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.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 特定目标2试图通过开发新方法，仪器和技术来改善蛋白质组分析。特定的重点领域包括改进处理磷蛋白组的方法；改进的相对定量和绝对定量的方法； MS灵敏度的进步；以及改善蛋白质鉴定的洗脱时间预测。 生物医学蛋白质中的一个非常普遍的问题是，在低抽象中存在的信息蛋白的行为，与高度丰富的蛋白质的复杂背景。实现此目标的策略包括降低分析样本复杂性的样本分级方法，以及仪器开发方法，以扩展精确测量的动态范围。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。该领域的进展是由具有非惯性和/或高度复杂样本以及对动态范围的需要的协作项目驱动的（D. Smith，Katze，Warren和Kulkarni）。 在去年，在资源中心开发了一些技术能力的中心进步，以促进磷蛋白组分析，包括一种特殊的自动化LC平台，该平台采用了较小的I.D.（（50€5）毛细管分析柱，带有集成的尖端和固体萃取型（SPE）（SPE）来进行样品清洁。这种新的LC系统为磷酸蛋白酶分析提供了高灵敏度和高吞吐能力。样品制备和固定的金属亲和色谱（imac）技术也进一步精炼。 鉴定磷酸化蛋白，给定蛋白质的磷酸化状态的变化以及对磷酸化特定位点的鉴定都是对生物学家的重要性问题。特别是，专注于Lamellipodia的磷光蛋白质组的Klemke协作项目在推动改进的磷蛋白质组学技术的发展方面发挥了至关重要的作用，应用了双步18O标记，IMAC和LC-MS分析。该中心的协作项目（Klemke，Rossie，Kohwi-Shigematsu，Stenoien和Squier）将继续提高灵敏度，定量准确性和磷光蛋白质覆盖范围的完整性。 该中心通过创建来自多个LC-MS实验的质量和时间特征的抽象概况，并执行针对性的LC-MS/MS实验，以侧重于改变抽象概况的特征，从而为全球靶向磷光蛋白质组分析开发了无标签的相对定量技术。中心研究人员将该技术应用于概念验证试验，以比较接受或不使用钙蛋白酶治疗的HELA细胞中蛋白质磷酸化的变化，这是PP1，PP2A的潜在抑制剂和其他磷酸蛋白磷酸酶（PPP）结构家族的抑制剂。该技术主要是针对鉴定磷酸肽的，这些磷酸肽显示出统计学上的显着变化，并支持磷酸肽测序的时必时间的过程。从该分析中揭示了定性和定量磷酸化变化，对于理解信号级联的动态操作同样重要。与合作者桑德拉·罗西（Sandra Rossie）博士的这项研究确定了1324年独特的肽和718种独特蛋白质的1769多个独特的磷酸化位点。对应于277种蛋白质的300多种蛋白质显示出对钙蛋白酶处理的反应显着变化，并且在处理的样品中检测到83种差异丰度的磷酸肽。这些蛋白质包括已知的PPP酶靶标的，以前未被鉴定为PPP酶调节的直接或间接靶标的蛋白质，以及以前未显示为磷酸化的已知和假定蛋白质。因此，这项研究揭示了新蛋白质靶标内的大量特定磷酸化位点，这些蛋白质靶标直接或间接地通过PPP酶活性改变。在中心的无标记目标分析中鉴定出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相当。无标签方法具有额外的优势，即除定量变化外，还揭示了定性的磷酸蛋白质组学变化，因为差异标记需要检测两个样品中的峰值。当解决信号转导诱导的磷酸化事件的变化时，这一点尤其重要。正在聘请使用自动LC卡车来识别PP5底物的类似策略，以与中心的当前AMT TAG方法结合使用。 中心研究人员将差异标签应用于定量的应用采用了与合作者理查德·克莱姆克（Richard Klemke）博士的改进方法。这种增强的稳定同位素标记方法用于定量磷酸蛋白质组学组合，胰蛋白酶催化16o/18o标签加上16o/18o-Methaneol酯化标签，用于定量，一种用于磷酸肽的宏观IMAC TRAP，用于磷酸化的磷酸化型含量，并具有单石capillary capillary compare insement Electrated Electrosed Electrosed Electrated Electray Electray Emitter。 LC分离和MS/MS之后是使用线性离子TRAP（LTQ）-FT质谱仪的中性损失依赖性MS/MS/MS，用于磷酸肽鉴定，以及用于解释MS/MS光谱的互补搜索算法。为了提高磷酸肽鉴定的置信度和吞吐量，应用了使用准确质量和反向数据库方法的参数。 MS灵敏度的进步两个决定MS灵敏度的主要因素是离子产生的效率以及离子向检测器传播的随后有效性。为了提高灵敏度，中心研究人员探索了利用电喷雾发射极中流速较低的ESI技术来增加离子产生。还探索了方法，即促进使用并行运行的多个发射器，并允许使用传统的高流速液体液体分离技术获得NL/min流速ESI（纳米-ESI）的优势。此外，通过合并多个高效的加热入口毛细血管，努力正在朝着减少ESI界面中的离子损失。与传统的撇渣器界面相比，这种入口修饰利用了离子漏斗的大离子捕获区域。 Thermo Electronic Corporation LTQ和LTQ-FT质谱仪中电子离子漏斗的实现使灵敏度提高了〜10倍。 与纳米ESI相关的优点包括：1）降低的流速减小了初始液滴大小，从而改善了脱溶剂并增加离子产生，2）每个分析物的可用费用更多，这也会增加离子的产生，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的流速，因此典型的HPLC分离无法实现低流量电喷雾所提供的高性能，该高速度电喷雾最为明显以下〜50 nl/min。该方法是将较高的流量分配在多个ESI发射器之间，从而使纳米ESI的优势从升高的流速分离中带来了优势。开发了一种新的ESI发射极发射器制造程序，并用于缩小整体LC柱的末端，从而提高了蛋白质组学测量的灵敏度和定量能力。基于二氧化硅的单片窄毛毛细管柱具有直接在色谱柱上制造的集成ESI发射器的狭窄毛细管柱，以提供高质量和稳健的Microspe-Nanolc-Esi-MS分析。集成的ESI发射器没有在LC分离中增加死量，并以〜10 nl/min的流速产生了更稳定的纳米电喷雾。集成的整体ESI发射器对堵塞更具抵抗力，还提供了良好的运行重复可重复性。中心研究人员正在使用ESI发射极制造方法来开发多个纳米ESI发射极，以提高较高流速液体分离的灵敏度和定量能力。 RPLC洗脱时间预测改善了中心内所有协作项目的蛋白质鉴定是导致蛋白质识别改善的发展。 2003年，该中心引入了一种人工神经元网络（ANN）方法，用于预测最初基于氨基酸组成的肽洗脱时间，后来扩展到包括部分肽序列信息。已经探索了各种方法，以提高相反期LC（RPLC）的肽洗脱时间预测精度。除了更复杂的ANN体系结构外，还检查了几种肽物理（肽长度，疏水性等）和依赖序列依赖性参数（肽序列，两亲性，最近的邻居等），这些参数已被证明会影响LC中的肽保留时间。为了评估序列依赖性参数，需要更大的训练数据集。结果，使用〜345,000个非冗余肽对网络进行了训练，这些肽是从总共20种不同生物体的12,059 LC-MS/MS分析中识别的，并且使用1303个确定的肽未包括在训练集中的1303个确定的肽，对模型的预测能力进行了测试。本模型通过人工神经元网络配置（1056输入，24个隐藏和一个输出节点）完全编码高达50个氨基酸的肽序列。与先前开发的保留时间预测算法相比，本模型可提供大约两个更好的结果。 与任何先前开发的预测因子不同，该模型现在能够准确预测异荷肽和异构肽的保留时间。这种能力可以更自信地识别异构体/异荷肽，否则准确的质量测量无法区分。在当前发展中，中心是其他分离维度的预测工具。为强阳离子交换（SCX）开发了一种预测能力，液态色谱肽分离已经开始，并且将随后对IMS和FAIMS分离的类似能力。