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

方法的 DVMT，导师

• 批准号: 7359099
• 负责人: RICHARD D SMITH
• 金额: $ 56.73万
• 依托单位: BATTELLE PACIFIC NORTHWEST LABORATORIES
• 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/7359099
• 关键词: DVMT; METHODS; INSTR; TECH; IMPROVED

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. The following six sections describe areas of progress under Specific Aim 2. ANN-based Predictor for Peptide Elution Time The artificial neural network (ANN)-based peptide elution time predictor was further improved this past year. The improvements were a result of a larger training test and a more complex ANN architecture. An ANN architecture that consisted of 1052 input nodes, 40 hidden nodes, and 1 output node was used to fully consider the amino acid residue sequence in each peptide, their length, and hydrophobic moment. The network was trained using 526,981 non-redundant peptides identified from a total of >15,000 LC-MS/MS analyses of >25 different organisms and the predictive capability of the model was tested using 2,752 confidently identified peptides that were not included in the training set. The model demonstrated an average elution time precision of <1.5% and a correlation between observed and predicted elution times of 0.973. The model is now able to accurately predict the elution time of both isomeric and isobaric peptides which is not possible by any other model. In comparison with models published by other groups, the present model is about 100% more precise. We believe that the present model has reached a point where only marginal improvements can be achieved in the future. As a result, we plan to shift our efforts to new predictive capabilities that will enhance our proteomic efforts such as the development of a predictor that can assign the likelihood of a peptide to be identified/detected by mass spectrometry. Furthermore, we plan to use fractionation techniques that separate peptides according to their isoelectric points in order to take advantage of the existent predictive adaptabilities for the calculation of the theoretical peptide isoelectric point. We plan to use these future developed capabilities in conjunction with elution time prediction and mass accuracy to increase the confidence of our peptide identifications. Developments in High Efficiency Reversed Phase LC Separations New developments in reversed phase (RP) LC separations have been achieved using 50 ¿m i.d. fused silica capillaries packed with micron-sized C18-bonded porous silica particles attaining peak capacities of 130-420. When these RPLC separations were combined with a linear ion trap mass spectrometer, ~1,000 proteins could be identified in 50 minutes based upon the identification of ~4,000 tryptic peptides; ~550 proteins in 20 minutes from ~1,800 peptides; and ~250 proteins in 8 minutes from ~700 peptides for a S. oneidensis tryptic digest. We found that 55% of the MS/MS spectra acquired during the entire analysis (and up to 100% of the MS/MS spectra acquired from the most data-rich zone) had sufficient quality for identifying peptides. The results indicate that such analyses using very fast (minutes) RPLC separations based on columns packed with micro-sized porous particles are primarily limited by the MS/MS analysis speed. We have explored the basis for ultrahigh-throughput proteomics measurements using high-speed RPLC combined with high accuracy mass spectrometric measurements. TOF and FTICR mass spectrometers were evaluated in conjunction with 0.8-¿m porous C18 particle-packed RPLC capillary columns (50 ¿m i.d.) for identifying peptides using the Accurate Mass and Time (AMT) tag approach. Peptide RPLC relative retention (elution) times were correlated to within 5% to elution times that differed by at least 25-fold in speed, which allowed peptides to be identified using AMT tags identified from much slower RPLC-MS/MS analyses. When coupled with RPLC, the mass spectrometers operated at fast spectrum acquisition speeds (e.g., 0.2 sec for TOF and either 0.3 or 0.6 sec for FTICR), and peptide mass measurement accuracies of better than ¿15 ppm were obtained. Ion population control during fast separations improved the mass accuracies obtained with FTICR, but the detection of low abundance species was somewhat suppressed in the fast analyses. The proteome coverage obtained using the AMT tag approach was limited by the separation peak capacity, the sensitivity of the MS, and the accuracy of both the mass measurements and the relative RPLC peptide elution times. Experimental results demonstrated that accuracies of 5% for the RPLC relative retention times and ¿15 ppm for mass measurements were sufficient for confident identification of >2800 peptides and >760 proteins from >13,000 different detected species. The approach allowed ~600 proteins from a S. oneidensis sample to be identified from assignment of ~2000 peptides in 150 sec. The TOF instrumentation was found to be advantageous for faster separations (of <120 sec), while FTICR MS was more effective for analysis times of >150 sec due to the improved mass accuracies achievable with longer spectrum acquisition times and better ion population control. The present work demonstrates the feasibility of very high throughput proteomics measurements and indicates additional significant improvements in throughput are achievable by further increasing the speed of high peak capacity separations, as well by increasing the measurement sensitivity and the accuracy of mass measurements. High-Throughput Proteome-Wide Analysis of Intact Proteins During the past year, we focused on the development of a novel capability for high-throughput proteome-wide analysis of intact proteins by combining bottom-up proteomics with high-accuracy FTICR intact protein mass measurements and targeted tandem mass spectrometry (MS/MS) employing variety of dissociation schemes (e.g., collisionally induced and electron capture dissociation; CID and ECD, respectively). The ultimate goal is to develop a tool set for comparative proteomics at the intact protein level that includes methods to accurately quantify changes in the levels of proteins and protein post-translational modifications (PTMs). Since protein MS/MS (particularly with ECD) is currently too slow to be effective for on-line separations, we opted to develop a profiling technique based on 2D separations coupled with FTICR MS (e.g., mass vs. retention time maps), and target exclusively discriminatory proteins for MS/MS; this methodology is novel in that it targets intact proteins (and not tryptic peptides), hence offering significant advantage for comparative proteomic measurements. We have an NCRR collaboration with Dr. Thomas Squier¿s group at PNNL to characterize the dynamics of protein modifications induced by reactive oxygen and nitrogen species in macrophage cells. Tyrosine phosphorylation is an essential part of cellular signaling, and nitrotyrosine formation could block or mimic phosphorylation. It has recently been argued that tyrosine nitration is also a dynamic process that leads to cellular signaling in radical rich environments, such as the mitochondria. The dynamic process of nitration and denitration, dependent on specific cellular conditions, lead some to speculate upon the existence of a denitrase enzyme. We have shown that the induction of radical generation in macrophages stimulates increased clearance of nitrated forms of the ubiquitous calcium sensor protein calmodulin and tentatively identified calmodulin as a substrate for putative denitrase in macrophages. We plan to follow up with an in-depth characterization of the changes in key complexes associated with oxidative stress; this research will shed light not only on the mechanisms of macrophage activation, but also on dynamics of protein nitration within the cell. We have an NCRR collaboration with Dr. Brian Thrall¿s group at PNNL to characterize the secreted proteome from a human mammary epithelial cell (HMEC) line and quantify changes in secreted proteins observed during phorbol 12-myristate 13-acetate (PMA) activation, a known tumor promoter and potent activator of protein secretion/shedding. Considering the significant role that excreted proteins play in the survival, proliferation, and differentiation of cells, it is important to develop methods that specifically detect, identify, and quantify these proteins. Intact protein studies will nicely complement already gathered ¿bottom-up¿ proteomics data. We have also initiated a new NCRR collaboration with Dr. Nahum Sonenberg at McGill University to study translational control mechanisms. A ¿top-down¿ mass spectrometric approach employing a 12T FTICR mass spectrometer equipped with ECD capability will be employed to characterize mouse brain-derived translational repressors that are subjected to developmentally regulated (e.g., adult vs. early postnatal brain) PTMs. These currently unknown PTMs may reveal a novel control mechanism for protein synthesis in the brain. Developments with Monolithic LC Columns In the past year high-efficiency 70 cm x 20 ¿m i.d. silica-based monolithic capillary LC columns have been prepared. The monolith appears as a porous network with ~3 ¿m pores. This denser sol-gel skeleton not only decreases the mass transfer resistance from the mobile phase to the stationary phase, but also increases the surface area of the silica skeleton, which determines the sample loading capacity of the column, as well as analyte retention. The columns at a mobile phase pressure of 5000 psi provide flow rates of ~40 nL/min at an optimum linear velocity of ~0.24 cm/s. The columns provide a separation peak capacity of ~420 in conjunction with both on-line coupling with micro solid phase extraction (SPE) and ESI-MS. The sensitivity of the monolithic columns for protein identification was evaluated using a BSA tryptic digest. A sample containing 15-attomole BSA tryptic digest in 10-¿L solution was loaded onto the on-line SPE 50 ¿m i.d. monolithic column, separated by the 20 ¿m i.d. monolithic column, and analyzed by ESI ion trap-MS/MS with peptide identification using the SEQUEST algorithm. As an example of the sensitivity achieved, three doubly charged tryptic peptides were confidently identified with high SEQUEST scores from 15-attomole of tryptically digested BSA. Application of the high sensitivity on-line microSPE-nanoLC-MS to the more complex S. oneidensis tryptically digested proteomic sample enabled identification of 855 proteins from a single 10 h nanoLC-MS/MS analysis. To further improve the sensitivity and reduce the ion suppression effect, 10-¿m-i.d. silica-based monolithic LC columns were fabricated with integral nanoESI emitters (i.e. from a single fused-silica capillary) and combined with a 50-¿m-i.d. SPE pre-column. The 25 cm long x 10-¿m-i.d. monolithic LC columns provide optimum flow rates of ~10 nL/min at pressures of ~1000 psi. A more hydrophilic SPE column (packed with more hydrophilic YMC ODS-AQ packing material) was used to reduce the effect of the dead volume between the SPE column and the analytical column on the separation. A 5-attomole BSA tryptic digest in 1-¿L solution was analyzed using the integrated monolithic column interfaced to a conventional ion trap (LCQ) for MS/MS. Multiple peptides were identified using the SEQUEST program. As an example of the high sensitivity achieved with a much more complex mixture of peptides, analysis of 100 ng of a tryptic digest of soluble S. oneidensis proteins allowed 1332 proteins to be confidently identified (from 5164 different peptides) in a 3 h analysis using a different linear ion trap (LTQ) for MS/MS. In addition, at a regime of 10 nL/min flow rates, the compound-to-compound variations in MS response are minimized and MS response is expected to vary more linearly with concentration. The good linear relationships between peptide MS responses and sample amount for BSA tryptic digests are consistent with this expectation. The use of an integrated 10-¿m-i.d. silica-based monolithic column has demonstrated good separation efficiency, as well as greatly improved ESI-MS sensitivity compared to columns of conventional dimensions. This combination of advances is anticipated to provide greater sensitivity for a broad range of proteomics applications, and facilitate the use of ¿label-free¿ quantitation methods by avoiding most contributions due to ion suppression effects. Developments on Multi-Nano-ESI Emitters The three main advantages to performing ESI at nL/min flow rates is (1) the reduced flow rate decreases initial droplet size improving desolvation, (2) there is more excess charge available per analyte, and (3) there is less charge competition improving quantitation. However, HPLC separations routinely use ?L/min flow rates. Over the past year our aim has been to split the higher flow into several smaller flows and create nano-ESI from each (creating nano-ESI from ?L/min flow rates). We are exploring two different approaches to accomplish this goal; (1) ESI emitters made from monolithic columns and (2) microfabricated, multi-emitter chips. The monolithic-based ESI emitters are made from a short section of an HPLC monolithic column. The porous monolith creates several flow paths (splits the high flow into several smaller flows) and the rough surface of the monolith at the emitter tip promotes the formation of multiple electrosprays (creating several nano-electrosprays from a ?L/min flow rate). Initial results show an ~10-fold increase in ESI current compared to a standard ESI emitter using a the same solution and flow rate. Additionally, the mapping of the current density of the ESI plume(s) shows increased ion production and the acquisition of mass spectra show increased peak intensities indicating the formation of multiple electrosprays from the emitters. This work has also led to an application of the technology with low-flow HPLC-MS/MS using a 10 ?m i.d. monolithic column with an incorporated monolithic ESI emitter for proteomic analyses. We are currently in the fabrication process of the multi-emitter chips. In contrast to the monolithic emitters, the chips have a discrete number of emitters and microfabricated channels to split the solution flow. Once the chips are completed, we will test, characterize, and compare them to the standard and monolithic emitters. In addition, this work is leading us to research better ways to transmit the resulting increased ion population into the first vacuum stage of the mass spectrometer which will give way to a significant improvement in instrument sensitivity. Progress on the Characterization of the Human Blood Plasma Proteome The blood plasma proteome has been widely recognized for its significant potential in providing diagnostic or therapeutic biomarkers for various diseases, as well as its potential contribution to personalized medicine; however, it also represents the most challenging mammalian proteome to be characterized due to the tremendous complexity and extraordinary dynamic range in protein concentrations. To address the challenge of plasma proteome characterization, we have developed a ¿divide-and-conquer¿ strategy that combines immunoaffinity depletion of the top 12 highly abundant plasma proteins, high efficiency enrichment of cysteinyl-peptides and N-linked glycopeptides, and two-dimensional LC-MS/MS analyses. In addition, a set of criteria were established to ensure the high confidence of plasma protein identifications based on a probability-based evaluation model. By applying this strategy to a pooled trauma patient plasma sample, we have achieved the highest confidence dataset of 3654 plasma proteins based upon 22,300 different peptide identifications to date with an overall dynamic range of detection of ~108. Among the 3654 proteins, 1494 proteins were identified by at least two peptides per protein (>99% confidence) and the other ~2100 proteins were identified with >90% confidence. The tremendous depth of plasma proteome coverage achieved by applying this approach demonstrates its potential for discovering candidate disease biomarkers for subsequent quantitative clinical applications.

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。特异性目标2旨在通过开发新的方法、仪器和技术来改进蛋白质组分析。以下六个部分描述具体目标2下的进展领域。基于人工神经网络的肽洗脱时间预测器在过去的一年中得到了进一步的改进。这些改进是更大的训练测试和更复杂的人工神经网络架构的结果。采用由1052个输入节点、40个隐藏节点和1个输出节点组成的ANN架构，充分考虑每个肽的氨基酸残基序列、长度和疏水力矩。该网络使用526,981个非冗余肽进行训练，这些非冗余肽是从>25种不同生物的>15,000个LC-MS/MS分析中鉴定出来的，并使用未包括在训练集中的2,752个确定的肽来测试模型的预测能力。该模型的平均洗脱时间精度<1.5%，实际洗脱时间与预测洗脱时间的相关性为0.973。该模型现在能够准确地预测同分异构体和等压肽的洗脱时间，这是任何其他模型都无法做到的。与其他小组发表的模型相比，本模型的精度提高了约100%。我们认为，目前的模式已经到了将来只能取得微小改进的地步。因此，我们计划将我们的工作转向新的预测能力，这将增强我们的蛋白质组学工作，例如开发一种预测器，可以分配肽的可能性，通过质谱识别/检测。此外，我们计划使用根据肽的等电点分离肽的分馏技术，以便利用现有的预测适应性来计算理论肽等电点。我们计划将这些未来开发的功能与洗脱时间预测和质量准确性结合使用，以增加我们肽鉴定的信心。在反相（RP） LC分离方面取得了新的进展，使用50¿m id的熔融二氧化硅毛细管填充微米尺寸的c18键合多孔二氧化硅颗粒，达到130-420的峰值容量。当这些RPLC分离与线性离子阱质谱仪相结合时，在鉴定约4,000个色氨酸的基础上，在50分钟内可鉴定约1,000个蛋白质；约1800多肽，20分钟约550个蛋白；在8分钟内从约700多肽中提取约250个蛋白质，用于单株链球菌的胰蛋白酶消化。我们发现在整个分析过程中获得的55%的MS/MS光谱（从数据最丰富的区域获得的MS/MS光谱高达100%）具有足够的质量用于鉴定肽。结果表明，基于填充微孔颗粒柱的快速（分钟）RPLC分离分析主要受到MS/MS分析速度的限制。我们已经探索了使用高速RPLC结合高精度质谱测量的超高通量蛋白质组学测量的基础。TOF和FTICR质谱仪与0.8-¿m多孔C18颗粒填充的RPLC毛细管柱（50¿m id）结合使用精确质量和时间（AMT）标签方法鉴定肽。肽段RPLC相对保留（洗脱）时间与5%的洗脱时间相关，其速度相差至少25倍，这允许使用从慢得多的RPLC-MS/MS分析中识别的AMT标签来鉴定肽段。当与RPLC耦合时，质谱仪以快速的光谱采集速度运行（例如，TOF为0.2秒，FTICR为0.3或0.6秒），并且获得了优于¿15 ppm的肽质量测量精度。快速分离过程中的离子种群控制提高了FTICR获得的质量精度，但在快速分析中对低丰度物种的检测受到一定抑制。使用AMT标签方法获得的蛋白质组覆盖范围受到分离峰容量、质谱灵敏度、质量测量和相对RPLC肽洗脱时间的准确性的限制。实验结果表明，RPLC相对保留时间的精确度为5%，质量测量的准确度为¿15 ppm，足以自信地鉴定来自>13,000种不同被检测物种的>2800个肽和>760个蛋白。该方法允许在150秒内从约2000个多肽中鉴定出约600个蛋白质。TOF仪器被发现有利于更快的分离（<120秒），而FTICR MS由于更长的光谱采集时间和更好的离子种群控制而提高了质量精度，因此在150秒的分析时间内更有效。目前的工作证明了非常高通量蛋白质组学测量的可行性，并表明通过进一步提高峰值容量分离的速度，以及通过提高测量灵敏度和质量测量的准确性，可以实现通量的进一步显著提高。在过去的一年里，我们专注于开发一种新的能力，通过将自下而上的蛋白质组学与高精度的FTICR完整蛋白质质量测量和采用多种解离方案（例如碰撞诱导和电子捕获解离；分别为CID和ECD）的靶向串联质谱（MS/MS）相结合，实现完整蛋白质的高通量蛋白质组分析。最终目标是开发一套完整蛋白质水平比较蛋白质组学的工具，包括精确量化蛋白质水平变化和蛋白质翻译后修饰（PTMs）的方法。由于蛋白质MS/MS（特别是ECD）目前太慢，无法有效地进行在线分离，我们选择开发一种基于二维分离和FTICR MS（例如，质量与保留时间图）的分析技术，并针对MS/MS专门针对歧视性蛋白质；这种方法是新颖的，因为它针对的是完整的蛋白质（而不是色氨酸肽），因此为比较蛋白质组学测量提供了显著的优势。我们与PNNL的Thomas Squier博士和他的团队进行了NCRR合作，以表征巨噬细胞中活性氧和活性氮诱导的蛋白质修饰动力学。酪氨酸磷酸化是细胞信号传导的重要组成部分，而硝基酪氨酸的形成可以阻断或模拟磷酸化。最近有人认为，酪氨酸硝化也是一个动态过程，在自由基丰富的环境（如线粒体）中导致细胞信号传导。硝化和脱硝的动态过程依赖于特定的细胞条件，这使一些人推测脱硝酶的存在。我们已经证明，在巨噬细胞中诱导自由基生成刺激了普遍存在的钙传感器蛋白钙调蛋白的硝化形式的清除增加，并初步确定了钙调蛋白是巨噬细胞中假定的脱氮酶的底物。我们计划进一步深入表征与氧化应激相关的关键复合物的变化；这项研究不仅将揭示巨噬细胞活化的机制，还将揭示细胞内蛋白质硝化的动力学。我们与PNNL的Brian Thrall博士的团队合作进行了NCRR的研究，以表征人类乳腺上皮细胞（HMEC）系的分泌蛋白质组，并量化在phorbol 12-肉豆蔻酸13-醋酸酯（PMA）激活过程中观察到的分泌蛋白质的变化，PMA是一种已知的肿瘤启动子和蛋白质分泌/脱落的有效激活剂。考虑到排泄蛋白在细胞存活、增殖和分化中发挥的重要作用，开发专门检测、鉴定和量化这些蛋白的方法是很重要的。完整蛋白质研究将很好地补充已经收集到的“自下而上”蛋白质组学数据。我们还与麦吉尔大学的Nahum Sonenberg博士发起了一项新的NCRR合作，研究翻译控制机制。采用配备ECD功能的12T FTICR质谱仪的自上而下质谱方法将被用于表征受发育调节（例如，成人与出生后早期大脑）PTMs影响的小鼠脑源性翻译抑制因子。这些目前未知的PTMs可能揭示了大脑中蛋白质合成的一种新的控制机制。在过去的一年里，高效的70厘米× 20微米硅基整体毛细管色谱柱已经制备出来。整体呈现为具有~3¿m孔的多孔网络。这种更致密的溶胶-凝胶骨架不仅降低了从流动相到固定相的传质阻力，而且增加了二氧化硅骨架的表面积，这决定了柱的样品装载能力，以及分析物的保留。在5000psi的流动相压力下，色谱柱的流量为~ 40nl /min，最佳线速度为~0.24 cm/s。该色谱柱结合微固相萃取（SPE）和ESI-MS的在线耦合，提供了~420的分离峰容量。用牛血清白蛋白色氨酸消化法评价整体柱对蛋白质鉴定的敏感性。将含有15原子BSA色氨酸消化液（10- L）的样品装上在线SPE 50 - m鉴别柱，用20 - m鉴别柱分离，采用ESI离子阱-质谱联用，采用SEQUEST算法进行多肽鉴定。作为灵敏度实现的一个例子，从15原子的胰蛋白酶消化的BSA中确定了三个双电荷的胰蛋白酶肽，并获得了高的SEQUEST分数。将高灵敏度在线microSPE-nanoLC-MS应用于更复杂的S. oneidensis tryptically酶解的蛋白质组学样品，可以在10小时的单次nanoLC-MS/MS分析中鉴定855种蛋白质。为进一步提高灵敏度，降低离子抑制效应，采用10-¿m-i.d。硅基单片LC柱由完整的纳米esi发射器（即从单个熔融二氧化硅毛细管）制成，并与50-¿m- id - d相结合。SPE pre-column。长25厘米，宽10厘米。单片LC柱在~1000 psi的压力下提供~10 nL/min的最佳流速。采用更亲水的固相萃取柱（填充更亲水的YMC ODS-AQ填料），减少固相萃取柱与分析柱之间的死体积对分离的影响。采用集成整体柱与传统离子阱（LCQ）连接，对1-¿L溶液中的5原子BSA色氨酸进行了分析。使用SEQUEST程序鉴定了多个多肽。作为用更复杂的多肽混合物实现高灵敏度的一个例子，使用不同的线性离子阱（LTQ）进行MS/MS，在3小时的分析中，使用100 ng的可溶性S. oneidensis蛋白的胰蛋白酶消化，可以自信地鉴定出1332种蛋白质（来自5164种不同的多肽）。此外，在10 nL/min流速下，化合物间质谱响应的变化最小，预计质谱响应与浓度的线性关系更大。BSA胰蛋白酶消化的肽质谱反应与样品数量之间良好的线性关系与这一预期一致。使用集成的10- m- id。硅基整体柱具有良好的分离效率，与常规尺寸柱相比，硅基整体柱的ESI-MS灵敏度大大提高。预计这一进展的结合将为广泛的蛋白质组学应用提供更高的灵敏度，并通过避免离子抑制效应的大部分贡献，促进“无标签”定量方法的使用。以nL/min流速进行ESI的三个主要优点是：(1)降低的流速减小了初始液滴尺寸，改善了脱溶；(2)每个分析物有更多的多余电荷可用；(3)电荷竞争减少，提高了定量。然而，HPLC分离通常使用？L/min流量。在过去的一年里，我们的目标是将较高的流程分成几个较小的流程，并从每个流程中创建纳米esi(从？L/min流量)。我们正在探索两种不同的方法来实现这一目标；(1)由整体柱制成的ESI发射器和(2)微加工的多发射器芯片。单片基ESI发射器由HPLC单柱的一小段制成。多孔单体形成了几个流动路径（将大流量分成几个小流量），发射器尖端的单体粗糙表面促进了多个电喷雾的形成(从一个？L/min流量)。初步结果表明，与使用相同溶液和流速的标准ESI发射器相比，ESI电流增加了约10倍。此外，ESI羽流的电流密度映射显示离子产生增加，质谱采集显示峰值强度增加，表明来自发射器的多个电喷雾形成。这项工作也导致了该技术的应用低流量HPLC-MS/MS使用10 ？m id整体柱与集成整体ESI发射器用于蛋白质组学分析。我们目前正处于多发射器芯片的制造过程中。与单片发射器相比，芯片具有离散数量的发射器和微制造通道来分裂溶液流。一旦芯片完成，我们将测试，表征，并将其与标准和单片发射器进行比较。此外，这项工作将引导我们研究更好的方法，将由此增加的离子群传输到质谱仪的第一真空级，这将大大提高仪器的灵敏度。血浆蛋白质组因其在各种疾病的诊断和治疗方面的巨大潜力以及对个性化医疗的潜在贡献而得到广泛认可。然而，由于蛋白质浓度的巨大复杂性和非凡的动态范围，它也代表了最具挑战性的哺乳动物蛋白质组。为了应对血浆蛋白质组表征的挑战，我们开发了一种“分而治之”的策略，结合了前12个高丰度血浆蛋白的免疫亲和耗尽，半胱氨酸肽和n -连接糖肽的高效富集，以及二维LC-MS/MS分析。此外，建立了一套基于概率的评价模型，以确保血浆蛋白鉴定的高置信度。通过将这一策略应用于创伤患者血浆样本，我们已经获得了迄今为止基于22,300种不同肽鉴定的3654种血浆蛋白的最高置信度数据集，总体动态检测范围为~108。在3654个蛋白中，1494个蛋白每蛋白至少鉴定出2个多肽（>99%置信度），其余约2100个蛋白每蛋白鉴定出>90%置信度。通过应用这种方法实现的血浆蛋白质组覆盖的巨大深度表明，它有潜力为后续定量临床应用发现候选疾病生物标志物。