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

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。 大规模蛋白质组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离，以最大限度地提高有用的MS数据的产量。这在几何上扩大了样品数量，极大地增加了处理负荷，通常涉及稀释(例如，高效液相色谱法)，通常要求样品浓度，并且通常需要多个步骤的样品处理，包括转移到不同的反应容器。这些步骤非常耗时，会导致样品丢失和潜在的污染。我们探索了使用一种新颖、简单、廉价(非机器人)的96孔阵列技术--BD MALDI浓缩器--来进行MALDI-MS分析的一锅到位样品制备。我们应用了这项技术，从一维和二维蛋白质LC直接沉积到靶点，用MALDI-TOF MS进行一维和二维蛋白质混合物的高效液相色谱分离。馏分可以直接收集到BD装置的井中，并使用优化的条件进行浓缩，在靶上/井中消化溶液，并将样品与基质共结晶。用Bruker Reflex IV MALDI-TOF MS获得MALDI质谱图，并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，一锅对目标/井内消化、浓缩和样品/基质共结晶很容易产生高质量的质谱图，从小到10fmol的多肽标准到高达200fmol的L起始溶液。通过将LC组分直接收集到96孔阵列浓缩器中，1D和2D-Protein-LC与MALDI-TOF MS耦合，实现了快速、高通量的蛋白质分离、消化、多肽基质共结晶和MALDI-TOF MS分析，只需最少的样品处理。该方法目前正在应用于资源中心和CPC的蛋白质组项目，正在对结果进行评估，以进一步完善程序。

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. 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.