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MALDI-TOF MS上，用于肽图谱。蛋白质标准品用BD MALDI浓缩器在溶液中消化，在真空下干燥，在不同条件下与基质共结晶。用Beckman PF2D系统进行蛋白质混合物的一维和二维高效液相色谱分离。将馏分直接收集到BD装置的井中，并使用优化的条件进行浓缩、目标上/井内溶液中的消化以及样品与基质的共结晶。用Bruker Reflex IV MALDI-TOF MS获得MALDI质谱图，并与用常规方法消化、浓缩和与基质共结晶的分级蛋白质混合物和多肽标准品进行比较。进一步将结果与收集到96孔板的LC组分以及1DSDS-PAGE和蛋白质凝胶内消化进行比较。 在优化的溶剂条件下，目标上/井内一锅消化、浓缩和样品/基质共结晶很容易产生MS分析，样品损失从1pmol蛋白质标准中最小，从高达200fmol的L起始溶液中的多肽标准样品损失少至10fmol。这相当于从pmo1蛋白和亚pmo1多肽浓度中很好地恢复了MS信号。这一方法被扩展到分析一维和二维RP-蛋白质-LC分离的蛋白质混合物。结果BD浓缩仪与凝胶内消化法分离的蛋白质标准品、1D和2D RP-Protein-LC分离的标准品比较，差异有统计学意义。虽然目前的RP-LC分离的分辨率低于1DSDS-PAGE，但由于系统自动化的能力，恢复的容易和程度得到了提高。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。今年出版了这一项目的三份手稿。

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.