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Accurate Mass Analysis of Singly-Charged Intact Proteins

单电荷完整蛋白质的精确质量分析

基本信息

批准号：
7924185
负责人：
Peter T. A. Reilly
金额：
$ 22.24万
依托单位：
WASHINGTON STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-01 至 2012-08-31
项目状态：
已结题

项目摘要

Our goal is analysis of singly-charged intact proteins and complexes with part-per-million mass accuracy up to one MDa. We have recently created an inlet and large-radius frequency-adjusted linear quadrupole ion trap that is capable of capturing large quantities (> 100 million ions) of massive (1-1000 kDa) singly-charged ions injected from the atmosphere into vacuum and holding them for on-demand injection into an awaiting mass analyzer. Trapping the ions before mass analysis removes the expansion-induced kinetic energy that causes the deterioration of mass accuracy, resolution and sensitivity as a function of increasing mass. Coupling our inlet and trapping system to an orthogonal time-of-flight mass spectrometer (with reflectron) will permit accurate mass analysis of intact proteins without loss of sensitivity and resolution at high mass. The following measures of performance will be determined for our instrument: 1. We will demonstrate that the sensitivity does not change significantly with increasing mass by measuring working curves (signal intensity versus concentration) for known proteins and peptides spanning the 1-200 kDa molecular weight range; 2. We will demonstrate part per million mass accuracy over the 20 to 1000 kDa range with know proteins, multimers of known proteins and immoglobulins; and 3. We will define the resolution over the entire range with known analytes. Resolution of 100,000 or better is expected for mass-to-charge ratios over 100 kDa. We will also design, implement and demonstrate a singly-charged ion source that may be coupled to chromatographic column output. We will then demonstrate that complex protein distributions can be measured by coupling our instrument to a chromatographic separation. Success in this endeavor will be defined by assigning accurate masses to the majority of expressed proteins in a bacterial lysate.

我们的目标是分析单电荷的完整蛋白质和百万分之一质量的复合物精度高达1 MDa。我们最近创造了一个入口和大半径的频率调整线性四极离子阱，能够捕获大量（> 1亿个离子）的大量（1-1000 kDa）单电荷离子从大气注入到真空中并保持它们用于按需注入到等待的质量分析仪中。在质量之前捕获离子分析消除了导致质量恶化的膨胀引起的动能准确度、分辨率和灵敏度随质量增加而变化。连接我们的入口和捕获系统与正交飞行时间质谱仪（带反射器）的连接将允许完整蛋白质的精确质量分析，而不会损失高质量下的灵敏度和分辨率。将为我们的仪器确定以下性能指标：1.我们将通过测量表明灵敏度不会随着质量的增加而显著变化，已知蛋白质和肽的工作曲线（信号强度与浓度） 1-200 kDa分子量范围; 2.我们将证明百万分之一的质量精度在20至1000 kDa范围内与已知蛋白质、已知蛋白质的多聚体和免疫球蛋白;和3.我们将使用已知分析物定义整个范围内的分辨率。对于超过100 kDa的质荷比，预计分辨率为100，000或更好。我们还将设计、实施和演示一个单电荷离子源，色谱柱输出。然后，我们将证明，复杂的蛋白质分布可以通过将我们的仪器连接到色谱分离来测量。胜任这奋进通过将精确的质量分配给大多数表达的蛋白质来定义，细菌裂解物。