Activated Ion Electron Capture Dissociation (AI ECD) of proteins: synchronization of infrared and electron irradiation with ion magnetron motion.
Here, we show that to perform activated ion electron capture dissociation (AI-ECD) in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer equipped with a CO(2) laser, it is necessary to synchronize both infrared irradiation and electron capture dissociation with ion magnetron motion. This requirement is essential for instruments in which the infrared laser is angled off-axis, such as the Thermo Finnigan LTQ FT. Generally, the electron irradiation time required for proteins is much shorter (ms) than that required for peptides (tens of ms), and the modulation of ECD, AI ECD, and infrared multiphoton dissociation (IRMPD) with ion magnetron motion is more pronounced. We have optimized AI ECD for ubiquitin, cytochrome c, and myoglobin; however the results can be extended to other proteins. We demonstrate that pre-ECD and post-ECD activation are physically different and display different kinetics. We also demonstrate how, by use of appropriate AI ECD time sequences and normalization, the kinetics of protein gas-phase refolding can be deconvoluted from the diffusion of the ion cloud and measured on the time scale longer than the period of ion magnetron motion.
Mikhailov
Victor A
VA
School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.
Cooper
Helen J
HJ
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D045304:Cytochromes c / Q000737:chemistry; D055590:Electromagnetic Phenomena; D004867:Equipment Design; D005583:Fourier Analysis; D006736:Horses; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D013997:Time Factors; D025801:Ubiquitin / Q000737:chemistry
D009211:Myoglobin; D010455:Peptides; D011506:Proteins; D025801:Ubiquitin; D045304:Cytochromes c