VC-MALDI-FTMS FOR 2D-PAGE GELS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 传统的二维聚丙烯酰胺凝胶电泳（2D-PAGE）具有许多众所周知的局限性。 它被限制在相当窄的质量和等电点范围内，它不能处理高度疏水的蛋白质或肽，高度碱性的蛋白质需要特别小心。 质量估计值经常因异常迁移和/或磷酸化、脱酰胺或糖基化而失真。 不久前，我们认识到，这些问题中的一些可以通过去除第二种凝胶并将等电聚焦凝胶与质谱联用来解决。[Loo，1996;Loo，1997;Loo，1997;Loo，2005] 我们研究复杂的蛋白质混合物（全细胞裂解物）的MALDI-MS，分离通过变性等电聚焦固定的pH梯度凝胶。 质量分析直接从干凝胶进行，提供与许多2-D凝胶分析直接相关的完整质量测量;例如，此外，我们最近将这些方法扩展到胰蛋白酶-地高辛-IEF-凝胶，然后直接从干燥凝胶进行肽MS和MS/MS。 这种扩展为我们的“虚拟2-D凝胶”增加了第三维信息。 原理验证实验证实，产生的胰蛋白酶肽可以通过MALDI-MS检测，甚至可以通过源后衰变进行测序。 尽管如此，质量校准的挑战是由不均匀的凝胶表面与我们的商业飞行时间仪器的电离源设计。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Traditional 2-dimentional polyacrylamide gel electrophoresis (2D-PAGE) has a number of well known limitations. It is limited to a fairly narrow range of masses and isoelectric points, it cannot handle highly highly hydrophobic proteins or peptides, and highly basic proteins require special care. Mass estimates are frequently distorted by anomalous migration and/or phosphorylation, deamidation, or glycosylation. We recognized some time ago that some of these problems could be solved by removing the second gel and coupling isoelectric focusing gels to mass spectrometry.[Loo, 1996;Loo, 1997;Loo, 1997;Loo, 2005] We examine complex protein mixtures (whole cell lysates) by MALDI-MS, separated by denaturing isoelectric focusing on immobilized pH gradient gels. Mass analysis is performed directly from dried gels, providing intact mass measurements that relate directly to many 2-D gel analyses; e.g., Western blotting, pulse-chase radiolabeling, etc. In addition, we have recently extended these methods to performing trypsin digestions in-IEF-gel, followed by peptide MS and MS/MS directly from dried gels. This extension adds a third dimension of information to our "virtual 2-D gels". Proof-of-principle experiments established that the tryptic peptides generated can be detected by MALDI-MS, and even sequenced by post-source decay. Nevertheless, mass calibration challenges are presented by the uneven gel surface in concert with our commercial time-of-flight instrument's ionization source design.