Accurate Mass Analysis of Singly-Charged Intact Proteins

单电荷完整蛋白质的精确质量分析

• 批准号: 8132756
• 负责人: Peter T. A. Reilly
• 金额: $ 26.73万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8132756
• 关键词: Accurate; Mass; Analysis; Singly; Charged

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完整的蛋白质和百万分之一质量的复合物