A Combination of Cold Ion Spectroscopy and Ion Mobility for the Study of Complex Peptides and Small Proteins

Abstract

We present in this work experiments that push the limits of cold ion spectroscopy to the study of complex peptides and small proteins in the gas phase. Although the low temperature attainable in a cold ion trap greatly simplifies the electronic spectra of large molecules, conformational heterogeneity can still be a significant source of congestion, complicating spectroscopic analysis. To overcome this hurdle, we propose an in tandem combination of ion mobility spectrometry and photofragment spectroscopy. This permits for conformational separation prior to laser interrogation. Towards this end, we describe a proof-of-principle experiment where we combine high- Field Asymmetric waveform Ion Mobility Spectrometry (FAIMS) with electronic spectroscopy. We demonstrate that using FAIMS to separate gas-phase peptide conformers before injecting them into a cold ion trap allows one to decompose a dense spectrum into contributions from different conformational families. In the inverse sense, cold ion spectroscopy can be used as a conformation-specific detector for ion mobility, allowing one to separate an unresolved mobility peak into contributions from different conformational families. The doubly protonated peptide bradykinin serves as a good test case for the marriage of these two techniques as it exhibits a considerable degree of conformational heterogeneity that results in a highly congested electronic spectrum. Our results demonstrate the feasibility and advantages of directly coupling ion mobility with spectroscopy and provide a diagnostic of conformational isomerization of this peptide after being produced in the gas phase by electrospray. In a second series of studies we show the potential but also the limitations of our spectroscopic approach for investigating small, naturally occurring proteins, by applying it to ubiquitin, a protein of 76 amino acids. We present electronic photofragment spectra of various protonation states of ubiquitin and we compare our results to literature data acquired by ion mobility spectrometry. These experiments show that the information obtained by laser interrogation of molecules of this size is limited and could be enhanced by a combination with ion mobility techniques. Although some of the results obtained may be important in characterizing small proteins in the gas phase, these experiments are at an early stage and the application of photofragment spectroscopy on proteins needs more investigation.