alves, S., Zenobi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+first+shot+phenomenon+in+MALDI+mass+spectrometry+of+protein+complexes.">Investigation of the first shot phenomenon in MALDI mass spectrometry of protein complexes.</a> The Analyst. 132, 199-207 (2007).</li><li>Beavis, R. C., Chaudhary, T., Chait, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-Cyano-4-hydroxycinnamic+Acid+as+a+Matrix+for+Matrix-assisted+Laser+Desorption+Mass+Spectrometry.">Alpha-Cyano-4-hydroxycinnamic Acid as a Matrix for Matrix-assisted Laser Desorption Mass Spectrometry.</a> Organic Mass Spectrometry. 27, 156-158 (1992).</li><li>Liu, Z., Schey, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+a+MALDI+TOF-TOF+mass+spectrometer+for+intact+protein+analysis.">Optimization of a MALDI TOF-TOF mass spectrometer for intact protein analysis.</a> Journal of the American Society for Mass Spectrometry. 16, 482-490 (2005).</li><li>Gorka, J., Bahr, U., Karas, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graphite+supported+preparation+(GSP)+of+alpha-cyano-4-hydroxycinnamic+acid+(CHCA)+for+matrix-assisted+laser+desorption/ionization+mass+spectrometry+(MALDI-MS)+for+peptides+and+proteins.">Graphite supported preparation (GSP) of alpha-cyano-4-hydroxycinnamic acid (CHCA) for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for peptides and proteins.</a> Journal of the American Society for Mass Spectrometry. 23, 1949-1954 (2012).</li><li>Laugesen, S., Roepstorff, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+of+two+matrices+results+in+improved+performance+of+MALDI+MS+for+peptide+mass+mapping+and+protein+analysis.">Combination of two matrices results in improved performance of MALDI MS for peptide mass mapping and protein analysis.</a> Journal of the American Society for Mass Spectrometry. 14, 992-1002 (2003).</li><li>Sparkman, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mass Spectrometry Desk Reference. , (2000).</li><li>Kussmann, M., Roepstorff, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+preparation+techniques+for+peptides+and+proteins+analyzed+by+MALDI-MS.">Sample preparation techniques for peptides and proteins analyzed by MALDI-MS.</a> Methods Mol Biol. 146, 405-424 (2000).</li><li>Wang, R., Chait, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-accuracy+mass+measurement+as+a+tool+for+studying+proteins.">High-accuracy mass measurement as a tool for studying proteins.</a> Current opinion in biotechnology. 5, 77-84 (1994).</li><li>Kirshenbaum, N., Michaelevski, I., Sharon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+large+protein+complexes+by+structural+mass+spectrometry.">Analyzing large protein complexes by structural mass spectrometry.</a> J. Vis. Exp. (40), e1954 (2010).</li><li>Hernandez, H., Robinson, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+the+stoichiometry+and+interactions+of+macromolecular+assemblies+from+mass+spectrometry.">Determining the stoichiometry and interactions of macromolecular assemblies from mass spectrometry.</a> Nature protocols. 2, 715-726 (2007).</li><li>Vestal, M. L., Juhasz, P., Martin, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+extraction+matrix-assisted+laser+desorption+time-of-flight+massspectrometry.">Delayed extraction matrix-assisted laser desorption time-of-flight massspectrometry.</a> Rapid Communications in Mass Spectrometry. 9, 1044-1050 (1995).</li></ol>

没有利益冲突的声明。

我们提出了一个详细的协议，以收购大完整的蛋白质高品质的质谱图与分子量大于100 kDa的大，用MALDI-TOF仪器。一些关于样品制备关键方面应慎重考虑，以提高质谱的质量。矩阵和高纯度的溶剂是至关重要的，因为所有存在于基质和溶剂中的杂质被浓缩在MALDI靶溶剂蒸发后。为了提高MALDI基质的纯度，可以使用经典的重结晶的方法（见上文）来纯化它们。我们也建议准备新鲜的矩阵解决方案（每周至少一次），以改善基体结晶，以避免基质降解。 基质沉积的方法是在光谱的最终质量非常关键的。如上所述，该薄层的方法是我们的​​方法øf选择12。此外，我们的优化方法来沉积第一层。当基质溶解在丙酮中，得到饱和的溶液中，使丙酮的溶剂的快速蒸发，同时也使得该第一层的尺寸非常难以控制。我们建议使用GELoader尖如您在matrix_acetone液浸让您快速地沉积在靶最小体积小刷子。在第一层的大小在某种程度上控制MALDI点的最终大小:一个小点（ 即 0.5-0.75毫米直径）是优选的，因为在这种情况下，样品浓度比大点的情况下更高。获得一个小点不同于以前在JOVE视频15提出了超薄层的方法。在Fenyo 等薄层溶液遍布使用吸管尖侧的MALDI靶。这种方法要求测试应满足特定条件的薄层（溶剂挥发的如速度）。如果不满足条件，在MALDI靶应清洗和一个新的薄层应来制备。这使得准备相当费力的一个新手。此外，样品/基质混合物在平板上沉积要求的过量溶剂的愿望使用真空管线和用TFA 15的洗涤步骤是必要的，使得Fenyo 等人制备了一点比我们的方法更困难。 基质与样品之间的体积比为完整蛋白质通过MALDI-TOF一个成功的分析很重要。测试不同的比率后，我们建议使用1:1的比例。不同的矩阵:样本比例可能危及光谱的质量，可能是由于不均匀的结晶。其中基质和样品存放的顺序也很重要。沉积所述基质薄层后，将样品沉积和后立即（在SAMP前乐点变得干燥）的CHCA_DHB混合物加入。此过程被定义为"夹心法"27，其中单独的样品上沉积2基质层之间。作为替代，本CHCA_DHB溶液和样品可以混合在0.5ml试管中，然后看准CHCA薄层12上。这将产生与导致高质量的光谱晶体的均匀层上的点。当与质量低于100 kDa的，DHB的浓度分析蛋白质可提高（ 如 60:40 DHB:CHCA），这可以产生更尖锐的峰，但可能影响测量灵敏度（ 即较不强烈的峰）。 对于正确的仪器校准，它是必不可少的沉积校准标准非常接近的样本点，允许一个获得更好的质量准确度。 "伪内部校准程序"先前建议15:采集样品光谱中，校准点我后秒激光拍摄的校准和信号被添加到样品谱图。这允许使用的校准峰你在内部校准样品谱图。 激光的能量也应仔细光谱采集过程中进行控制。在大多数情况下，更高的能量提高了灵敏度，但是降低了分辨率。我们建议使用最小可能的激光能量而不会影响收购的灵敏度。此外，为了改善光谱质量，人们可以获取每个样本点更多的投篮。一般情况下，更多的投篮您获得（1000-2000张），信号的强度就越高。当用激光击中当场，需要找到合适的位置（ 即 "甜蜜点"），因为采样是不是均匀分布。您可以在同一区域拍摄，直到该信号增加，然后尝试找到含有样品的另一个领域。 在矩阵和SOLV的结论，高纯度已废除，用于样品和目标矩阵的沉积方法，用于校准，激光能量的强度，收购时间（张/现货数量）的标准位置是一个应该考虑的时候的关键因素通过MALDI-TOF MS分析完整蛋白质。 作者投稿 LS和EBE设计实验，进行质谱实验，分析数据，并写了稿子。

结构生物学依赖于生产高质量的蛋白1，因此，需要被加上有效和可靠的方法用于蛋白质分析2,3。在我们的质谱结构 ​​生物学的研究所内（MS）的实验室，我们需要确认蛋白质的一级序列，评估突变和翻译后修饰的存在下，蛋白质的降解，样品均一性，以及同位素标记的质量（ 例如，氘化蛋白质核磁共振研究4）。由于结构生物学家利用有限的蛋白水解从灵活的部分区分刚性结构域，我们需要使用MS可靠地表征这种截短蛋白。 当生物分子是由MS分析，两种可能的方法是利用电离轻声这么重的和不稳定的分子。电喷雾电离（ESI）直接从离子化的分子液相5;基质辅助激光解吸电离（MALDI）要求的生物分子共结晶具有紫外线吸收的有机分子（ 即基质分子）6。 ESI - 飞行时间质谱（TOF）MS连接到液相色谱法已经成为常规技术为完整蛋白质的分析，因为它允许质量测定精度高（≤50ppm的）。然而，这是很容易的样品缓冲液的组合物（特别是盐和洗涤剂）和污染物（ 即聚合物）有时是难以消除，导致分析物的信号的抑制。 MALDI-TOF MS表示一个有效的替代:ESI-MS，因为它的性能的影响较小缓冲液组分，去污剂和污染物，并且允许进行序列验证完整蛋白质量的测定以足够的精度（≤500ppm以下）。 protei后Ñ​​消化，MALDI-TOF MS还可以用于通过所谓的"肽质量指纹"来分析所获得的进一步的一级序列确认的肽。 在我们的手中，质量测定完整蛋白质，它比100 kDa的大和异构（由于修改或截断）的，是不是通过ESI-MS更容易通过MALDI-MS。这是由于目前缺乏在ESI谱重叠电荷态分布。此外，由于MALDI过程是不依赖于被分析物的大小7，这样的方法产生高灵敏度的，当生物分子质量是高于100 kDa的。完整蛋白的显着的分析，进行了使用20世纪80年代末和90年代8-11的开始之间的国产仪器。 MALDI-TOF也可以用作筛选工具来评估蛋白质样品的质量，因为它需要进行样品制备更少的时间和更不容易受到interferenCES由于常见的杂质（ 如盐）。后的第一，快速通过MALDI-MS的评价中，样品可以进一步通过ESI-TOF分析，以确定更高精度的质量。此外，电离生成包含比ESI费用更少离子，因此获取和解读MALDI数据更简单。这使得学生在结构生物学工作刚刚经过短暂培训，以分析其重组蛋白。 两个主要因素影响的MALDI光谱的质量:基质和用于基质沉积（ 如干燥液滴8和薄层12-16,17,18）的技术。一个单一的有机基质[ 例如，芥子酸（SA），19-21或α-氰基-4 -羟基肉桂酸（α-CHCA）14,22,23]通常用于完整蛋白质和交联的蛋白质复合物的质谱检查。用α-CHCA，蔡特集团此前提出的p一个详细的协议之前的可溶性蛋白和膜蛋白14,15的MALDI分析修配业的超薄层。最近，戈尔卡等人所说明的石墨基涂层的目标，以提高使用的α-CHCA作为基质24的肽和蛋白质的MALDI分析。 在这里，我们提出了一个简单的协议，用于完整蛋白质的MALDI-TOF质谱分析，利用两个矩阵的混合物:2,5 -二羟基苯甲酸（DHB）和α-CHCA 25。我们系统地评估了DHB-CHCA组合的表现相比，SA和α-CHCA基质，用于完整蛋白质的控制。该基质混合物允许更高的分辨率（ 即蛋白质峰有更清晰的）。另外，强烈的多个电荷的离子的存在下（ 即 M +2 H 2 +，M 3 H 3 +等）可以实现更精确的质量测定，因为轴向MALDI-TOF仪器的分辨率更高的过充较低质量（M / Z）26。这是用于测定分子量的蛋白质比100kDa的较大的特别有用的。 更高的灵敏度也达到了使用DHB-CHCA混合物（0.5皮摩尔蛋白质的发现对MALDI靶）。 正如我们上面提到的，应该使用MALDI仪器时必须考虑的一个重要因素是基质沉积。劳格森等人提出的第一次25采用DHB-CHCA混合物，利用干滴沉积。然而，我们观察到更好的效果（ 例如，更高的灵敏度），当我们使用其中第一层是由α-CHCA溶解在丙酮中形成的薄层的方法。该薄层的方法12,27意味着矩阵晶体的均匀基质上的MALDI靶，这是以前15所述的视频朱庇特的形成。然后，将样品沉积在该基质上，最后附加基质沉积（见下文）。在这篇文章中，我们还说明了如何存放样品的MALDI靶，却怎么也清理目标15，再结晶，并准备矩阵。 总括而言，我们的目标是提供所有必要的信息用于分析完整蛋白质的科学家（特别是结构生物学家）谁需要评估在一个快速和简单的方式产生的蛋白质的质量，谁不那么熟悉的MALDI-TOF MS。据预测在1990年代中期，28，MS已经对生物学研究的一个增加的影响，因为它的辅助功能生物学家有所增加。我们希望我们提供的信息将是使MALDI-TOF访问生物学家和科学家，谁愿意开始使用质谱分析非常有用。

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>Trizma hydrochloride</td>
			<td>Sigma</td>
			<td>T3253</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trizima</td>
			<td>Sigma</td>
			<td>T6066</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Micro Bio-Spin 6 chromatography columns</td>
			<td>Bio-Rad</td>
			<td>732-6221</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vivaspin 500</td>
			<td>Sartorius</td>
			<td>VS0122</td>
			<td>various molecular weight cut off</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fluka</td>
			<td>14262</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fluka</td>
			<td>02860</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KIMTECH SCIENCE Precision Wipes Tissue Wipers</td>
			<td>Kimberly Clark</td>
			<td>05511</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&alpha;-cyano-4-hydroxycinnamic acid</td>
			<td>Sigma Aldrich</td>
			<td>C8982</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma Aldrich</td>
			<td>650501</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2,5-dihydroxybenzoic acid</td>
			<td>Fluka</td>
			<td>39319</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>GELoader Tips</td>
			<td>Eppendorf</td>
			<td>0030 001.222</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma Aldrich</td>
			<td>34998</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Fluka</td>
			<td>09676</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid</td>
			<td>Sigma Aldrich</td>
			<td>302031</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Protein Standard II</td>
			<td>Bruker Daltonics</td>
			<td>207234</td>
			<td>It contains Trypsinogen, Protein A, Serum Albumin-Bovine</td>
		</tr>
		<tr>
			<td>Immunoglobulin G</td>
			<td>ABSciex</td>
			<td>GEN602151</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Water purifier PURELAB Ultra Analytic</td>
			<td>ELGA LabWater</td>
			<td>89204-052</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Autoflex MALDI-TOF instrument</td>
			<td>Bruker Daltonics</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MALDI-TOF target</td>
			<td>Bruker Daltonics</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

matrix-assisted laser desorption/ionization time of flight (maldi-tof) mass spectrometric analysis of intact proteins larger than 100 kda

有效地确定群众蛋白是许多生物的研究（ 例如，用于结构生物学研究）的关键。精确质量测定允许一个评估蛋白质一级序列的正确性，突变和/或翻译后修饰，可能的蛋白质降解，样品均一性，以及同位素掺入的情况下，标记的程度（ 例如，的13 C标记的存在）。 电喷雾电离（ESI）质谱（MS）是广泛用于质量测定变性蛋白，但其效率受样品缓冲液的组成。特别地，盐，去垢剂和污染物的存在严重损害由ESI-MS蛋白质分析的有效性。基质辅助激光解吸/电离（MALDI）质谱是一个有吸引力的替代方案，由于其耐盐性和数据采集和解释的简单性化。此外，大众的决心大型异构蛋白（大于100 kDa的）是由于没有重叠的高电荷态分布，存在于ESI光谱更容易通过MALDI-MS。 在这里，我们提出了由飞行（TOF）的MALDI-时间来分析蛋白质大于100 kDa的更大的访问方法。我们示出了使用两个矩阵（ 即 2,5 -二羟基苯甲酸，α-氰基-4 -羟基肉桂酸）的混合物中，并在薄层的方法作为样品的沉积方法的实用的优点。我们还讨论了采集时间矩阵和溶剂纯度的关键作用，用于校准的标准，，激光的能量，和。总体而言，我们提供必要的信息，初学，用于分析完整的蛋白质比为100kDa，通过MALDI-MS较大。

使用两个不同的矩阵，2沉积方法和相同的激光强度（ 图1）:;我们分析了一个完整的蛋白质（123386沓分子量染色体区域维修1蛋白，CRM1）。 SA和CHCA_DHB混合物被用作基质。的混合物，得到更高质量的质谱中的信噪比和（ 图1A和B）的灵敏度方面。特别是，我们可以检测到0.5皮摩尔蛋白的沉积在MALDI靶（ 图1C）。使用SA（ 图1D）的蛋白质相同数量几乎检测不到。此外，使用基质的混合物，该基质的沉积方法的选择是"薄层"的方法（ 图1A）。使用"干滴"的方法，我们没有发现任何蛋白质与质量高于100 kDa的（数据未显示）。利用CHCA_DHB混合物（ 图1A和1C），多电荷的protei离子N的观察，使质量测定具有更高的精度，因为在轴向MALDI-TOF仪器的峰值分辨率是成反比的M / Z 26。 我们还分析了单体的β-半乳糖苷（116300道尔顿）（ 图2）。该CHCA_DHB谱均高于SA光谱灵敏度和分辨率方面更好。此外，多电荷离子（M 2 +，M 3 +，M 4 +）的存在下使我们能够确认该蛋白质的质量以更高的精度（ 图2A和2C）。使用薄层的方法，我们比较了CHCA_DHB混合物，SA和α-CHCA单独的表现为免疫球蛋白，IgG的分析（148500道尔顿）（ 图3）。当我们关联的不同数据，信号蛋白含量较高，并与在CHCA_DHB光谱更好的分辨率。得出结论，使用CHCA_DHB混合物和薄层沉积方法的<html>展现在利用MALDI TOF仪器所采集的大量完整的蛋白质质谱的质量显著改善。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/50635/50635fig1highres.jpg" src="/files/ftp_upload/50635/50635fig1.jpg" /> 图1。 123386沓:完整的染色体区域的维修1蛋白，（CRM1），分子量的MALDI-TOF分析。 。使用DHB_CHCA组合为矩阵B）量A）1皮摩尔CRM1的分析:1皮摩尔;矩阵:SA C）量:0.5皮摩尔;矩阵:DHB_CHCA D）金额:0.5皮摩尔;矩阵:SA。峰的信号，表示在任意强度单位刻度，是归一化至最大存在于每个谱值。 PG"SRC ="/ files/ftp_upload/50635/50635fig2.jpg"/&gt; 图2。完整的β-半乳糖苷酶（116,300道尔顿）的MALDI-TOF分析。 A）金额:20皮摩尔;矩阵DHB_CHCA B）金额:20皮摩尔;矩阵:SA C）量:5皮摩尔;矩阵:DHB_CHCA D）金额:5皮摩尔;矩阵:SA。 <img alt="图3" fo:content-width="3.5in" fo:src="/files/ftp_upload/50635/50635fig3highres.jpg" src="/files/ftp_upload/50635/50635fig3.jpg" /> 图3。一个完整的免疫球蛋白，IgG抗体（148,500道尔顿）的MALDI-TOF分析;金额:1.7皮摩尔使用CHCA_DHB将得到的谱更为激烈:比SA光谱（最大8200任意单位，AU）（最大:1,200盟）和α-CHCA光谱（最大:4500金）

Watch this Scientific Journal Video about Matrix-assisted Laser Desorption/Ionization Time of Flight MALDI-TOF Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa at JoVE.com

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

精确质量测定代表蛋白质的调查过程中的重要一步。基质辅助激光解吸/电离（MALDI）质谱（MS）可用于这种确定和其主要优点是耐受的盐，去垢剂和污染物。在这里，我们示出了用于蛋白质大于100 kDa的通过MALDI-MS较大的分析可访问的方法。

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

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

matrix-assisted-laser-desorptionionization-time-flight-maldi-tof-mass

Research

JoVE Journal

Chemistry

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

80.3K 次观看.  Université J. Fourier. 精确质量测定代表蛋白质的调查过程中的重要一步。基质辅助激光解吸/电离（MALDI）质谱（MS）可用于这种确定和其主要优点是耐受的盐，去垢剂和污染物。在这里，我们示出了用于蛋白质大于100 kDa的通过MALDI-MS较大的分析可访问的方法。 

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

Profiling the Triacylglyceride Contents in Bat Integumentary Lipids by Preparative Thin Layer Chromatography and MALDI-TOF Mass Spectrometry

The mammalian integument includes sebaceous glands that secrete an oily material onto the skin surface. Sebum production is part of the innate immune system that is protective against pathogenic microbes. Abnormal sebum production and chemical composition are also a clinical symptom of specific skin diseases. Sebum contains a complex mixture of lipids, including triacylglycerides, which is species-specific. The broad chemical properties exhibited by diverse lipid classes hinder the specific determination of sebum composition. Analytical techniques for lipids typically require chemical derivatizations that are labor-intensive and increase sample preparation costs. This paper describes how to extract lipids from mammalian integument, separate broad lipid classes by thin-layer chromatography, and profile the triacylglyceride contents using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This robust method enables a direct determination of the triacylglyceride profiles among species and individuals, and it can be readily applied to any taxonomic group of mammals.

Mammalian integument contains solvent-extractable lipids that can provide chemical compositions characteristic of individual species. This paper presents a routine method for separating broad lipid classes isolated from integumentary tissues using thin layer chromatography and determining the triacylglyceride profile by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

用制备薄层色谱和MALDI-TOF质谱分析的三酸甘油酯含量的蝙蝠外皮血脂

The mammalian integument includes sebaceous glands that secrete an oily material onto the skin surface. Sebum production is part of the innate immune ...

科研

化学

Mass Spectrometric Approaches to Study Protein Structure and Interactions in Lyophilized Powders

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for characterizing lyophilized formulations of protein therapeutics. Labeling followed by suitable proteolytic digestion allows the protein structure and interactions to be mapped with peptide-level resolution. Since the protein structural elements are stabilized by a network of chemical bonds from the main-chains and side-chains of amino acids, specific labeling of atoms in the amino acid residues provides insight into the structure and conformation of the protein. In contrast to routine methods used to study proteins in lyophilized solids (e.g., FTIR), ssHDX-MS and ssPL-MS provide quantitative and site-specific information. The extent of deuterium incorporation and kinetic parameters can be related to rapidly and slowly exchanging amide pools (Nfast, Nslow) and directly reflects the degree of protein folding and structure in lyophilized formulations. Stable photolytic labeling does not undergo back-exchange, an advantage over ssHDX-MS. Here, we provide detailed protocols for both ssHDX-MS and ssPL-MS, using myoglobin (Mb) as a model protein in lyophilized formulations containing either trehalose or sorbitol.

Here, we present detailed protocols for solid-state amide hydrogen/deuterium exchange mass spectrometry (ssHDX-MS) and solid-state photolytic labeling mass spectrometry (ssPL-MS) for proteins in solid powders. The methods provide high-resolution information on protein conformation and interactions in the amorphous solid-state, which may be useful in formulation design.

质谱途径研究蛋白质结构和相互作用的冻干粉

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for ...

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental exposure in a non-invasive manner. In this work, we present a protocol to characterize the exhaled VOCs in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry (Sec-nanoESI-HRMS). The homemade Sec-nanoESI source was readily set up based on a commercial nanoESI source. Hundreds of peaks were observed in the background-subtracted mass spectra of exhaled breath, and the mass accuracy values are -4.0-13.5 ppm and -20.3-1.3 ppm in the positive and negative ion detection modes, respectively. The peaks were assigned with accurate elemental composition according to the accurate mass and isotopic pattern. Less than 30 s is used for one exhalation measurement, and it takes approximately 7 min for six replicated measurements.

A protocol for characterizing chemical composition of exhaled breath in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry is demonstrated.

利用二次 Nanoelectrospray 电离耦合高分辨质谱的实时呼吸分析

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental ...

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) Mass Spectrometry

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group analysis as matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). This tutorial demonstrates methods for optimization of the sample preparation, spectral acquisition, and data analysis of synthetic polymers using MALDI-TOF MS. Critical parameters during sample preparation include the selection of the matrix, identification of an appropriate cationization salt, and tuning the relative proportions of the matrix, cation, and analyte. The acquisition parameters, such as mode (linear or reflector), polarization (positive or negative), acceleration voltage, and delay time, are also important. Given some knowledge of the chemistry involved to synthesize the polymer and optimizing both the data acquisition parameters and the sample preparation conditions, spectra should be obtained with sufficient resolution and mass accuracy to enable the unambiguous determination of the end groups of most homopolymers (masses below 10,000) in addition to the repeat unit mass and the overall molecular weight distribution. Though demonstrated on a limited set of polymers, these general techniques are applicable to a much wider range of synthetic polymers for determining mass distributions, though end group determination is only possible for homopolymers with narrow dispersity.

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight MALDI-TOF Mass Spectrometry

A protocol for the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) characterization of synthetic polymers is described including the optimization of sample preparation, spectral acquisition, and data analysis.

基质辅助激光解吸电离飞行时间 (MALDI) 质谱法表征合成聚合物

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group ...

An Efficient Sample Preparation Method to Enhance Carbohydrate Ion Signals in Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Sample preparation is a critical process in mass spectrometry (MS) analysis of carbohydrates. Although matrix-assisted laser desorption/ionization (MALDI) MS is the method of choice in carbohydrate analysis, poor ion signal and data reproducibility of carbohydrate samples continue to be severe problems. For quantitative analysis of carbohydrates, an effective analytical protocol providing superior data quality is necessary. This video demonstrates sample preparation protocols to improve signal intensity and minimize data variation of carbohydrates in MALDI-MS. After drying and crystallization of sample droplets, the crystal morphology is reformed by methanol before mass spectrometric analysis. The enhancement in carbohydrate signal is examined with MALDI imaging mass spectrometry (IMS). Experimental results show that crystal reformation adjusts crystalline structures and redistributes carbohydrate analytes. In comparison with the dried droplet preparation method in conventional MALDI-MS, reforming carbohydrate crystal morphologies with methanol shows significantly better signal intensity, ion image distribution, and data stability. Since the protocols demonstrated herein do not involve changes in sample composition, they are generally applicable to various carbohydrates and matrixes.

A protocol for enhancing carbohydrate ion signals in MALDI mass spectrometry by reforming crystalline structures during sample preparation processes is demonstrated.

基质辅助激光解吸/电离质谱中增强碳水化合物离子信号的高效样品制备方法

Sample preparation is a critical process in mass spectrometry (MS) analysis of carbohydrates. Although matrix-assisted laser desorption/ionization (MALDI) ...

Imaging Corrosion at the Metal-Paint Interface Using Time-of-Flight Secondary Ion Mass Spectrometry

Corrosion developed at the paint and aluminum (Al) metal-paint interface of an aluminum alloy is analyzed using time-of-flight secondary ion mass spectrometry (ToF-SIMS), illustrating that SIMS is a suitable technique to study the chemical distribution at a metal-paint interface. The painted Al alloy coupons are immersed in a salt solution or exposed to air only. SIMS provides chemical mapping and 2D molecular imaging of the interface, allowing direct visualization of the morphology of the corrosion products formed at the metal-paint interface and mapping of the chemical after corrosion occurs. The experimental procedure of this method is presented to provide technical details to facilitate similar research and highlight pitfalls that may be encountered during such experiments.

Time-of-flight secondary ion mass spectrometry is applied to demonstrate the chemical mapping and corrosion morphology at the metal-paint interface of an aluminum alloy after being exposed to a salt solution compared with a specimen exposed to air.

利用飞行时二次离子质谱法成像金属颜料界面的腐蚀

Corrosion developed at the paint and aluminum (Al) metal-paint interface of an aluminum alloy is analyzed using time-of-flight secondary ion mass ...

A Two-Step Pyrolysis-Gas Chromatography Method with Mass Spectrometric Detection for Identification of Tattoo Ink Ingredients and Counterfeit Products

Tattoo inks are complex mixtures of ingredients. Each of them possesses different chemical properties which have to be addressed upon chemical analysis. In this method for two-step pyrolysis online coupled to gas chromatography mass spectrometry (py-GC-MS) volatile compounds are analyzed during a first desorption run. In the second run, the same dried sample is pyrolyzed for analysis of non-volatile compounds such as pigments and polymers. These can be identified by their specific decomposition patterns. Additionally, this method can be used to differentiate original from counterfeit inks. Easy screening methods for data evaluation using the average mass spectra and self-made pyrolysis libraries are applied to speed up substance identification. Using specialized evaluation software for pyrolysis GS-MS data, a fast and reliable comparison of the full chromatogram can be achieved. Since GC-MS is used as separation technique, the method is limited to volatile substances upon desorption and after pyrolysis of the sample. The method can be applied for quick substance screening in market control surveys since it requires no sample preparation steps.

This method for two-step pyrolysis online coupled to gas chromatography with mass spectrometric detection and data evaluation protocol can be used for multi-component analysis of tattoo inks and discrimination of counterfeit products.

一种用于检测纹身油墨成分和假冒产品的两步热解-气相色谱法

Tattoo inks are complex mixtures of ingredients. Each of them possesses different chemical properties which have to be addressed upon chemical analysis. ...

Analysis of Complex Molecules and Their Reactions on Surfaces by Means of Cluster-Induced Desorption/Ionization Mass Spectrometry

Desorption/Ionization Induced by Neutral SO2 Clusters (DINeC) is employed as a very soft and efficient desorption/ionization technique for mass spectrometry (MS) of complex molecules and their reactions on surfaces. DINeC is based on a beam of SO2 clusters impacting on the sample surface at low cluster energy. During cluster-surface impact, some of the surface molecules are desorbed and ionized via dissolvation in the impacting cluster; as a result of this dissolvation-mediated desorption mechanism, low cluster energy is sufficient and the desorption process is extremely soft. Both surface adsorbates and molecules of which the surface is composed of can be analyzed. Clear and fragmentation-free spectra from complex molecules such as peptides and proteins are obtained. DINeC does not require any special sample preparation, in particular no matrix has to be applied. The method yields quantitative information on the composition of the samples; molecules at a surface coverage as low as 0.1 % of a monolayer can be detected. Surface reactions such as H/D exchange or thermal decomposition can be observed in real-time and the kinetics of the reactions can be deduced. Using a pulsed nozzle for cluster beam generation, DINeC can be efficiently combined with ion trap mass spectrometry. The matrix-free and soft nature of the DINeC process in combination with the MSn capabilities of the ion trap allows for very detailed and unambiguous analysis of the chemical composition of complex organic samples and organic adsorbates on surfaces.

Neutral SO2 clusters of low kinetic energy (&#60; 0.8 eV/constituent) are used to desorb complex surface molecules such as peptides or lipids for further analysis by means of mass spectrometry using an ion trap mass spectrometer. No special sample preparation is required, and real-time observation of reactions is possible.

聚类诱导解吸/电电质谱法对复杂分子及其在表面的反应分析

Desorption/Ionization Induced by Neutral SO2 Clusters (DINeC) is employed as a very soft and efficient desorption/ionization technique for mass ...

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

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

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

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

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

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

Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of protein therapeutics and the determination of the conformational transition pathways critical for biological functions, ranging from molecular recognition to enzymatic catalysis. Hydrogen/deuterium exchange (HDX) reaction coupled with top-down mass spectrometric (MS) analysis provides a means to characterize protein higher-order structures and dynamics in a conformer-specific manner. The conformational resolving power of this technique is highly dependent on the efficiencies of separating protein states at the intact protein level and minimizing the residual non-deuterated protic content during the HDX reactions. 
Here we describe a capillary electrophoresis (CE)-based variant of the HDX MS approach that aims to improve the conformational resolution. In this approach, proteins undergo HDX reactions while migrating through a deuterated background electrolyte solution (BGE) during the capillary electrophoretic separation. Different protein states or proteoforms that coexist in solution can be efficiently separated based on their differing charge-to-size ratios. The difference in electrophoretic mobility between proteins and protic solvent molecules minimizes the residual non-deuterated solvent, resulting in a nearly complete deuterating environment during the HDX process. The flow-through microvial CE-MS interface allows efficient electrospray ionization of the eluted protein species following a rapid mixing with the quenching and denaturing modifier solution at the outlet of the sprayer. The online top-down MS analysis measures the global deuteration level of the eluted intact protein species, and subsequently, the deuteration of their gas-phase fragments. This paper demonstrates this approach in differential HDX for systems, including the natural protein variants coexisting in milk.

Presented here is a protocol for a capillary electrophoresis-based hydrogen/deuterium exchange (HDX) approach coupled with top-down mass spectrometry. This approach characterizes the difference in higher-order structures between different protein species, including proteins in different states and different proteoforms, by conducting concurrent differential HDX and electrophoretic separation.

基于毛细管电泳的氢/氘交换，用于自上而下的质谱法对蛋白质进行构象表征

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of ...