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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td colspan="4">Section 1</td>
		</tr>
		<tr>
			<td>Tissue homogenizer: Bullet blender + beads</td>
			<td>Next Advance</td>
			<td>BB24-AU</td>
			<td>http://www.nextadvance.com/api/index.cfm/products.info/c/421/Bullet-Blender</td>
		</tr>
		<tr>
			<td>Compartmental Extraction kit</td>
			<td>Millipore</td>
			<td>2145</td>
			<td>http://www.emdmillipore.com/US/en/product/Compartment-Protein-Extraction-Kit,MM_NF-2145</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I</td>
			<td>Sigma-Aldrich</td>
			<td>DN25</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/dn25?lang=en&#38;region=US</td>
		</tr>
		<tr>
			<td>Ribonuclease A</td>
			<td>Qiagen</td>
			<td>19101</td>
			<td>http://www.qiagen.com/products/catalog/sample-technologies/dna-sample-technologies/plasmid-dna/rnase-a/</td>
		</tr>
	</tbody>
</table>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td colspan="4">Section 3</td>
		</tr>
		<tr>
			<td>HPLC-grade water</td>
			<td>Sigma-Aldrich</td>
			<td>34877</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sial/34877?lang=en&#38;region=US</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma-Aldrich</td>
			<td>U4883</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/u4883?lang=en&#38;region=US</td>
		</tr>
		<tr>
			<td>Dithiotreitol (DTT)</td>
			<td>Thermo Scientific</td>
			<td>20291</td>
			<td>http://www.piercenet.com/product/dithiothreitol-dtt</td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate (NH4HCO3)</td>
			<td>Sigma-Aldrich</td>
			<td>9830</td>
			<td>http://www.sigmaaldrich.com/catalog/product/fluka/09830?lang=en&#38;region=US</td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>Sigma-Aldrich</td>
			<td>A3221</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/a3221?lang=en&#38;region=US</td>
		</tr>
		<tr>
			<td>Peptide -N-Glycosidase F (PNGaseF)</td>
			<td>New England Biolabs</td>
			<td>P0704S</td>
			<td>https://www.neb.com/products/p0704-pngase-f</td>
		</tr>
		<tr>
			<td>Endoproteinase LysC, mass spectrometry-grade&#160;</td>
			<td>Wako</td>
			<td>125-05061</td>
			<td>http://www.wako-chem.co.jp/english/labchem/product/life/Lys-C/index.htm</td>
		</tr>
		<tr>
			<td>Trypsin, mass spectrometry-grade&#160;</td>
			<td>Promega</td>
			<td>V5111</td>
			<td>https://www.promega.com/products/mass-spectrometry/proteases-and-surfactants/sequencing-grade-modified-trypsin/</td>
		</tr>
		<tr>
			<td>Trifluoro-acetic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>T6508</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sial/t6508?lang=en&#38;region=US</td>
		</tr>
		<tr>
			<td>Acetonitrile, mass spectrometry-grade&#160;</td>
			<td>Thermo Scientific</td>
			<td>51101</td>
			<td>http://www.piercenet.com/product/acetonitrile-acn-lc-ms-grade</td>
		</tr>
		<tr>
			<td>Desalting columns: Oasis HLB 1 cc, 10 mg Sorbent per Cartridge</td>
			<td>Waters</td>
			<td>186000383</td>
			<td>http://www.waters.com/waters/partDetail.htm?partNumber=186000383</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

Research

JoVE Journal

Biology

26.6K 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

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI样品制备：超薄层的方法

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

科研

生物学

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N2) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (&Omega;). 
Recently, IM spectrometry was coupled with mass spectrometry and a traveling-wave (T-wave) Synapt ion mobility mass spectrometer (IM-MS) was released. Integrating mass spectrometry with ion mobility enables an extra dimension of sample separation and definition, yielding a three-dimensional spectrum (mass to charge, intensity, and drift time). This separation technique allows the spectral overlap to decrease, and enables resolution of heterogeneous complexes with very similar mass, or mass-to-charge ratios, but different drift times. Moreover, the drift time measurements provide an important layer of structural information, as &Omega; is related to the overall shape and topology of the ion. The correlation between the measured drift time values and &Omega; is calculated using a calibration curve generated from calibrant proteins with defined cross-sections1.
The power of the IM-MS approach lies in its ability to define the subunit packing and overall shape of protein assemblies at micromolar concentrations, and near-physiological conditions1. Several recent IM studies of both individual proteins2,3 and non-covalent protein complexes4-9, successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available)10. We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical &Omega; values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field.

Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.

T波离子迁移质谱蛋白复合物分析的基本实验程序

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. ...

Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like experimental conditions. Laser ablation electrospray ionization (LAESI) is one such development and is particularly well-suited for the investigation of water-containing specimens. LAESI utilizes a mid-infrared laser beam (2.94 &mu;m wavelength) to excite the water molecules of the sample. When the ablation fluence threshold is exceeded, the sample material is expelled in the form of particulate matter and these projectiles travel to tens of millimeters above the sample surface. In LAESI, this ablation plume is intercepted by highly charged droplets to capture a fraction of the ejected sample material and convert its chemical constituents into gas-phase ions. A mass spectrometer equipped with an atmospheric-pressure ion source interface is employed to analyze and record the composition of the released ions originating from the probed area (pixel) of the sample. A systematic interrogation over an array of pixels opens a way for molecular imaging in the microprobe analysis mode. A unique aspect of LAESI mass spectrometric imaging is depth profiling that, in combination with lateral imaging, enables three-dimensional (3D) molecular imaging. With current lateral and depth resolutions of ~100 &mu;m and ~40 &mu;m, respectively, LAESI mass spectrometric imaging helps to explore the molecular structure of biological tissues. Herein, we review the major elements of a LAESI system and provide guidelines for a successful imaging experiment.

Laser ablation electrospray ionization (LAESI) is an atmospheric-pressure ion source for mass spectrometry. In the imaging mode, a mid-infrared laser probes the distributions of molecules across a tissue section or a biofilm. This technique presents a new approach for diverse bioanalytical studies carried out under native experimental conditions.

常压LAESI质谱生物组织和生物膜的分子成像

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like ...

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

在大气压力质谱直接分析单细胞

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

加上质谱肽免疫亲和富集的蛋白质定量

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

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

利用同位素标记的质谱剖析巯基氧化还原蛋白质

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

完整的工作流程为组蛋白翻译后修饰的分析采用自下而上质谱法：从组蛋白提取数据分析

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

一种快速，为隔离，功能可扩展的研究方法，以及细胞衍生的细胞外基质的分析

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

完整、全小鼠乳腺的分离及细胞外基质表达及腺体形态学分析

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly expanded our understanding of certain diseases. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to unravel alterations in the molecular mechanisms and protein signaling pathways in the vascular beds in health and disease. However, sample preparation steps prior to MS analyses are crucial to obtain reproducible results and in-depth elucidation of the complex proteome. This is particularly important for preparation of ocular microvessels, where the amount of sample available for analyses is often limited and thus, poses a challenge for optimum protein extraction. This article endeavors to provide an efficient, rapid and robust protocol for sample preparation from an exemplary retrobulbar ocular vascular bed employing the porcine short posterior ciliary arteries. The present method focuses on protein extraction procedures from both the supernatant and pellet of the sample following homogenization, sample cleaning with centrifugal filter devices prior to one-dimensional gel electrophoresis and peptide purification steps for label-free quantification in a liquid chromatography-electrospray ionization-linear ion trap-Orbitrap MS system. Although this method has been developed specifically for proteomics analyses of ocular microvessels, we have also provided convincing evidence that it can also be readily employed for other tissue-based samples.

Proteome characterization of ocular microvascular beds is pivotal for in-depth understanding of many ocular pathologies in humans. This study demonstrates an effective, rapid, and robust method for protein extraction and sample preparation from small blood vessels employing the porcine short posterior ciliary arteries as model vessels for mass-spectrometry-based proteomics analyses.

基于性光谱的眼微血管蛋白质组学样品制备

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly ...