Abstract
Ultraviolet photodissociation (UVPD) of gas-phase proteins has attracted increased attention in recent years. This growing interest is largely based on the fact that, in contrast to slow heating techniques such as collision induced dissociation (CID), the cleavage propensity after absorption of UV light is distributed over the entire protein sequence, which can lead to a very high sequence coverage as required in typical top-down proteomics applications. However, in the gas phase, proteins can adopt a multitude of distinct and sometimes coexisting conformations, and it is not clear how this three-dimensional structure affects the UVPD fragmentation behavior. Using ion mobility-UVPD-mass spectrometry in conjunction with molecular dynamics simulations, we provide the first experimental evidence that UVPD is sensitive to the higher order structure of gas-phase proteins. Distinct UVPD spectra were obtained for different extended conformations of 11(+) ubiquitin ions. Assignment of the fragments showed that the majority of differences arise from cis/trans isomerization of one particular proline peptide bond. Seen from a broader perspective, these data highlight the potential of UVPD to be used for the structural analysis of proteins in the gas phase.
Highlights
Understanding the structure and dynamics of proteins is one of the greatest challenges in the life sciences
Ultraviolet photodissociation (UVPD) of gasphase proteins has attracted increased attention in recent years. This growing interest is largely based on the fact that, in contrast to slow heating techniques such as collision induced dissociation (CID), the cleavage propensity after absorption of UV light is distributed over the entire protein sequence, which can lead to a very high sequence coverage as required in typical top-down proteomics applications
Using ion mobility−UVPD−mass spectrometry in conjunction with molecular dynamics simulations, we provide the first experimental evidence that UVPD is sensitive to the higher order structure of gas-phase proteins
Summary
Understanding the structure and dynamics of proteins is one of the greatest challenges in the life sciences. Techniques to study proteins in the absence of solvent, that is, in the gas phase, have emerged as promising additions that provide complementary information. Most of these methods rely on gentle ionization techniques such as electrospray ionization (ESI) followed by mass spectrometry (MS), and together with methods to isolate, energize, and fragment gas-phase molecules, MS is today the work-horse technique in “omics” research.[1,2]. MS by itself provides only indirect information about higher order structure such as the hydrogen bonding network and the folding of the molecule To investigate such structural aspects, MS can be combined with optical spectroscopy or ion mobility spectrometry (IMS) methods. IMS methods on the other hand can yield complementary information on the overall shape of the molecule in the form of the absolute collision cross section (CCS).[4−6] Further, IMS allows one to separate species of the same mass-to-charge (m/z) ratio that exhibit multiple conformations
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