Practical Mass Spectrometry Upgrade for Identifying Fragile Protein Modifications by ECD

现代质谱仪的速度、分辨率和高质量准确度已经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中的一个主要挑战。 质谱用于磷酸化蛋白质组学的关键弱点在于用于诱导的方法 断裂，因为磷酰基键是蛋白质中最不稳定的化学键之一， 通过目前基于碰撞的碎片化方法以复杂的方式。另一种分裂 一种称为电子捕获解离（ECD）的方法已经建立，可以产生特殊的光谱， 磷酸肽，但目前仅在昂贵的FTICR质谱仪中可行。根本 对ECD的限制是提供足够的低能电子以有效地使肽片段化。我们有两 已发布专利，保护一项新技术，该技术使一种实用的ECD电池能够使用精心雕刻的磁性材料， 场来限制电子。ECD电池的长度仅为两厘米，并且可以容易地并入到电池中。 几乎所有的质谱仪限制我们的ECD细胞适应的主要因素是效率 限制为5-10%的双电荷磷酸肽。这引起了人们对低灵敏度的担忧。 丰度肽。然而，肽现在只在细胞中飞行一次。我们的第一阶段建议增加 通过反射离子使其多次通过ECD池来提高这种效率。为此，我们将 Orbitrap质谱仪已成为蛋白质组学中使用最广泛的仪器。 其独特的设计允许在不改变Orbitrap本身任何组件的情况下集成ECD单元。 在第一阶段要回答的可行性问题是：如何最好地纳入ECD细胞通过肽 和蛋白质通过细胞多次，以增加破碎效率？挑战在于避免 由于肽离子在反射时散射而失去灵敏度。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来验证用于量化翻译后修饰的细胞 蛋白质组学方法支持信包括从发现者的幼儿发展，发明者的 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 升级套件，用于目前运行的数千个轨道器。随着技术赢得认可， 公司将开发新一代的质谱仪利用额外的信息 由ECD提供。以适度的成本采用我们的技术将加速许多NIH 研究人员探索疾病机制以及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.