DVMT OF METHODS, INSTR & TECH FOR IMPROVED PROTEOME & PROTEIN COMPLEX ANAL

方法的 DVMT，导师

• 批准号: 7602860
• 负责人: RICHARD D SMITH
• 金额: $ 58.76万
• 依托单位: BATTELLE PACIFIC NORTHWEST LABORATORIES
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7602860
• 关键词: Address; Affect; Affinity Chromatography; Algorithms; Amino Acids; Anus; Area; Automobile Driving; Behavior; Biological Neural Networks; Blood capillaries; Cations; Charge; Clinical; Complex; Computer Architectures; Computer Retrieval of Information on Scientific Projects Database; Data Set; Databases; Detection; Development; Device or Instrument Development; Dimensions; Effectiveness; Electrons; Electrospray Ionization; Enzymes; Esterification; Event; Family; Fractionation; Funding; Goals; Grant; Heating; Hela Cells; High Pressure Liquid Chromatography; Hydrophobicity; Institution; Ions; Isomerism; Label; Lead; Length; Liquid substance; Measurement; Metals; Methanol; Methods; Modeling; Modification; Numbers; Operative Surgical Procedures; Organism; Output; Peptides; Performance; Phase; Phosphopeptides; Phosphorylation; Phosphorylation Site; Play; Preparation; Procedures; Process; Production; Protein Phosphatase 2A Regulatory Subunit PR53; Protein phosphatase; Proteins; Proteome; Proteomics; Range; Rate; Regulation; Relative (related person); Reproducibility; Research; Research Personnel; Resistance; Resources; Role; Running; Sampling; Signal Transduction; Silicon Dioxide; Solid; Source; Stable Isotope Labeling; Statistically Significant; System; Techniques; Technology; Testing; Time; Training; Trypsin; United States National Institutes of Health; base; calyculin; capillary; concept; desire; detector; enzyme activity; improved; inhibitor/antagonist; instrumentation; mass spectrometer; member; nano; nano-electrospray; protein aminoacid sequence; research study; response; size; tissue-factor-pathway inhibitor 2; tool; transmission process

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. Specific Aim 2 seeks to improve proteome analyses through the development of new methods, instrumentation, and techniques. Specific areas of focus included improved methods for dealing with the phosphoproteome; improved methods for relative and absolute quantitation; advances in MS sensitivity; and elution time prediction for improved protein identification. A very common problem in biomedical proteomics is the desire to track the behavior of informative proteins that are present in low abundance, against a complex background of highly abundant proteins. Strategies for achieving this goal include both sample fractionation approaches to reduce the complexity of the sample analyzed, and instrument development approaches to extend the dynamic range of accurate measurements. These capabilities are the key to any study that involves discovery proteomics for clinical samples. Progress in this area has been driven by collaborative projects that feature non-conventional and/or highly complex samples and the need for dynamic range (D. Smith, Katze, Warren, and Kulkarni). Center advances for analyzing the phosphoproteome  Several technical capabilities have been developed during the last year at the Resource Center to facilitate phosphoproteome analysis, including a special automated LC platform that employs a smaller i.d., (50 ¿m) capillary analytical column with integrated tip and solid phase extraction (SPE) column for sample cleaning. This new LC system provides a high sensitivity and high throughput capability for phosphoproteome analysis. Sample preparation and Immobilized Metal Affinity Chromatography (IMAC) techniques were also further refined. Identification of phosphorylated proteins, changes in the phosphorylation state of a given protein, and identification of specific sites of phosphorylation are all questions of high importance to biologists. In particular, the Klemke collaborative project that focuses on the phosphoproteome of lamellipodia has played a crucial role in driving the development of improved techniques for phosphoproteomics, applying the combined dual-step 18O labeling, IMAC, and LC-MS analysis. Continued improvements in the sensitivity, quantitative accuracy, and completeness of phosphoproteome coverage will continue to be driven by the collaborative projects in the Center (Klemke, Rossie, Kohwi-Shigematsu, Stenoien, and Squier). The Center developed a label-free relative quantitation technology for global targeted phosphoproteome analysis by creating abundance profiles of mass and time features from multiple LC-MS experiments, and performing targeted LC-MS/MS experiments focusing on features of changing abundance profiles. Center researchers applied this technology as a proof-of-concept trial to compare changes in protein phosphorylation in HeLa cells treated with or without calyculin, a potent inhibitor of PP1, PP2A and other members of the phosphoprotein phosphatase (PPP) structural family. This technology was mainly directed toward the identification of phosphopeptides that showed statistically significant changes, and for facilitating the very time-consuming process of phosphopeptide sequencing. Both qualitative and quantitative phosphorylation changes, which are equally important for understanding the dynamic operation of signaling cascades, were revealed from this analysis. This study with collaborator Dr. Sandra Rossie identified more than 1769 unique phosphorylation sites in 1324 unique peptides and 718 unique proteins. More than 300 phosphopeptides corresponding to 277 proteins showed significant changes in response to calyculin treatment, and 83 phosphopeptides of differential abundance were detected in treated samples. These proteins included known targets for PPP enzymes, proteins not previously identified as direct or indirect targets for PPP enzyme regulation, and known and putative proteins not previously shown to be phosphorylated. This study thus reveals a large number of specific phosphorylation sites within new protein targets that are directly or indirectly altered by PPP enzyme activity. The number of changing targets identified in the Centers label-free targeted analysis is comparable to those determined in quantitative studies using differential labeling. The label-free approach has the added advantage of revealing qualitative phosphoproteomic changes in addition to quantitative changes, as differential labeling requires detection of peaks in both samples. This is particularly important when addressing changes in signal transduction-induced phosphorylation events. A similar strategy is being employed to identify PP5 substrates using the automated LC cart for phosphoproteomics combined with the Centers current AMT tag approach. The application of differential labeling for quantitation by Center investigators utilized a modified approach with collaborator Dr. Richard Klemke. This application of an enhanced stable isotope labeling approach for quantitative phosphoproteomics combines trypsin-catalyzed 16O/18O labeling plus 16O/18O-methanol esterification labeling for quantitation, a macro-IMAC trap for phosphopeptide enrichment, and a monolithic capillary column with integrated electrospray emitter. LC separation and MS/MS is followed by neutral loss-dependent MS/MS/MS for phosphopeptide identification using a linear ion trap (LTQ)-FT mass spectrometer and complementary searching algorithms for interpreting MS/MS spectra. To improve the confidence and throughput of phosphopeptide identifications, parameters utilizing accurate mass and reverse database approaches were applied. Advances in MS sensitivity  Two of the major factors that determine MS sensitivity are the efficiency of ion production and the subsequent effectiveness of the transmission of the ions to the detector. To improve sensitivity, Center investigators explored ESI technologies that utilize lower flow rates in the electrospray emitter to increase ion production. Approaches have also been explored that facilitate the use of multiple emitters that operate in parallel and allow the advantages of nL/min flow rate ESI (nano-ESI) to be obtained with traditional higher flow rate liquid separation techniques. In addition, efforts are moving towards reducing ion losses in the ESI interface by incorporating multiple highly efficient heated inlet capillaries. Such inlet modifications take advantage of the large ion capture region of the ion funnel compared to the traditional skimmer interface. The implementation of an electrodynamic ion funnel in Thermo Electron Corporation LTQ and LTQ-FT mass spectrometers improved the sensitivity by ~10 fold. The advantages associated with nano-ESI include: 1) the reduced flow rate decreases initial droplet size, which improves desolvation and increases ion production, 2) there is more excess charge available per analyte, which also increases ion production, and 3) there is less charge competition among different species, which improves quantitation. However, since capillary LC generally employs flow rates greater than ~1 ¿L/min, typical HPLC separations do not achieve the high performance afforded by low flow rate electrospray, which is most pronounced below ~50 nL/min. The approach is to divide the higher flow among multiple ESI emitters, thus enabling the advantages of nano-ESI from elevated flow rate separations. A new ESI emitter emitter fabrication procedure was developed and used to taper the ends of monolithic LC columns, which increased the sensitivity and quantitative ability of proteomic measurements. Silica-based monolithic narrow-bore capillary columns with integrated ESI emitters fabricated directly on the column were developed to provide high quality and robust microSPE-nanoLC-ESI-MS analyses. The integrated ESI emitters added no dead volume to the LC separation, and produced more stable nano-electrosprays at flow rates of ~10 nL/min. The integrated monolithic ESI emitter is more resistant to clogging and also provides good run-to-run reproducibility. Center researchers are using the ESI emitter fabrication method to develop a multiple nano-ESI emitter for improving the sensitivity and quantitative ability of higher flow rate liquid separations. RPLC elution time prediction for improved protein identification  Of benefit to all collaborative projects within the Center are developments that lead to improvements in protein identification. In 2003, the Center introduced an artificial neural network (ANN) method for predicting peptide elution times that was originally based on amino acid composition and later extended to include partial peptide sequence information. Various approaches have been explored for increasing peptide elution time prediction accuracy in reversed phase LC (RPLC). In addition to more complex ANN architectures, several peptide physicochemical (peptide length, hydrophobicity, etc.) and sequence-dependent parameters (peptide sequence, amphipathicity, nearest neighbor, etc.) were examined that have been shown to affect the peptide retention time in LC. To evaluate the sequence-dependent parameters, a much larger training dataset was necessary. As a result, the network was trained using ~345,000 non-redundant peptides identified from a total of 12,059 LC-MS/MS analyses of more than 20 different organisms, and the predictive capability of the model was tested using 1303 confidently identified peptides that were not included in the training set. The present model fully encodes the sequence of peptides up to 50 amino acid residues through an artificial neural network configuration of 1056 input, 24 hidden, and one output node. When compared to previously developed retention time prediction algorithms, the present model provides approximately two-fold better results. Unlike any of the previously developed predictors, this model is now able to accurately predict the retention times of both isobar and isomer peptides. Such capability allows more confident identification of isomer/isobar peptides otherwise indistinguishable by accurate mass measurements. Under current development in the Center are predictive tools for other separation dimensions. A predictive capability developed for strong cation exchange (SCX) liquid chromatographic peptide separations has begun and similar capabilities for IMS and FAIMS separations will follow.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 列出的机构是。 对于中心来说，它不一定是研究者的机构。 具体目标 2 旨在通过开发新方法、仪器和技术来改进蛋白质组分析。用于改进蛋白质鉴定。 生物医学蛋白质组学中一个非常常见的问题是希望在高丰度蛋白质的复杂背景下跟踪低丰度蛋白质的行为。实现这一目标的策略包括两种样品分级分离方法，以降低样品的复杂性。这些功能是任何涉及临床样本发现蛋白质组学的研究的关键，这些进展是由非传统和/或高度协作项目推动的。复杂的样品和需要动态范围（D. Smith、Katze、Warren 和 Kulkarni）。 中心在分析磷酸化蛋白质组方面取得的进展 去年，资源中心开发了多项技术能力，以促进磷酸化蛋白质组分析，包括一个特殊的自动化 LC 平台，该平台采用较小内径（50 µm）的毛细管分析柱，具有集成的尖端和固体用于样品清洗的相萃取 (SPE) 柱 这种新型 LC 系统为磷酸化蛋白质组分析提供了高灵敏度和高通量能力。 （IMAC）技术也得到了进一步完善。 磷酸化蛋白质的鉴定、给定蛋白质磷酸化状态的变化以及磷酸化特定位点的鉴定对于生物学家来说都是非常重要的问题，特别是专注于板状伪足磷酸化蛋白质组的 Klemke 合作项目发挥了至关重要的作用。推动磷酸蛋白质组学改进技术的发展，应用组合双步 18O 标记、IMAC 和 LC-MS 分析，不断提高灵敏度。磷酸化蛋白质组覆盖的准确性和完整性将继续由中心的合作项目（Klemke、Rossie、Kohwi-Shigematsu、Stenoien 和 Squier）推动。 该中心开发了一种用于全球靶向磷酸化蛋白质组分析的无标记相对定量技术，通过多次 LC-MS 实验创建质量和时间特征的丰度曲线，并针对中心研究人员丰度曲线变化的特征进行靶向 LC-MS/MS 实验。应用该技术作为概念验证试验，以比较使用或不使用花萼蛋白（PP1、PP2A 和磷蛋白其他成员的有效抑制剂）处理的 HeLa 细胞中蛋白质磷酸化的变化该技术主要针对显示显着变化的磷酸肽的鉴定，以及促进非常耗时的磷酸化肽测序过程，这对于理解磷酸化变化同样重要。该研究合作者 Sandra Rossie 博士在 1324 个独特的肽和 718 个独特的肽中发现了超过 1769 个独特的磷酸化位点。对应于 277 种蛋白质的 300 多种磷酸肽在花萼蛋白处理后表现出显着变化，并且在处理的样品中检测到了 83 种不同丰度的磷酸肽，这些蛋白质包括 PPP 酶的已知靶标，这些蛋白质以前未被识别为直接或间接靶标。 PPP 酶调节，以及先前未显示被磷酸化的已知和假定蛋白质因此，本研究揭示了新蛋白质靶标中的大量特定磷酸化位点，这些位点直接或间接被 PPP 酶改变。该中心的无标记靶向分析中确定的变化目标的数量与使用差异标记的定量研究中确定的目标数量相当，无标记方法除了揭示定量变化外，还具有揭示定性磷酸化蛋白质组变化的优势。差异标记需要检测两个样品中的峰，这在解决信号转导诱导的磷酸化事件的变化时尤其重要，使用自动化 LC 车结合磷酸化蛋白质组学来识别 PP5 底物。中心当前的 AMT 标记方法。 中心研究人员与合作者 Richard Klemke 博士合作采用了一种改进的方法进行差异标记定量。这种增强型定量稳定同位素标记方法在磷酸蛋白质组学中的应用结合了胰蛋白酶催化的 16O/18O 标记和 16O/18O-甲醇酯化标记。定量、用于磷酸肽富集的宏 IMAC 陷阱以及带有集成电喷雾发射器的整体毛细管柱。 LC 分离和 MS/MS 之后是中性损失依赖性 MS/MS/MS，使用线性离子阱 (LTQ)-FT 质谱仪和补充搜索算法来解释 MS/MS 谱图，以提高可信度和通量。在磷酸肽鉴定中，应用了利用精确质量和反向数据库方法的参数。 MS 灵敏度的进步 MS 灵敏度的两个主要因素是离子产生的效率以及随后离子传输到检测器的有效性，中心研究人员探索了在电喷雾中利用较低流速的 ESI 技术。还探索了提高离子产量的方法，以促进使用并行操作的多个发射器，并允许 nL/min 流速 ESI（纳米 ESI）与传统的更高流速液体分离技术的优点。此外，与传统的撇渣器接口相比，人们正在努力通过合并多个高效加热入口毛细管来减少 ESI 接口中的离子损失。 Thermo Electron Corporation LTQ 和 LTQ-FT 质谱仪中的漏斗将灵敏度提高了约 10 倍。 纳米 ESI 的优点包括：1) 流速降低，初始液滴尺寸减小，从而改善去溶剂化并增加离子产生，2) 每个分析物有更多可用的多余电荷，这也增加了离子产生，3)不同物种之间的电荷竞争较少，从而改善了定量，但是，由于毛细管 LC 通常采用大于 ~1 ¿ L/min，典型的 HPLC 分离无法实现低流速电喷雾所提供的高性能，在低于 ~50 nL/min 时最为明显。该方法是将较高的流量分配给多个 ESI 发射器，从而发挥纳米技术的优势。 - 提高流速分离的 ESI 开发了一种新的 ESI 发射器制造程序，并用于使整体 LC 柱的末端变细，从而提高了基于二氧化硅的蛋白质组测量的灵敏度和定量能力。开发了直接在柱上制造的带有集成 ESI 发射器的整体窄孔毛细管柱，可提供高质量和稳定的 microSPE-nanoLC-ESI-MS 分析。集成 ESI 发射器不会增加 LC 分离的死体积，并产生更稳定的纳米。 - 电喷雾流速约为 10 nL/min。集成的整体 ESI 发射器更耐堵塞，并且还提供良好的运行重现性。正在使用 ESI 发射器制造方法开发多纳米 ESI 发射器，以提高高流速液体分离的灵敏度和定量能力。 RPLC 洗脱时间预测可改进蛋白质鉴定 蛋白质鉴定方面的发展使中心内的所有合作项目受益匪浅。2003 年，该中心引入了一种人工神经网络 (ANN) 方法，用于预测最初基于的肽洗脱时间。除了更复杂的 ANN 架构之外，还探索了多种方法来提高反相 LC (RPLC) 中肽洗脱时间的预测精度。检查了影响 LC 中肽保留时间的序列相关参数（肽长度、疏水性等）和序列相关参数（肽序列、两亲性、最近邻等）。因此，需要更大的训练数据集，使用从 20 多种不同生物体的总共 12,059 次 LC-MS/MS 分析中鉴定出的约 345,000 个非冗余肽来训练网络。使用未包含在训练集中的 1303 个可靠识别的肽来测试模型的预测能力。本模型通过 1056 个输入、24 个隐藏的人工神经网络配置完全编码多达 50 个氨基酸残基的肽序列。 ，和一个输出节点 与之前开发的保留时间预测算法相比，本模型提供了大约两倍的结果。 与之前开发的任何预测器不同，该模型现在能够准确预测同分异构体和异构体肽的保留时间，这种能力可以更可靠地识别异构体/同分异构体肽，否则在中心当前的开发中无法区分。是用于其他分离维度的预测工具。针对强阳离子交换 (SCX) 液相色谱肽分离开发的预测功能已经开始，随后还将推出针​​对 IMS 和 FAIMS 分离的类似功能。