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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。大规模蛋白质组学分析需要高通量样品制备技术。然而，高度复杂的混合物需要在质谱分析之前进行多维分馏，以最大限度地提高有用的质谱数据的产量。这成倍地增加了样品数量，极大地增加了处理负荷，通常涉及稀释（例如，HPLC），通常要求样品浓度，并且通常需要多个步骤的样品处理，包括转移到不同的反应容器。这些步骤耗时，导致样品损失和潜在的污染。我们探索了一种新颖、简单、廉价（非机器人）的96孔阵列技术，即BD MALDI浓缩器，用于进行MALDI- ms分析的单罐靶样制备。我们将该技术应用于1D和2D蛋白LC直接到靶点的沉积，通过MALDI- tof ms进行肽定位。蛋白质标准品使用BD MALDI浓缩器在溶液中靶上/井中消化，在真空下干燥，并在不同条件下与基质共结晶。采用Beckman PF2D系统对蛋白混合物进行1D和2D-HPLC分离。馏份直接收集到BD装置的孔中，利用优化后的条件进行浓缩、靶/孔内溶出、样品与基质共结晶。用Bruker Reflex IV MALDI- tof质谱仪获得MALDI质谱，将结果与分离蛋白混合物和经常规方法消化、浓缩并与基质共结晶的肽标准品进行比较。结果进一步比较了96孔板的LC部分收集和1D SDS-PAGE，然后凝胶内消化蛋白质。在优化的溶剂条件下，一锅靶/井内消化，浓缩和样品/基质共结晶容易产生质谱分析，从1 pmol蛋白质标准品到10 fmol肽标准品，从高达200¿l的起始溶液中，样品损失最小。这相当于从皮摩尔蛋白和亚皮摩尔肽浓度中恢复良好的质谱信号。该方法扩展到分析1D和2D RP-protein-LC分离的蛋白质混合物。结果BD浓缩器与SDS-PAGE分离的蛋白标准品和1D、2D RP-protein-LC分离的蛋白标准品凝胶消解效果良好。虽然目前RP-LC分离的分辨率低于1D SDS-PAGE，但回收率和自动化系统的能力都得到了提高。通过将LC部分直接收集到96孔阵列浓缩器中，将1D和2d蛋白LC与MALDI-TOF MS耦合，实现了快速、高通量的蛋白质分离、消化、肽基质共结晶和MALDI-TOF MS分析，只需最少的样品处理。

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 picomolar protein and sub picomolar 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.