VC-MALDI-FTMS FOR 2D-PAGE GELS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 传统的二维聚丙烯酰胺凝胶电泳法(2D-PAGE)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多肽，并且高度碱性的蛋白质需要特别照顾。质量估计经常受到异常迁移和/或磷酸化、脱酰胺化或糖基化的影响。我们早在一段时间前就认识到，其中一些问题可以通过去除第二个凝胶并将等电聚焦凝胶耦合到质谱学中来解决。 我们用MALDI-MS检测复杂的蛋白质混合物(全细胞裂解产物)，通过变性等电聚焦在固定的pH梯度凝胶上分离。直接从干凝胶中进行质量分析，提供与许多二维凝胶分析直接相关的完整质量测量；例如，Western blotting、脉冲追逐放射性标记等。此外，我们最近将这些方法扩展到在IEF-Gel中进行胰酶消化，然后直接从干凝胶中进行多肽MS和MS/MS。这个扩展为我们的“虚拟2-D凝胶”增加了第三个维度的信息。原理验证实验证明，所产生的胰蛋白酶多肽可以通过MALDI-MS检测到，甚至可以通过源后衰变进行测序。然而，与我们的商用飞行时间仪器的电离源设计相一致，不平坦的凝胶表面带来了质量校准的挑战。

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.