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

用于高通量蛋白质组学的定向消化的 2D-LC MALDI

• 批准号: 7369252
• 负责人: KEVIN W CHEN
• 金额: $ 0.07万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7369252
• 关键词: 2D; LC; MALDI; TARGET; DIGESTION

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.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一，该子项目和研究者 (PI) 可能已从其他 NIH 来源获得主要资助，因此可以在其他 CRISP 机构中得到体现。列出的是该中心，该中心不一定是研究人员的机构，需要高通量样品制备技术，但是，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产量。 MS 数据呈几何级数增加，极大地增加了处理负荷，通常涉及稀释（例如 HPLC），通常需要样品浓缩，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。我们探索了使用一种新颖、简单、廉价（非机器人）96 孔阵列技术 BD MALDI 浓缩器来进行一锅式目标样品制备。 MALDI-MS 分析。我们已应用该技术，通过 MALDI-TOF MS 直接沉积至目标蛋白，并使用 BD MALDI 在溶液中对目标/孔内消化蛋白质标准品。浓缩器，真空干燥并在不同条件下与基质共结晶，使用 Beckman PF2D 系统进行蛋白质混合物的 1D 和 2D-​​HPLC 分级。直接收集到 BD 装置的孔中，并使用优化的条件来浓缩、消化溶液中的目标/孔内样品，并使用 Bruker Reflex IV 获得基质与 MALDI 质谱共结晶。将 MALDI-TOF MS 结果与已通过常规方法消化、浓缩并与基质共结晶的分级蛋白质混合物和肽标准品进行比较，并将结果与​​收集到 96 孔中的 LC 级分进行比较。板和 1D SDS-PAGE，然后在优化的溶剂条件下对蛋白质进行一锅定点/孔内消化、浓缩和样品/基质共结晶，轻松进行 MS 分析，并将样品损失降至最低。 pmol 蛋白质标准品和低至 10 fmol 的肽标准品（高达 200 ¿）这相当于从皮摩尔蛋白质和亚皮摩尔肽浓度中很好地恢复了 MS 信号，该方法被扩展到使用 BD 浓缩器对蛋白质混合物进行分析，结果与凝胶内分析结果相比较。通过 SDS-PAGE 分离的蛋白质标准品以及通过 1D 和 2D RP-蛋白质-LC 分离的标准品的消化，而当前 RP-LC 分离的分辨率低于 1D 获得的分辨率。 SDS-PAGE 的简便性和回收程度得到了提高，通过将 LC 组分直接收集到 96 孔阵列浓缩器中，将 1D 和 2D 蛋白质 LC 与 MALDI-TOF MS 耦合的自动化能力也得到了提高。能够以最少的样品处理实现快速、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。