Structure-elucidation of human CCL5 by integrating trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) with Structure Relaxation Approximation (SRA) analysis

Abstract

Ion mobility spectrometry-mass spectrometry offers the potential to characterize structures of transient protein assemblies and protein isoforms by means of their orientationally-averaged momentum transfer cross-sections. A commonly observed phenomenon is the compaction of a protein in the ion mobility measurement, that is, the cross section measured for the protein by ion mobility spectrometry is smaller than the cross section expected for its native structure. Consequently, this compaction means that at least some structural changes of the protein must have occurred during the ion mobility measurement. A major challenge is then to identify which aspects of the solution structure are retained in the ion mobility measurement and which ones are not. Here, we apply our recently developed Structure Relaxation Approximation (SRA) method in conjunction with trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) to probe compaction of the human protein chemokine (C-C motif) ligand 5 (also CCL5). Ion mobility spectra are recorded for various charge states and solution conditions of CCL5 under both “soft” and collisionally-activated conditions. Our data show that the SRA reproduces the overall trends in the experimental spectra: (1) the compaction of the CCL5 structure as seen in the experiments; (2) the general increase in the cross section for the various charge states; and (3) the increase in cross section after collisional-activation. The SRA attributes the compaction of the CCL5 structure mainly to the folding of the unstructured N-terminus onto the central Greek key motif of CCL5. By contrast, the SRA indicates that native residue-residue contacts present in the NMR structure are largely retained. Additionally, our analysis indicates that accurate treatment of proton transfer processes during the structural relaxation process would significantly improve the structural interpretation of ion mobility data by the SRA.