公开(公告)号：US07166441B2

公开(公告)日：2007-01-23

申请号：US10327342

申请日：2002-12-20

申请人： Timothy K. Nadler ,  Kenneth G. Parker ,  George J. Vella ,  Barrie G. Wagenfeld ,  Yulin Huang ,  Robert J. Lotti

发明人： Timothy K. Nadler ,  Kenneth G. Parker ,  George J. Vella ,  Barrie G. Wagenfeld ,  Yulin Huang ,  Robert J. Lotti

IPC分类号： C12Q1/37

CPC分类号： G01N33/6848 ,  G01N33/6851

摘要： Polypeptides are electroblotted through a digestion membrane to a composite capture membrane that can be directly analyzed using mass spectrometry. The molecular weights of the fragments generated by the digestion membrane are then used to identify the polypeptide from which they originated. The digestion membrane contains an immobilized protease such as trypsin, which cleaves the electroblotted polypeptides into fragments during electroblotting with such high enzyme cleavage capacity and efficiency that one pass of the polypeptide through the membrane is sufficient. The peptide fragments are collected onto a composite capture membrane that is chemically treated, for example by adding a mixture of nitrocellulose and MALDI matrix, so as to absorb peptides near the surface to facilitate desportion, thereby increasing the sensitivity of subsequent analysis by MALDI-TOF mass spectrometry. A wide variety of application are disclosed including identifying proteins separated on a gel or within a tissue sample.

摘要翻译： 多肽通过消化膜电渗透到复合捕获膜，可以使用质谱直接分析。 然后将消化膜产生的片段的分子量用于鉴定它们起源的多肽。 消化膜含有固定化的蛋白酶，例如胰蛋白酶，其在电印迹期间将电印迹多肽切割成片段，其具有如此高的酶切割能力和多肽通过膜的一次通过足够的效率。 将肽片段收集到化学处理的复合捕获膜上，例如通过加入硝酸纤维素和MALDI基质的混合物，以吸收表面附近的肽以促进分离，从而提高随后的MALDI-TOF分析的灵敏度 质谱。 公开了各种各样的应用，包括鉴定在凝胶上或组织样品中分离的蛋白质。