Practical Mass Spectrometry Upgrade for Identifying Fragile Protein Modifications by ECD

现代质谱仪的速度、分辨率和高质量精度使蛋白质组学发生了革命性的变化， 尤其是用于确定控制大多数细胞过程的脆弱的翻译后修饰。 准确鉴定和定量磷酸化位点仍然是蛋白质组学中的一个主要挑战。 对于磷酸蛋白质组学来说，质谱学的主要弱点在于用来诱导 碎裂，因为磷酸键是蛋白质中最不稳定的化学键之一，会丢失 通过当前基于冲突的分段方法以复杂的方式。另一种分割方式 被称为电子俘获解离(ECD)的方法被广泛应用于产生特殊的光谱 目前，只有在昂贵的FTICR质谱仪中才能实现。最基本的 ECD的局限性在于提供了足够的低能电子来有效地碎裂多肽。我们有两个 颁发的专利保护了一项新技术，使实用的ECD单元能够使用精心雕刻的磁性 限制电子的电场。ECD电池只有两厘米长，并且可以很容易地结合到 几乎所有的质谱仪。限制我们ECD电池适应的主要因素是效率是 对于双电荷的磷酸肽，限制在5-10%。这引发了人们对Low失去敏感度的担忧 丰富的多肽。然而，多肽现在只在细胞中飞行一次。我们的第一阶段建议增加 这种效率是通过反射离子使其多次通过ECD电池来实现的。为此，我们将 重点关注Orbitrap质谱仪，它已经成为蛋白质组学最广泛使用的工具。 其独特的设计允许集成ECD单元，而无需更改Orbitrap本身的任何组件。 第一阶段需要回答的可行性问题是：如何最好地结合ECD细胞来传递多肽 和蛋白质多次通过细胞来提高碎裂效率？挑战是要避免 由于多肽离子在反射时的散射而失去灵敏度。在第二阶段，我们将与早期- 采用者通过自上而下和自下而上两种方式验证单元以量化翻译后修改 蛋白质组学方法。其中包括ECD的发现者、ECD的发明者 Orbitrap和两位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 为目前正在运行的数以千计的轨道诱捕器升级套件。随着这项技术获得认可，我们的 该公司将利用这些额外信息开发新一代质谱仪 由ECD提供。以适中的成本采用我们的技术将加速许多NIH的能力 研究人员将探索疾病机制并确定诊断和治疗生物标志物 更高的准确性、更快的速度和更少的错误。

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.