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.
Sensitive, accurate, and resolved mass analysis of singly charged intact proteins and complexes is not currently feasible above roughly 20 kDa. There is also no easy method to comprehensively measure protein distributions and compare them. This project will prove that these goals are achievable.
|Chen, Huijuan; Lee, Jeonghoon; Reilly, Peter T A (2012) High-resolution ultra-high mass spectrometry: increasing the m/z range of protein analysis. Proteomics 12:3020-9|
|Wang, Xinyu; Chen, Huijuan; Lee, Jeonghoon et al. (2012) Increasing the Trapping Mass Range to m/z = 10(9)-A Major Step Toward High Resolution Mass Analysis of Intact RNA, DNA and Viruses. Int J Mass Spectrom 328-329:28-35|
|Lee, Jeonghoon; Chen, Huijuan; Liu, Tiancheng et al. (2011) High resolution time-of-flight mass analysis of the entire range of intact singly-charged proteins. Anal Chem 83:9406-12|
|Lee, Jeonghoon; Reilly, Peter T A (2011) Limitation of time-of-flight resolution in the ultra high mass range. Anal Chem 83:5831-3|
|Lee, Jeonghoon; Marino, Maxwell A; Koizumi, Hideya et al. (2011) Simulation of Duty Cycle-Based Trapping and Ejection of Massive Ions Using Linear Digital Quadrupoles: the Enabling Technology for High Resolution Time-of-Flight Mass Spectrometry in the Ultra High Mass Range. Int J Mass Spectrom 304:36-40|