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的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 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。 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。我们已经应用了这种技术，从1D和2D蛋白质LC直接沉积到目标，通过MALDI-TOF MS进行肽图谱分析。蛋白质标准品使用BD MALDI浓缩器在溶液中靶向/孔内消化，在真空下干燥，并在不同条件下与基质共结晶。用Beckman PF 2D系统进行蛋白质混合物的1D和2D-HPLC分级分离。 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。将结果与通过常规方法消化、浓缩并与基质共结晶的分级分离的蛋白质混合物和肽标准品进行比较。将结果进一步与LC级分收集到96孔板中以及与1D SDS-PAGE随后进行蛋白质凝胶内消化进行比较。 在优化的溶剂条件下，一锅靶上/孔内消化、浓缩和样品/基质共结晶容易地产生MS分析，其中从1 pmol蛋白质标准品中损失最少的样品，从高达200 μ l的起始溶液中损失少至10 fmol的肽标准品。这相当于从pMol蛋白质和亚pMol肽浓度良好地回收MS信号。该方法被扩展到分析通过1D和2D RP-蛋白质-LC分离的蛋白质混合物。 使用BD浓缩器的结果与通过SDS-PAGE分离的蛋白质标准品的凝胶内消化以及通过1D和2D RP-蛋白质-LC分离的标准品进行了很好的比较。虽然目前的RP-LC分离的分辨率小于用1D SDS-PAGE获得的分辨率，但回收的容易性和程度得到了提高，因为系统自动化的能力也得到了提高。 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偶联，使得能够以最少的样品处理进行快速、高通量的蛋白质分级分离、消化、肽基质共结晶和MALDI-TOF MS分析。今年出版了该项目产生的三份手稿。

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. Protein standards were digested in-solution on-target/in-well using the BD MALDI Concentrator, dried under vacuum and co-crystallized with matrix under differing conditions. 1D and 2D-HPLC fractionation of protein mixtures was conducted with a Beckman PF2D system. Fractions were collected directly into the wells of the BD device, and optimized conditions were used to concentrate, digest in-solution on-target/in-well, and co-crystallize the samples with matrix. MALDI mass spectra were obtained with a Bruker Reflex IV MALDI-TOF MS. Results were compared, with fractionated protein mixtures and peptide standards that had been digested, concentrated and co-crystallized with matrix by conventional methods. Results were further compared with LC fraction collection into 96 well plates and with 1D SDS-PAGE followed by in-gel digestion of proteins. One-pot on-target/in-well digestion, concentration and sample/matrix co-crystallization under optimized solvent conditions readily yielded MS analyses with minimal sample loss from 1 pmol protein standards and as little as 10 fmol of peptide standards from up to 200 ¿l starting solution. This amounted to good recovery of MS signal from pMol protein and sub pMol peptide concentrations. This methodology was expanded to analyze protein mixtures separated by 1D and 2D RP-protein-LC. Results using the BD Concentrator compared well with in-gel digestion of protein standards separated via SDS-PAGE and with standards separated by 1D and 2D RP-protein-LC. While the resolution of the current RP-LC separation is less than that obtained with 1D SDS-PAGE, the ease and degree of recovery is enhanced as is the ability to automate the system. 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 enabled rapid, high-throughput protein fractionation, digestion, peptide matrix co-crystallization, and MALDI-TOF MS analyses with minimal sample handling. Three manuscripts resulting from this project were published this year.