Name: enrichment of extracellular matrix proteins from tissues and digestion into peptides for mass spectrometry analysis
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The authors would like to thank Drs. Amanda Del Rosario and Guillaume Carmona for critical reading of the manuscript. This work was supported by grants from the Department of Defense&#160;DOD Innovator Award (W81XWH-14-1-0240&#160;to ROH); the National Cancer Institute (U54 CA126515/CA163109); the Broad Institute of MIT and Harvard; the Howard Hughes Medical Institute, of which ROH is an Investigator; and in part by the Support Grant from the National Cancer Institute to the Koch Institute for Integrative Cancer Research at MIT (P30-CA14051). AN received postdoctoral fellowships from the Howard Hughes Medical Institute and the Ludwig Center for Cancer Research at MIT.

注:该过程可以在新鲜或快速冷冻的组织进行。我们建议灌流用PBS高度血管化的组织在夹层的时间内消除红血细胞和血浆蛋白质。我们不建议进行程序上的固定组织的固定（ 即化学交联）干扰脱细胞，也可以显著妥协随后质谱分析。整个过程中，我们推荐使用低滞留管和枪头，以最大限度地提高蛋白质和肽恢复。 1.脱细胞组织或肿瘤的注:在开始，制备试剂和添加蛋白酶抑制剂（设置有箱蛋白提取试剂盒），以每个缓冲器的所需体积。所有的缓冲液和样品应保持在冰上，除了缓冲器的CS的实验的持续时间需要被保持在室温，以防止SDS雨itation。 注:该协议使用在不同的pH值和含有不同量的盐和洗涤剂依次提取胞内蛋白质和富集不溶ECM蛋白（ 图1，表1的缓冲器的一系列培养;另见讨论用于替代使用的这里的商业试剂盒进行详细说明）。试剂的下面给出的容积为组织或肿瘤（ 见表1）的100毫克，需要进行适当调整。 <ol><li>均质化100毫克组织在500μl缓冲液C的含有使用组织匀浆直至组织被完全破坏，可以得到一个均匀的悬浮液蛋白酶抑制剂。 注:加入脱氧核糖核酸酶I（终浓度:200微克/毫升，根据制造商的说明重组）和核糖核酸酶A（终浓度:20微克/毫升，根据制造商的说明重组）到缓冲区N. </li><li>细胞内可溶性蛋白的连续提取<ol><li>提取胞浆蛋白。孵育管旋转20分钟的匀浆在4℃下。保存匀浆用于随后免疫印迹分析（见预期结果部分和图2）的一小等分试样（10微升至20微升）。 </li><li>离心匀浆在16000×g离心20分钟，在4℃。收集上清液在一个干净的管中，这将构成Western印迹分析的胞质（C）的分数。闪光冻结该馏分并储存在-80℃。 </li><li>洗净，悬浮于400μl缓冲液的沉淀W¯¯含有蛋白酶抑制剂孵育的样品上管旋转20分钟，在4℃。离心匀浆在16000×g离心20分钟，在4℃。弃去上清液。 </li><li>要提取，核蛋白，悬浮在150微升缓冲存储器N含有蛋白酶抑制剂的颗粒，deoxyribonuclea本身I和核糖核酸酶A和孵化在管旋转样品30分钟，在4℃。 </li><li>离心样品在16000×g离心30分钟，在4℃，在一个干净的管回收上清。重复此步骤一次:离心样品的第二次后，将上清液添加到先前上清液:这将构成Western印迹分析的核（N）的比例。闪光冻结该馏分并储存在-80℃。然后，执行洗涤按照步骤1.2.3。 </li><li>提取膜蛋白，重悬在含蛋白酶抑制剂的100微升缓冲液M中的沉淀，并培育样品上管旋转30分钟，在4℃。在4℃下离心样品在16000×g离心30分钟</li><li>收集在一个干净的试管上清液:这将构成Western印迹分析的膜（M）的分数。闪光冻结该馏分并储存在-80℃。 </li><li>为了提取细胞骨架蛋白，悬浮颗粒在200微升缓冲液的CS含有蛋白酶抑制剂孵育的样品上管旋转30分钟，在室温。 注意，该粒料不会完全溶解。我们建议扰乱小球通过上下抽吸直至观察粒料的中断。 </li><li>离心样品在16000×g离心30分钟，在RT。收集在一个干净的管中的上清液。注意这一点在颗粒（见视频）的规模进一步显着下降。 </li><li>重悬在150微升缓冲液C含蛋白酶抑制剂的小球，并培育的样品上管旋转20分钟，在4℃。离心样品在16000×g离心20分钟，在4℃。 </li><li>收集上清液，并将其添加到先前上清液:这将构成Western印迹分析细胞骨架（CS）的级分。闪光冻结该馏分并储存在-80℃。 </li><li>执行额外的清洗。悬浮颗粒在500微升PBS含有蛋白酶抑制剂次孵育上管旋转的样品进行5分钟，在4℃。离心样品在16000×g离心5分钟，在4℃。弃去上清液。重复此步骤三次。 注意:需要通过大量的洗涤​​前的蛋白质消化去除成肽（见第3节）的洗涤剂的所有痕迹。在这一点上，对ECM-富集粒料可以是快速冷冻并保存在-80℃。需要注意的是颗粒的大小将取决于不溶性（ECM）蛋白的起始材料的量和脱细胞的效率。 </li></ol></li></ol> 2.监测脱细胞/ ECM富集的质量通过SDS-PAGE和Western Blot <ol><li>总的混合组织提取10-20微升等份和中间馏分Laemmli缓冲液含有100毫米DTT的50微升等分。含重悬100毫米DTT的ECM富集，不溶部分的3倍Laemmli缓冲液。 注:他浓度升高DTT和SDS的协助ECM的蛋白质，是相对不溶性的溶解。 </li><li>通过SDS-PAGE分离蛋白质，并转移到硝酸纤维素膜上。 </li><li>使用抗体来监测蛋白质展示各种胞质，核，膜，细胞骨架和细胞外基质组分的执行免疫印迹（见代表结果部分， 表2和图2）。 </li></ol> 3.在溶液消化蛋白质的肽质谱分析注:SDS的脱细胞程序和去除后得到的沉淀物是不溶性ECM蛋白对于通过质谱进一步分析这些蛋白质需要被消化成肽高度浓缩。需要注意的是，由于细胞外基质蛋白的不溶性的结果，因此不可能在这个步骤测量样品的蛋白浓度。因此，我们提供的试剂量来消化ECM-ENRICHED样品到基于对ECM富集粒料（ 表3）的尺寸（mm）或干重的肽。碳酸氢铵，脲，DTT，碘乙酰胺和三氟乙酸的溶液都需要新鲜配制。 <ol><li>通过加入8M尿素的合适体积到ECM富集沉淀重悬的ECM富集样品，并添加DTT以10mM的终浓度（ 见表3）。连续搅拌孵育1400 rpm离心2小时，在37℃。 注意:在这一点上的ECM蛋白不会完全溶解，可见的大蛋白颗粒不应通过离心或过滤将其丢弃。该悬挂将清除后，去糖基化和消化（见视频）。 </li><li>烷化<ol><li>在准备HPLC级水的碘乙酰胺的解决方案。冷却样品至室温，碘乙酰胺加至25mM的终浓度。有关完整的烷基化，数码地面电视:碘乙酰胺的比例应为1间:2。5和1:3。 </li><li>孵育在黑暗中在室温30分钟。 </li></ol></li><li>去糖基化: 注:去糖基化是需要去除糖侧链与鉴定肽用N修饰的干扰-连接糖基化。 <ol><li>稀释至2M尿素用100mM碳酸氢铵pH为8.0，并添加PNGaseF适量（ 见表3）。连续搅拌孵育1400 rpm离心2小时，在37℃。 </li></ol></li><li>消化<ol><li>添加的Lys-C和连续搅拌，在37℃孵育1400离心2小时。加入胰蛋白酶和连续搅拌孵育在1400转O / N在37℃。 注:ECM丰富的悬架后开始在8M尿素重建初期出现混浊后O / N消化（见视频）明确。 </li><li>添加胰蛋白酶的第二等分试样的样品，孵育连续搅拌在1400转速下额外的2小时，在37℃下&lt;/ LI&gt; </ol></li><li>酸化<ol><li>经消化完成后，通过酸化用新鲜制备的50％三氟乙酸（TFA）的样品灭活胰蛋白酶。样品应达到的pH &lt;2，我们建议增加1-1.5微升的50％TFA在一个时间和使用1μl的肽溶液的测量使用pH试纸（见视频）将溶液的pH值。 </li><li>离心酸化的样品在16000×g离心5分钟，在RT。收集在干净的低保持管上清。此时，该肽溶液可以储存在-20℃。 </li></ol></li><li>脱盐 注意:此最后步骤是在质谱设施根据自己的优选的方法，通常进行。 <ol><li>之前蛋白质组学分析，脱盐洗脱新鲜制备的60％乙腈，0.1％三氟乙酸，然后浓缩，在真空浓缩器将样品和肽。悬浮肽新鲜配制3％acetonitrILE，0.1％三氟乙酸8。 注意:脱盐后，将肽溶液的浓度可通过分光光度法测定（见预期结果部分）。 </li><li>现在分析样品通过质谱法，根据该质谱设施的最佳程序一次。 注:我们鼓励感兴趣的研究人员利用质谱参阅其他出版物8表征的ECM的组成- 10和网站，提供进一步的详细说明，包括LC-MS / MS参数，质谱数据搜索蛋白质鉴定和数据分析用在硅片 matrisome标注工具，我们developped 8。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

修改虽然我们采用这个确切的过程，从十组织和肿瘤类型8充实ECM - 10，协议的修改应在下列情况下可以考虑: 1）检测中间馏分ECM的蛋白质。 从不同的组织或肿瘤类型的细胞外基质可能会有不同的萃取性/不溶性，如以上所讨论为纤连蛋白和层粘连蛋白。例如，它被认为是纤维化组织或重塑的组织的细胞外基质翻转非常动态，因而人们可以观察到这些组织的比例较高的ECM蛋白的更容易萃取的12。这取决于ECM蛋白被检测到的级分，我们建议减少步骤的孵育时间导致ECM蛋白的提取或省略该步骤。 2）Detection的细胞内成分的在ECM富集粒料一个显著比例。在一些组织或肿瘤类型的比例的细胞:细胞外基质是特别高（ 例如，肝，脾，非纤维组织增生性肿瘤）。在这种情况下，通过细胞内蛋白的ECM的富集级分的显著污染（特别是细胞骨架蛋白和/或组蛋白）可以观察到。耗尽细胞内蛋白质有效地，我们建议重复两次在缓冲液M和/或缓冲液的CS（均包含洗涤剂，这通常耗尽丰富细胞内蛋白）温育。另一种替代方法是使用另一种缓冲器，具有较高的洗涤剂浓度，需要提醒的是，这可能导致相对更可溶ECM蛋白的耗尽以及（见下段）。 3）替代使用商业试剂盒。 我们无法，专有的原因，得到的缓冲器的精确组成来回m为房室蛋白提取试剂盒的供应商。不过，我们已在使用表自制的缓冲器，确定洗涤剂（NP-40，脱氧胆酸钠和SDS）的浓度进行类似拔牙根据我们自己的经验1注。最近的一项研究也强调脱细胞缓冲剂的pH保持ECM蛋白13的重要性。 该技术的局限性这里提出的方法依赖于以下事实ECM蛋白本质上比大多数胞内蛋白质更不溶性的。然而，脱细胞的方法在这里所描述肯定提取ECM中存在的可溶性组分，如某些生长因子或细胞外基质重塑酶。虽然ECM相关蛋白紧密结合的ECM蛋白通过在制备的样品的蛋白质组学所述检测，这种方法可能过于严格，充分轮廓的组合物matrisome相关蛋白质。 该技术相对于其它方法的意义这里介绍过的其它方法的方法的优点是，它可以根据所关注的ECM的性质:可以省略中间步骤或重复以防止ECM蛋白的损失或增加分别污染细胞内蛋白质的耗尽。这种方法也只使用被冲洗掉，以防止它们与随后的肽制剂和质谱干扰清净剂的最小量。最后，这里描述的消化ECM的富含蛋白质制剂成肽的方法还具有不需要的蛋白是可溶的，并且可以在"粗"的ECM富氧馏份进行的优点。 利用离液剂（如盐酸胍）替代脱细胞的方法 研究的ECM通过质谱法的组合物已bEEN文献报道（在14中综述），并已用于结合质谱软骨15,16，心脏17，乳腺18和血管19和肾小球20基底膜的ECM组合物的特征。 建议采用胰蛋白酶消化进出细胞脱细胞的方法不应该被使用，如果ECM被富集用于随后蛋白质组学分析，如胰蛋白酶消化将导致局部细胞外基质的消化和ECM蛋白质和肽的损失。同样，如果胶原酶消化要用于帮助组织破坏，这将需要仔细监测，因为它会导致ECM的消化和ECM蛋白质和肽的损失。 在溶液主场迎战凝胶消化？ ECM蛋白是交联的和高度不溶，并且即使当在3×Laemmli缓冲液再悬浮（含有6％SDS）和100mM的DTT，独立婆奥利在SDS凝胶上。因此，在凝胶内消化的是不是一个优选的方法。 未来的应用值得注意的是，虽然没有在这里讨论的，每个所述脱细胞过程中收集的中间馏分的组成也可以通过质谱法进行分析。研究非常小的样品（ 即，人活检）或时，这可能是特别有价值的信息时所需的其他细胞组分。

细胞外基质（ECM）是交联的和糖基化的蛋白质，提供了体系结构支持和锚固细胞和定义，部分，组织"生物力学特性1,2的复合小梁。 ECM蛋白还发信号给细胞直接通过它们的受体（ 例如，整联，syndecans 等 ）或通过调节生长因子信号3。将ECM因此提供生物物理和生物化学线索是细胞过程如增殖，存活，分化，分 ​​化，迁移等的主要调节该ECM起着生理，发育和衰老4关键作用。此外，一些病症，如心血管疾病，纤维化，肌肉骨骼疾病，癌症，是由引起的，或导致，ECM的改变。此外，ECM有助于干细胞小生境的维护和确定关键ECM分子次在支持干性将在组织工程和再生医学5直接应用。然而，尽管它的重要性，ECM一直保持，直到最近，未勘探6。 在硅片分析表明，该matrisome，定义为ECM和ECM相关蛋白的合奏，包括几百种基因同时在人类和小鼠基因组1,7,8的产品。然而，ECM蛋白的不溶性阻碍体内外的正常和病理标本基质的组合物的系统的特性。我们最近表明，这种不溶性可以变成优势，并用来丰富ECM蛋白8 - 10。我们和其他人进一步证实，质谱是选择的方法的ECM 8的组合物的特征- 10，13。 我们这里描述了由连续孵育在不同pH值和盐和洗涤剂浓度的一个缓冲的过程脱细胞。这个过程的结果在细胞质，核，膜和细胞骨架蛋白的提取（或消耗）和ECM蛋白的富集。然后，我们将介绍如何消化ECM-富含蛋白制剂成肽进行后续分析通过质谱分析。 使用的程序的详细和在这里示出，我们已经成功地富集和特征通过质谱法从十个不同组织和肿瘤类型的细胞外基质:正常鼠肺8，人和鼠结肠8,9，人肝脏9，人结肠直肠肿瘤和衍生肝转移9，黑素瘤异种移植物8，乳腺肿瘤异种移植物10，鼠胰岛和胰岛素瘤鼠（纳巴等人 ，未发表）。不同matrisom的比较西文透露，可以进一步用作潜在的诊断或预后标志物组织 - 和肿瘤特异性的ECM签名。 我们认为，此过程可以应用到其他标本没有或相对较小的修改。

<table><tbody><tr><td>Tissue homogenizer: Bullet blender + beads</td><td>Next Advance</td><td>&lt;a href=&quot;http://www.nextadvance.com/api/index.cfm/products.info/c/421/Bullet-Blender&quot;&gt;BB24-AU&lt;/a&gt;</td><td></td></tr><tr><td>Compartmental Extraction kit</td><td>Millipore</td><td>&lt;a href=&quot;http://www.emdmillipore.com/US/en/product/Compartment-Protein-Extraction-Kit,MM_NF-2145&quot;&gt;2145&lt;/a&gt;</td><td></td></tr><tr><td>Deoxyribonuclease I</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/dn25?lang=en&amp;amp;region=US&quot;&gt;DN25&lt;/a&gt;</td><td></td></tr><tr><td>Ribonuclease A</td><td>Qiagen</td><td>&lt;a href=&quot;http://www.qiagen.com/products/catalog/sample-technologies/dna-sample-technologies/plasmid-dna/rnase-a/&quot;&gt;19101&lt;/a&gt;</td><td></td></tr><tr><td>HPLC-grade water</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sial/34877?lang=en&amp;amp;region=US&quot;&gt;34877&lt;/a&gt;</td><td></td></tr><tr><td>Urea</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/u4883?lang=en&amp;amp;region=US&quot;&gt;U4883&lt;/a&gt;</td><td></td></tr><tr><td>Dithiotreitol (DTT)</td><td>Thermo Scientific</td><td>&lt;a href=&quot;http://www.piercenet.com/product/dithiothreitol-dtt&quot;&gt;20291&lt;/a&gt;</td><td></td></tr><tr><td>Ammonium bicarbonate (NH&lt;sub&gt;4&lt;/sub&gt;HCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/fluka/09830?lang=en&amp;amp;region=US&quot;&gt;9830&lt;/a&gt;</td><td></td></tr><tr><td>Iodoacetamide</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/a3221?lang=en&amp;amp;region=US&quot;&gt;A3221&lt;/a&gt;</td><td></td></tr><tr><td>Peptide-&lt;em&gt;N&lt;/em&gt;-Glycosidase F (PNGaseF)</td><td>New England Biolabs</td><td>&lt;a href=&quot;https://www.neb.com/products/p0704-pngase-f&quot;&gt;P0704S&lt;/a&gt;</td><td></td></tr><tr><td>Endoproteinase LysC, mass spectrometry-grade&amp;nbsp;</td><td>Wako</td><td>&lt;a href=&quot;http://www.wako-chem.co.jp/english/labchem/product/life/Lys-C/index.htm&quot;&gt;125-05061&lt;/a&gt;</td><td></td></tr><tr><td>Trypsin, mass spectrometry-grade&amp;nbsp;</td><td>Promega</td><td>&lt;a href=&quot;https://www.promega.com/products/mass-spectrometry/proteases-and-surfactants/sequencing-grade-modified-trypsin/&quot;&gt;V5111&lt;/a&gt;</td><td></td></tr><tr><td>Trifluoro-acetic Acid</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sial/t6508?lang=en&amp;amp;region=US&quot;&gt;T6508&lt;/a&gt;</td><td></td></tr><tr><td>Acetonitrile, mass spectrometry-grade&amp;nbsp;</td><td>Thermo Scientific</td><td>&lt;a href=&quot;http://www.piercenet.com/product/acetonitrile-acn-lc-ms-grade&quot;&gt;51101&lt;/a&gt;</td><td></td></tr><tr><td>Desalting columns: Oasis HLB 1&amp;nbsp;cc, 10 mg Sorbent per Cartridge</td><td>Waters</td><td>&lt;a href=&quot;http://www.waters.com/waters/partDetail.htm?partNumber=186000383&quot;&gt;186000383&lt;/a&gt;</td><td></td></tr></tbody></table>

enrichment of extracellular matrix proteins from tissues and digestion into peptides for mass spectrometry analysis

细胞外基质（ECM）是交联的蛋白质的复合小梁，提供生物物理和生物化学线索是细胞增殖，存活，迁移等主要调节ECM的在发展和多样化的病理学的重要作用，包括心血管和肌肉骨骼疾病，纤维化和癌症。因此，表征的正常和患病组织的ECM的组合物可能导致新的预后和诊断标志物和潜在的新的治疗目标的识别。然而，ECM蛋白的性质（尺寸大，交联和共价结合，高度糖基化）已经呈现的ECM有挑战性的生化分析。为了克服这个难题，我们开发了一种方法，从新鲜或冷冻的组织和肿瘤发生ECM蛋白的不溶性的优点丰富的ECM。我们在这里描述的细节，包括顺序I脱细胞过程在不同的pH值和盐和洗涤剂浓度的缓冲剂ncubations和的结果在1）的细胞内（细胞质，核，膜和细胞骨架）蛋白和ECM蛋白质的2）的富集的提取。然后，我们描述了如何和去糖基化ECM消化富含蛋白制剂成肽进行后续分析通过质谱分析。

脱细胞过程的质量控制脱细胞的效率可以通过免疫印迹分析各级分的蛋白质含量来监测。 表的诊断价值2列出的蛋白质，以评估脱细胞过程的质量。 图2A示出在胞质的中间馏分高效提取（GAPDH ），核（组蛋白），膜（β1整合）和细胞骨架（肌动蛋白）的蛋白质，而没有ECM蛋白（胶原蛋白I）中这些级分被检测（ 图2）。反过来，最终沉淀富集了ECM蛋白，基本上耗尽细胞内蛋白（ 图2A）。图2B呈现满意的细胞内蛋白耗竭（无组蛋白是在ECM富级分中检测），虽然，肌动蛋白仍然可以在被检测的在ECM-丰富的分数和单体Ⅰ型胶原的枯竭- 表观分子量〜110 kDa的，推定相应于未组装Ⅰ型胶原蛋白 - 可以在CS馏分被观察到。 我们还定期监测额外ECM蛋白如纤连蛋白和层粘连蛋白，虽然，在一些组织中，这些组件可部分溶解在较早的级分8-10。例如，纤连蛋白，也会发生可溶性血浆纤连蛋白未结合到ECM中。萃取前的组织的灌注降低血浆纤维结合蛋白的浓度，但并不总是消除它。在一些组织中，层粘连蛋白被发现松散与细胞表面或在中间馏分的ECM和提取相关联。如果发生这种情况，可以通过改变提取条件（见讨论）来解决。 需要注意的是ECM蛋白和胞内组分的伴随耗尽的富集是基于蛋白在不同的缓冲器中的相对溶解度。钍是不同组织中的不同 - 在某些情况下，组蛋白和肌动蛋白更容易提取比其他。还要注意的是，虽然组蛋白预期在N级分（ 图2B）被提取，我们经常看到在M或CS分数（ 图2A和B）组蛋白的更完全耗尽。 表示肽浓度预期消化后，酸化和脱盐获得的肽溶液的浓度可通过分光光度法或者通过测量所述肽溶液的吸光度使用280纳米波长对应于色氨酸，酪氨酸，或使用对应于该肽的吸收的205纳米的波长进行测量债券。 我们测量从三个鼠肺样本脱细胞获得的肽溶液的浓度（82毫克，100毫克和100毫克，分别）编写，J红色的并行，并且从每个获得:424纳克/微升，450纳克/微升，和肽分别580纳克/微升。 ECM肽的通过质谱法鉴定 ECM富集蛋白质样品，如下所述制备的组合物的质谱分析，结果表明，信号强度&gt; 70％对应于ECM和ECM相关蛋白8-10。 表1体积从房室提取试剂盒试剂decellularize组织或肿瘤的100毫克（湿重）的。此表列出的成分和用于进行100毫克组织或肿瘤的脱细胞的每个缓冲器的体积。蛋白酶抑制剂的鸡尾酒被提供为50倍溶液中，需要被添加到每个缓冲器2。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 从车厢蛋白提取试剂盒 </td><td> 对于组织100毫克体积 </td><td> 成分（基于微孔数据表猫＃2145 1） </td></tr><tr><td>缓冲液C </td><td> 500微升</td><td> HEPES（pH值7.9 3）， 氯化镁 ，氯化钾，美国东部时间4，蔗糖，甘油，钒酸钠5 </td></tr><tr><td>缓冲区W¯¯ </td><td> 400微升</td><td> HEPES（pH值7.9）， 氯化镁 ，氯化钾，EDTA，蔗糖，甘油，钒酸钠</td></tr><tr><td>缓冲区n </td><td> 150微升×2 </td><td> HEPES（pH值7.9）， 氯化镁 ，氯化钠，EDTA，甘油，钒酸钠</td></tr><tr><td>缓冲区W¯¯ </td><td> 400微升</td><td> HEPES（pH值7.9）， 氯化镁 ，氯化钾，EDTA，蔗糖，甘油，钒酸钠</td></tr><tr><td>缓冲区m </td><td> 100微升</td><td> HEPES（pH值7.9）， 氯化镁 ，氯化钾，EDTA，蔗糖，甘油，脱氧胆酸钠（DOC）6，NP-40 6 </sup&gt;，原钒酸钠</td></tr><tr><td>缓冲区CS </td><td> 200微升</td><td> PIPES（pH6.8）中， 氯化镁 ，氯化钠，EDTA，蔗糖，十二烷基硫酸钠（SDS），7，钒酸钠</td></tr><tr><td>缓冲液C </td><td> 150微升</td><td> HEPES（pH值7.9）， 氯化镁 ，氯化钾，EDTA，蔗糖，甘油，钒酸钠</td></tr><tr><td> 1X PBS </td><td> 500微升/洗</td><td> - </td></tr></tbody></table> 注意事项 : 1对于专有的原因，我们无法获得该套件的供应商的缓冲区的确切成分，但我们在这里包括使用自制的缓冲区进行类似拔牙根据我们自己的经验的一些注意事项。 2蛋白酶抑制剂:最好是包括各种抗半胱氨酸，丝氨酸和苏氨酸的肽酶，丝氨酸酯酶，二价阳离子依赖型金属蛋白酶等酶抑制剂的许多市售的蛋白酶抑制剂的鸡尾酒存在。 3 pH高于7.0是溶酶体蛋白酶的有效抑制剂。 4 EDTA（通常用于为2mM）是二价阳离子依赖型蛋白酶的有效抑制剂。 5原钒酸钠是磷酸酶抑制剂。一个典型的有效浓度是0.5-5毫米。 6 NP40在0.1％-0.5％的足以溶解大多数膜脂质。 NP40和DOC的组合-通常使用在相同浓度（ 例如 ，0.5％的每一个） -通常被用作一个更严格的提取仍然留下了许多蛋白质-蛋白质相互作用的完整。 7 SDS是更严格的离子型去污剂（CMC 0.1％）。它也可以结合使用其它两种洗涤剂SDS / NP40 / DOC 0.1 / 0.5 / 0.5％作为中间严紧缓冲。 表2.诊断蛋白质监测脱细胞过程的质量。该表列出的蛋白质，是每个亚细胞区室（细胞溶质，细胞核，质膜，细胞骨架和细胞外基质），可用于监测脱细胞过程的质量和的效率的特性的例子ECM-富集。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 内室 </td><td> 诊断性蛋白质 </td></tr><tr><td>胞质蛋白</td><td>甘油醛-3-磷酸脱氢酶（GAPDH） </td></tr><tr><td>核蛋白</td><td>组蛋白，核纤层蛋白，核孔蛋白</td></tr><tr><td>膜蛋白</td><td>整合，转铁蛋白受体</td></tr><tr><td>细胞骨架蛋白</td><td>肌动蛋白，微管蛋白，波形蛋白</td></tr><tr><td>基底膜细胞外基质蛋白</td><td> IV型胶原蛋白，Nidogens，层粘连蛋白</td></tr><tr><td>间质细胞外基质蛋白（质） </td><td>胶原蛋白I，III型胶原，胶原蛋白VI，纤连蛋白</td></tr></tbody></table>有关抗体的建议，请参阅我们的出版物8-10。 表3.音量试剂消化ECM-丰富的样本为肽。该表列出了用于悬浮ECM-的蛋白样品，减少，烷基化，和去糖基化蛋白质消化成样品前质谱分析多肽试剂。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 试剂 </td><td> 准备 </td><td> 终浓度/量为1mm厚粒料（〜5-10毫克干重） </td><td> 1毫米厚的小球体积（〜5-10毫克干重） </td></tr><tr><td>碳酸氢铵（NH 4 HCO 3） </td><td>在HPLC级水中的100mM溶液</td><td> - </td><td> - </td></tr><tr><td>尿素</td><td>在100mM碳酸氢铵的8M溶液</td><td> 8M的</td><td> 50微升</td></tr><tr><td>二硫</td><td>在500mM的重建在HPLC级水</td><td> 10毫</td><td> 1微升</td></tr><tr><td>碘乙酰胺</td><td>在500mM的重建在HPLC级水</td><td> 25毫米</td><td> 2.5微升</td></tr><tr><td>肽ñ-Glycosidase F（PNGaseF） </td><td>在500 U /μL商业解决方案</td><td> 1000ü </td><td> 2微升</td></tr><tr><td>内切酶的lysC，质谱级</td><td>在0.5微克/微升重组在HPLC级水</td><td> 1微克</td><td> 2微升</td></tr><tr><td>胰蛋白酶，质谱级（ 第1轮 ） </td><td>在0.5微克/微升的商业解决方案</td><td> 3微克</td><td> 6微升</td></tr><tr><td>胰蛋白酶，质谱级（ 第2轮 ） </td><td>在0.5微克/微升的商业解决方案</td><td> 1.5微克</td><td> 3微升</td></tr><tr><td>三氟乙酸（TFA） </td><td>在HPLC级水中的50％溶液</td><td> - </td><td> 2-5微升</td></tr><tr><td>乙腈（ 洗脱 ） </td><td>用0.1％TFA的60％的溶液在HPLC级水</td><td> - </td><td> 500微升</td></tr><tr><td>乙腈（ 重建 ） </td><td>用0.1％TFA的3％溶液在HPLC级水</td><td> - </td><td> 100微升</td></tr></tbody></table><img alt="图1" src="/files/ftp_upload/53057/53057fig1highres.jpg" width="600" /> 图1方案的实验程序的。该protoco示意图工作流l至decellularize组织（第1），控制所述脱细胞的质量，并评估对ECM富集（第2部分）和消化ECM的富集蛋白质样品进入之前质谱分析（第3节）的肽。 <img alt="图2" src="/files/ftp_upload/53057/53057fig2highres.jpg" width="500" /> 图2.质量控制通过Western印迹的脱细胞过程的蛋白质印迹对小鼠肺（A）和人乳腺癌异种移植物（B）中使用以下抗体样品上进行:兔抗肌动蛋白（克隆14-1）和兔抗-β1整合在我们的实验室产生的抗体;兔抗胶原蛋白I，小鼠抗GAPDH和兔抗泛组蛋白抗体来自Millipore的。下列初级抗体温育后，洗涤膜并孵育在HRP缀合的山羊抗兔或山羊抗谋的存在从杰克逊免疫研究实验室本身的二级抗体。最后，将膜洗涤和温育在西方闪电™化学发光试剂（珀金埃尔默LAS）。 *表示对ECM富馏分的最小残留组蛋白污染。 **表示的单体（推定未组装）Ⅰ型胶原蛋白的在CS分数部分耗尽。 ***表示ECM丰富的部分残留肌动蛋白污染。用抗胶原抗体检测的涂片表示不同程度的翻译后修饰（ 例如，交联，糖基化）。

Watch this Scientific Journal Video about Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis at JoVE.com

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

从富集组织及消化细胞外基质蛋白的成肽质谱分析

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

enrichment-extracellular-matrix-proteins-from-tissues-digestion-into

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

26.9K 次观看.  Massachusetts Institute of Technology. This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry. 

视频: Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

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

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass determination allows one to evaluate the correctness of protein primary sequences, the presence of mutations and/or post-translational modifications, the possible protein degradation, the sample homogeneity, and the degree of isotope incorporation in case of labelling (e.g. 13C labelling).
Electrospray ionization (ESI) mass spectrometry (MS) is widely used for mass determination of denatured proteins, but its efficiency is affected by the composition of the sample buffer. In particular, the presence of salts, detergents, and contaminants severely undermines the effectiveness of protein analysis by ESI-MS. Matrix-assisted laser desorption/ionization (MALDI) MS is an attractive alternative, due to its salt tolerance and the simplicity of data acquisition and interpretation. Moreover, the mass determination of large heterogeneous proteins (bigger than 100 kDa) is easier by MALDI-MS due to the absence of overlapping high charge state distributions which are present in ESI spectra.
Here we present an accessible approach for analyzing proteins larger than 100 kDa by MALDI-time of flight (TOF). We illustrate the advantages of using a mixture of two matrices (i.e. 2,5-dihydroxybenzoic acid and &#945;-cyano-4-hydroxycinnamic acid) and the utility of the thin layer method as approach for sample deposition. We also discuss the critical role of the matrix and solvent purity, of the standards used for calibration, of the laser energy, and of the acquisition time. Overall, we provide information necessary to a novice for analyzing intact proteins larger than 100 kDa by MALDI-MS.

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

Accurate mass measurement represents an important step during the investigation of proteins. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) can be used for such determination and its key advantage is the tolerance to salts, detergents and contaminants. Here we illustrate an accessible approach for the analysis of proteins larger than 100 kDa by MALDI-MS.

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 ...

科研

化学

Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). Using proteomics, we have previously identified more than 150 ECM and ECM-associated proteins in cardiovascular tissues. Notably, many ECM proteins are glycosylated. This post-translational modification affects protein folding, solubility, binding, and degradation. We have developed a sequential extraction and enrichment method for ECM proteins that is compatible with the subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of intact glycopeptides. The strategy is based on sequential incubations with NaCl, SDS for tissue decellularization, and guanidine hydrochloride for the solubilization of ECM proteins. Recent advances in LC-MS/MS include fragmentation methods, such as combinations of higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD), which allow for the direct compositional analysis of glycopeptides of ECM proteins. In the present paper, we describe a method to prepare the ECM from tissue samples. The method not only allows for protein profiling but also the assessment and characterization of glycosylation by MS analysis.

This paper describes a methodology to prepare cardiovascular tissue samples for MS analysis that allows for (1) the analysis of ECM protein composition, (2) the identification of glycosylation sites, and (3) the compositional characterization of glycan forms. This methodology can be applied, with minor modifications, to the study of the ECM in other tissues.

细胞外基质的Glycoproteomics:使用质谱联用完整糖肽分析的方法

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). ...

生物化学

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

生物标志物发现的介孔二氧化硅薄膜的低分子量蛋白质富集

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

生物工程

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

使用膜内消化技术评估蛋白质与蛋白质相互作用

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, including RNA processing, signal transduction, DNA damage response, miRNA biogenesis, and translation.
In recent years, thanks to biochemical and analytical developments, mass spectrometry (MS)-based proteomics has emerged as the most effective strategy to characterize the cellular methyl-proteome with single-site resolution. However, identifying and profiling in vivo protein R-methylation by MS remains challenging and error-prone, mainly due to the substoichiometric nature of this modification and the presence of various amino acid substitutions and chemical methyl-esterification of acidic residues that are isobaric to methylation. Thus, enrichment methods to enhance the identification of R-methyl-peptides and orthogonal validation strategies to reduce False Discovery Rates (FDR) in methyl-proteomics studies are required.
Here, a protocol specifically designed for high-confidence R-methyl-peptides identification and quantitation from cellular samples is described, which couples metabolic labeling of cells with heavy isotope-encoded Methionine (hmSILAC) and dual protease in-solution digestion of whole cell extract, followed by off-line High-pH Reversed Phase (HpH-RP) chromatography fractionation and affinity enrichment of R-methyl-peptides using anti-pan-R-methyl antibodies. Upon high-resolution MS analysis, raw data are first processed with the MaxQuant software package and the results are then analyzed by hmSEEKER, a software designed for the in-depth search of MS peak pairs corresponding to light and heavy methyl-peptide within the MaxQuant output files.

Protein Arginine (R)-methylation is a wide-spread post-translational modification regulating multiple biological pathways. Mass spectrometry is the best technology to globally profile the R-methyl-proteome, when coupled to biochemical approaches for modified peptide enrichment. The workflow designed for the high confidence identification of global R-methylation in human cells is described here.

基于质谱的蛋白质组学方法用于全局和高置信度的蛋白质R-甲基化分析

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, ...

A Spin-Tip Enrichment Strategy for Simultaneous Analysis of N-Glycopeptides and Phosphopeptides from Human Pancreatic Tissues

Mass spectrometry can provide deep coverage of post-translational modifications (PTMs), although enrichment of these modifications from complex biological matrices is often necessary due to their low stoichiometry in comparison to non-modified analytes. Most enrichment workflows of PTMs on peptides in bottom-up proteomics workflows, where proteins are enzymatically digested before the resulting peptides are analyzed, only enrich one type of modification. It is the entire complement of PTMs, however, that leads to biological functions, and enrichment of a single type of PTM may miss such crosstalk of PTMs. PTM crosstalk has been observed between protein glycosylation and phosphorylation, the two most common PTMs in human proteins and also the two most studied PTMs using mass spectrometry workflows. Using the simultaneous enrichment strategy described herein, both PTMs are enriched from post-mortem human pancreatic tissue, a complex biological matrix. Dual-functional Ti(IV)-immobilized metal affinity chromatography is used to separate various forms of glycosylation and phosphorylation simultaneously in multiple fractions in a convenient spin tip-based method, allowing downstream analyses of potential PTM crosstalk interactions. This enrichment workflow for glyco- and phosphopeptides can be applied to various sample types to achieve deep profiling of multiple PTMs and identify potential target molecules for future studies.

Post-translational modifications (PTMs) change protein structures and functions. Methods for the simultaneous enrichment of multiple PTM types can maximize coverage in analyses. We present a protocol using dual-functional Ti(IV)-immobilized metal affinity chromatography followed by mass spectrometry for the simultaneous enrichment and analysis of protein N-glycosylation and phosphorylation in pancreatic tissues.

一种同时分析人胰腺组织中N-糖肽和磷酸肽的自旋尖端富集策略

Mass spectrometry can provide deep coverage of post-translational modifications (PTMs), although enrichment of these modifications from complex biological ...

单克隆抗体药品中宿主细胞蛋白的富集

Host Cell Protein Analysis using Enrichment Beads Coupled with Limited Digestion

Host cell proteins (HCPs) are impurities that can adversely affect therapeutic proteins, even in small quantities. To evaluate the potential risks associated with drug products, methods have been developed to identify low-abundance HCPs. A crucial approach for developing a sensitive HCP detection method involves enriching HCPs while simultaneously removing monoclonal antibodies (mAbs) before analysis, utilizing liquid chromatography-mass spectrometry (LC-MS).
This protocol offers detailed instructions for enriching host cell proteins using commercially available proteome enrichment beads. These beads contain a diverse library of hexapeptide ligands with specific affinities for different proteins. The protocol also incorporates limited digestion and subsequent peptide detection using nano LC-MS/MS. By employing these techniques, HCPs with low abundance can be enriched over 7000-fold, resulting in an impressive detection limit as low as 0.002 ppm. Significantly, this protocol enables the detection of 850 HCPs with a high level of confidence using a NIST mAb. Moreover, it is designed to be user-friendly and includes a video demonstration to assist with its implementation. By following these steps, researchers can effectively enrich and detect HCPs, enhancing the sensitivity and accuracy of risk assessment for drug products.

作者聚焦：使用富集微珠和有限消化检测药品中低丰度的宿主细胞蛋白 

A protocol is presented for enriching host cell proteins (HCPs) from drug products (DP) and detecting peptides using proteome enrichment beads. The method is demonstrated using an in-house manufactured monoclonal antibody (mAb) drug substance (DS), which is a well-characterized reference material for evaluating and comparing different methods in terms of performance.

使用富集微球与有限消化相结合的宿主细胞蛋白分析

Host cell proteins (HCPs) are impurities that can adversely affect therapeutic proteins, even in small quantities. To evaluate the potential risks ...

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

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their significance to both cell physiology and immunology, much remains to be discovered about novel lipoprotein forms, how they are synthesized, and the effect of the various forms on host immunity. To enable thorough studies on lipoproteins, this protocol describes a method for bacterial lipoprotein enrichment and preparation of N-terminal tryptic lipopeptides for structural determination by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Expanding on an established Triton X-114 phase partitioning method for lipoprotein extraction and enrichment from the bacterial cell membrane, the protocol includes additional steps to remove non-lipoprotein contaminants, increasing lipoprotein yield and purity. Since lipoproteins are commonly used in Toll-like receptor (TLR) assays, it is critical to first characterize the N-terminal structure by MALDI-TOF MS. Herein, a method is presented to isolate concentrated hydrophobic peptides enriched in N-terminal lipopeptides suitable for direct analysis by MALDI-TOF MS/MS. Lipoproteins that have been separated by Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) are transferred to a nitrocellulose membrane, digested in situ with trypsin, sequentially washed to remove polar tryptic peptides, and finally eluted with chloroform-methanol. When coupled with MS of the more polar trypsinized peptides from wash solutions, this method provides the ability to both identify the lipoprotein and characterize its N-terminus in a single experiment. Intentional sodium adduct formation can also be employed as a tool to promote more structurally informative fragmentation spectra. Ultimately, enrichment of lipoproteins and determination of their N-terminal structures will permit more extensive studies on this ubiquitous class of bacterial proteins.

The enrichment of bacterial lipoproteins using a non-ionic surfactant phase partitioning method is described for direct use in TLR assays or other applications. Further steps are detailed to prepare N-terminal tryptic lipopeptides for structural characterization by mass spectrometry.

质谱法富集细菌脂蛋白及 n-端脂肽的制备结构测定

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

从富集组织及消化细胞外基质蛋白的成肽质谱分析

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此视频中的章节

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

细胞外基质的Glycoproteomics:使用质谱联用完整糖肽分析的方法

生物标志物发现的介孔二氧化硅薄膜的低分子量蛋白质富集

使用膜内消化技术评估蛋白质与蛋白质相互作用

基于质谱的蛋白质组学方法用于全局和高置信度的蛋白质R-甲基化分析

一种同时分析人胰腺组织中N-糖肽和磷酸肽的自旋尖端富集策略

使用富集微球与有限消化相结合的宿主细胞蛋白分析

使用富集微球与有限消化相结合的宿主细胞蛋白分析

使用富集微球与有限消化相结合的宿主细胞蛋白分析

质谱法富集细菌脂蛋白及 n-端脂肽的制备结构测定