Abstract
We have investigated the photoionization and photofragmentation yields of gas-phase multiply protonated melittin cations for photon energies at the K-shell absorption edges of carbon, nitrogen, and oxygen. Two similar experimental approaches were employed. In both experiments, mass selected [melittin+qH]q+ (q=2–4) ions were accumulated in radiofrequency ion traps. The trap content was exposed to intense beams of monochromatic soft X-ray photons from synchrotron beamlines and photoproducts were analyzed by means of time-of-flight mass spectrometry. Mass spectra were recorded for fixed photon energies, and partial ion yield spectra were recorded as a function of photon energy. The combination of mass spectrometry and soft X-ray spectroscopy allows for a direct correlation of protein electronic structure with various photoionization channels. Non-dissociative single and double ionization are used as a reference. The contribution of both channels to various backbone scission channels is quantified and related to activation energies and protonation sites. Soft X-ray absorption mass spectrometry combines fast energy deposition with single and double ionization and could complement established activation techniques.Graphical ᅟ
Highlights
Tandem mass spectrometry is one of the essential techniques for biomolecular structure determination by means of analysis of the molecular fragmentation pattern
In a previous work on soft Xray spectroscopy of cryogenic (T ~ 10 K) multiply protonated melittin at the C K-edge, we have recently found a correlation between melittin structure and electron impact ionization from within [21], i.e., inelastic scattering of photoelectrons along their trajectories through the photoabsorbing molecule
From a soft X-ray spectroscopy point of view, it is most straightforward to look into the data for non-dissociative single and double photoionization, i.e., the partial ion yields of [melittin+qH](q+1)+ and [melittin+qH](q+2)+ as a function of photon energy
Summary
Tandem mass spectrometry is one of the essential techniques for biomolecular structure determination by means of analysis of the molecular fragmentation pattern. We will further investigate this effect for protonated melittin close to room temperature and at all three relevant K-edges of carbon, nitrogen, and oxygen It is the goal of the article to investigate soft X-rayinduced protein fragmentation in greater detail in order to assess the potential of the technique for analytical mass spectrometry. Typical photoexposure times of several 100 ms lead to photoabsorption in 5–10% of the trapped protonated proteins This way, more than 90% of the photoproduct ions are due to single-photon absorption events. Key features of the experiments are the large trap capacity, allowing for efficient recording of soft X-ray absorption spectra and the presence of a buffer gas, quenching slow fragmentation channels. After phase space compression in the RF ion funnel, ion currents typically exceed 1 nA and more than 100 pA of mass-selected [melittin+3H]3+ ions can be generated
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