Advancing Top-down Analysis of the Human Proteome Using a Benchtop Quadrupole-Orbitrap Mass Spectrometer

作者：Fornelli Luca; Durbin Kenneth R; Fellers Ryan T; Early Bryan P; Greer Joseph B; LeDuc Richard D; Compton Philip D; Kelleher Neil L

来源：Journal of Proteome Research, 2017, 16(2): 609-618.

DOI：10.1021/acs.jproteome.6b00698

• top-down proteomics
• gas-phase fractionation
• Orbitrap
• quadrupole
• data-dependent acquisition
• false-discovery rate
• mass spectrometry
• proteoform
• AUTOPILOT
• medium/high

Over the past decade, developments in high resolution mass spectrometry have enabled the high throughput analysis of intact proteins from complex proteomes, leading to the identification of thousands of proteoforms. Several previous reports on top-down proteomics (TDP) relied on hybrid ion trap Fourier transform mass spectrometers combined with data-dependent acquisition strategies. To further reduce TDP to practice, we use a quadrupole-Orbitrap instrument coupled with software for proteoform-dependent data acquisition to identify and characterize nearly 2000 proteoforms at a 1% false discovery rate from human fibroblasts. By combining a 3 m/z isolation window with short transients to improve specificity and signal-to-noise for proteoforms >30 kDa, we demonstrate improving proteome coverage by capturing 439 proteoforms in the 30-60 kDa range. Three different data acquisition strategies were compared and resulted in the identification of many proteoforms not observed in replicate data-dependent experiments. Notably, the data set is reported with updated metrics and tools including a new viewer and assignment of permanent proteoform record identifiers for inclusion of highly characterized proteoforms (i.e., those with C-scores >40) in a repository curated by the Consortium for Top-Down Proteomics.

• 出版日期2017-2
• 单位西北大学