Practical Mass Spectrometry Upgrade for Identifying Fragile Protein Modifications by ECD

通过 ECD 识别脆性蛋白质修饰的实用质谱升级

基本信息

批准号：
9253957
负责人：
Valery G. Voinov
金额：
$ 22.5万
依托单位：
E-MSION, INC.
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-01-01 至 2017-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9253957
关键词：
Address Adoption Amides Arthritis Back Biological Businesses Cell physiology Cells Charge Cleaved cell Complex Computer software Computers Data Diabetes Mellitus Diagnosis Disease Dissociation Electrons Engineering Feedback Fourier transform ion cyclotron resonance Generations Goals Heart Diseases Inflammation Infrared Rays International Ions Legal patent Length Letters Location Malignant Neoplasms Marketing Mass Spectrum Analysis Measures Methodology Methods Modernization Modification Molecular Weight Nerve Degeneration Pattern Peptide Fragments Peptides Phase Phosphopeptides Phosphorylation Phosphorylation Site Post-Translational Protein Processing Power Sources Process Protein Fragment Proteins Proteomics Research Personnel Resolution Sales Serine Small Business Innovation Research Grant Speed Technology Testing Threonine Time Tyrosine Phosphorylation Ubiquitin United States National Institutes of Health Work base chemical bond cost cost effective density design diagnostic biomarker electron energy fly improved instrument lens magnetic field mass spectrometer metabolomics new technology operation phosphoproteomics prevent quantum therapeutic biomarker tool virtual voltage

项目摘要

The speed, resolution and high mass accuracy of modern mass spectrometers have revolutionized proteomics, particularly for determining fragile post-translational modifications that control most cellular processes. Accurate identification and quantitation of phosphorylation sites remain a major challenge in proteomics. The key weakness with mass spectrometry for phospho-proteomics lies in the methods used to induce fragmentation, because phosphoryl bonds are among the most labile chemical bonds in proteins and are lost in complex ways by current collision-based fragmentation approaches. An alternative fragmentation methodology called electron capture dissociation (ECD) is well established to produce exceptional spectra of phosphopeptides, but is currently feasible only in expensive FTICR mass spectrometers. The fundamental limitation to ECD is providing enough low-energy electrons to efficiently fragment peptides. We have two issued patents protecting a new technology enabling a practical ECD cell that uses carefully sculpted magnetic fields to confine electrons. The ECD cell is only two centimeters in length and can be readily incorporated into virtually any mass spectrometer. The major factor limiting adaption with our ECD cell is that the efficiency is limited to 5-10% for doubly charged phosphopeptides. This raise concerns about the loss of sensitivity for low abundance peptides. However, peptides now fly just once through the cell. Our Phase-I proposes to increase this efficiency by reflecting ions to make multiple passages through the ECD cell. For this purpose, we will focus on Orbitrap mass spectrometers, which have become the most widely used instruments for proteomics. Their unique design allows integration of the ECD cell without changing any component in the Orbitrap itself. The feasibility question to be answered in Phase-I is: how to best incorporate the ECD cell to pass peptides and proteins through the cell multiple times to increase fragmentation efficiency? The challenge is to avoid losing sensitivity because of peptide ions scattering as they are reflected. In Phase-II, we will work with early- adopters to validate the cell for quantifying post-translational modifications by both top-down and bottom-up proteomic approaches. Supporting letters are included from the discoverer of ECD, the inventor of the Orbitrap, and two internationally known leaders of proteomics. Phase-III will be to provide cost-effective upgrade kits for the thousands of Orbitraps currently in operation. As the technology wins acceptance, our company will develop new generations of mass spectrometers capitalizing on the additional information provided by ECD. The adoption of our technology for a modest cost will accelerate the ability of many NIH investigators to probe disease mechanisms as well as identify diagnostic and therapeutic biomarkers with increased accuracy, greater speed and fewer mistakes.

现代质谱仪的速度，分辨率和高质量精度已彻底改变了蛋白质组学，特别是用于确定控制大多数细胞过程的脆弱的翻译后修饰。磷酸化位点的准确鉴定和定量仍然是蛋白质组学的主要挑战。磷酸蛋白质学的质谱质谱的关键弱点在于用于诱导的方法碎裂，因为磷酸键是蛋白质中最不稳定的化学键之一，并且丢失通过当前基于碰撞的碎片方法复杂的方式。另一种破碎称为电子捕获解离（ECD）的方法论已经很好地确定，以产生特殊的光谱磷酸肽，但目前仅在昂贵的FTICR质谱仪中可行。基本 ECD的局限性提供了足够的低能电子来有效地碎片肽。我们有两个发行的专利保护了一项新技术，实现了使用精心雕刻磁性的实用ECD单元局限电子的字段。 ECD单元的长度仅为两厘米，可以很容易地纳入几乎所有质谱仪。通过我们的ECD单元进行适应的主要因素是效率是双电荷磷酸肽的限制为5-10％。这引起了人们对低敏感性丧失的担忧丰富的肽。但是，肽现在仅通过细胞飞行一次。我们的I期建议增加通过反映离子以通过ECD细胞进行多个段落的效率。为此，我们将专注于Orbitrap质谱仪，这些质谱仪已成为蛋白质组学最广泛使用的仪器。它们的独特设计允许集成ECD单元格，而无需更改Orbitrap本身中的任何组件。在I期中要回答的可行性问题是：如何最好地纳入ECD细胞以通过肽多次通过细胞的蛋白质以提高碎裂效率？挑战是避免由于肽离子的散射反射而失去敏感性。在第三阶段，我们将与早期合作采用者可以通过自上而下和自下而上蛋白质组学方法。支持信件包括来自ECD的发现者， Orbitrap和两名国际知名的蛋白质组学领导者。第三阶段将提供具有成本效益的升级目前正在运行的数千个Orbitrap的套件。随着技术的胜利，我们的公司将开发新一代的质谱仪，利用其他信息由ECD提供。我们的技术以适度的成本采用将加速许多NIH的能力研究人员探究疾病机制，并确定具有诊断性和治疗性生物标志物提高准确性，更高的速度和更少的错误。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Improved Protein and PTM Characterization with a Practical Electron-Based Fragmentation on Q-TOF Instruments.

DOI：
10.1021/jasms.0c00482
发表时间：
2021-08-04
期刊：
Journal of the American Society for Mass Spectrometry
影响因子：
3.2
作者：
Beckman JS;Voinov VG;Hare M;Sturgeon D;Vasil'ev Y;Oppenheimer D;Shaw JB;Wu S;Glaskin R;Klein C;Schwarzer C;Stafford G
通讯作者：
Stafford G

Direct Determination of Antibody Chain Pairing by Top-down and Middle-down Mass Spectrometry Using Electron Capture Dissociation and Ultraviolet Photodissociation.

DOI：
10.1021/acs.analchem.9b03129
发表时间：
2020-01-07
期刊：
Analytical chemistry
影响因子：
7.4
作者：
Shaw JB;Liu W;Vasil Ev YV;Bracken CC;Malhan N;Guthals A;Beckman JS;Voinov VG
通讯作者：
Voinov VG

Top-Down Characterization of Denatured Proteins and Native Protein Complexes Using Electron Capture Dissociation Implemented within a Modified Ion Mobility-Mass Spectrometer.