2D-LC MALDI WITH ON-TARGET DIGESTION FOR HIGH-THROUGHPUT PROTEOMICS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 大规模的蛋白质组学分析需要高通量的样品制备技术。 然而，高度复杂的混合物需要在MS分析之前进行多维分馏，以最大限度地提高有用MS数据的产量。 这在几何上扩大了样品数量，显著地增强了处理负荷，通常涉及稀释（例如，HPLC），通常要求样品浓度，并且通常需要多个样品处理步骤，包括转移到不同的反应容器中。这些步骤耗时，导致样品损失和潜在污染。我们已经探索了使用一种新颖的，简单的，廉价的（非机器人）96孔阵列技术，BD MALDI浓缩器，进行一锅靶样品制备MALDI-MS分析。我们已经应用这种技术与沉积从1D和2D蛋白质LC直接到目标的肽映射MALDI-TOF MS。1D和2D-HPLC分馏的蛋白质混合物进行了贝克曼PF 2D系统。 可以将级分直接收集到BD装置的威尔斯孔中，并使用优化的条件来浓缩、在溶液中在靶上/在孔中消化，并使样品与基质共结晶。用Bruker Reflex IV MALDI-TOF MS获得MALDI质谱。将结果与其他样品处理方法进行比较。在优化的溶剂条件下，一锅靶上/孔内消化、浓缩和样品/基质共结晶容易从高达200 μ l的起始溶液中的低至10 fmol的肽标准品产生良好质量的质谱。通过将LC级分直接收集到96孔阵列浓缩器中，将1D和2D蛋白质-LC与MALDI-TOF MS耦合，可实现快速、高通量的蛋白质分级、消化、肽基质共结晶和MALDI-TOF MS分析，只需最少的样品处理。该方法目前正应用于资源和CPC的蛋白质组学项目，并正在评估结果，以进一步改进程序。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Large-scale proteomic analyses necessitate high-throughput sample preparation techniques. However, highly complex mixtures require multi-dimensional fractionation prior to MS analysis to maximize the yield of useful MS data. This geometrically expands sample numbers, dramatically intensifying processing load, commonly involves dilution (e.g., HPLC), often demanding sample concentration, and typically requires multiple steps of sample handling, including transfer to different reaction vessels. These steps are time consuming, lead to sample losses and potential contamination. We have explored the use of a novel, simple, inexpensive (non-robotic) 96-well array technology, the BD MALDI Concentrator, to conduct one-pot on-target sample preparation for MALDI-MS analysis. We have applied this technology with deposition from 1D and 2D protein LC direct-to-target for peptide mapping by MALDI-TOF MS. 1D and 2D-HPLC fractionation of protein mixtures is conducted with a Beckman PF2D system. Fractions can be collected directly into the wells of the BD device, and optimized conditions are used to concentrate, digest in-solution on-target/in-well, and co-crystallize the samples with matrix. MALDI mass spectra are obtained with a Bruker Reflex IV MALDI-TOF MS. Results are compared to other sample handling methods. One-pot on-target/in-well digestion, concentration and sample/matrix co-crystallization under optimized solvent conditions readily yields good quality mass spectra from as little as 10 fmol of peptide standards from up to 200 ¿l starting solution. The coupling of 1D and 2D-protein-LC to MALDI-TOF MS through the collection of LC fractions directly into the 96-well array concentrator enables rapid, high-throughput protein fractionation, digestion, peptide matrix co-crystallization, and MALDI-TOF MS analyses with minimal sample handling. The method is now being applied to proteomic projects of the Resource and the CPC and the results are being evaluated to generate further refinements of the procedures.