Comment on Effective Temperature and Structural Rearrangement in Trapped Ion Mobility Spectrometry

Abstract

Metrics & MoreArticle Recommendations I on mobility spectrometry-mass spectrometry offers the potential to reveal structures of biological molecules not amenable to established biophysical methods. It is, however, still controversial if ion mobility truly reveals biologically relevant structures. This is because the measurement takes place in the gas phase, but it is not known for how long the native structure survives in this environment. 1 Ion mobility spectrometry is thus a nonequilibrium method 2 and successful only to the extent that native structures are metastable within the measurement time scale. 3,4 Ergo, the critical question is how much "heat" do ions take up from electric fields in an ion mobility spectrometer?In ref 5, Morsa et al. investigated ion heating in a trapped ion mobility spectrometer (TIMS). The authors claim that "Using the lowest possible transmission voltages [...the authors] obtained vibrational effective temperatures T eff,Vib of 512 K" for a thermometer ion which accounts for a "high [axial electric field] E/N above the low-field limit", from which the authors conclude that "the ion transport regimes in traditional [drift-tube ion mobility spectrometry] and modern [...] TIMS instrumentations differ therefore advising caution when comparing data obtained on the different platforms."If true, these claims made in ref 5 would have far-reaching, adverse implications on the use of TIMS for structural studies: if the ion separation process in TIMS truly heated molecules to ∼500 K, then ion mobilities would be unreliable and biological molecules would structurally denature in TIMS.Indeed, ref 5 expresses such concerns by "questioning the adequacy of the strategy [of keeping the ions stationary employed in TIMS to improve the mobility resolving power] for native mass spectrometry applications targeting fragile analytes associated with low activation energy barrier E a such as small molecules or noncovalent complexes."These claims, however, stand in stark contrast to experiences made by us and others that ion mobilities obtained from TIMS measurements are accurate 6,7 and, when carefully tuned, spectra from TIMS and tandem-TIMS resemble "soft" drifttube spectra for fragile noncovalent peptide assemblies, 8 monomeric proteins, 2,9−11 and even indicate solvent to be trapped inside protein assemblies. 12 Here, we clarify (a) that the ion heating to ∼500 K reported in ref 5 is not intrinsic to TIMS and (b) that TIMS enables native mass spectrometry applications:1. When discussing the low-field limit and vibrational ion heating in ion mobility spectrometry, all forces that

Trapped Ion Mobility Spectrometry and Parallel Accumulation–Serial Fragmentation in Proteomics

“…In the TIMS tunnel, operating at a high rf amplitude or overfilling the TIMS analyzer with too many ions can lead to heating J o u r n a l P r e -p r o o f effects 47,58 . However, when operated properlykeeping potentials low and not overfilling -TIMS can be a powerful tool for a broad range of native applications 59,60 . Further opportunities may arise from the combination of ion fragmentation in or before TIMS 61,62 , followed by a MS 3 analysis of the mobility-separated fragment ions with PASEF.…”

Surface-Induced Dissociation of Protein Complexes Selected by Trapped Ion Mobility Spectrometry

“…41 This raises concern when performing nMS experiments, in which maintaining the native-like structure and avoiding unintentional activation is of critical importance. 21,43,44 It has since been clarified that that ion heating likely occurs due to non-intrinsic properties of TIMS (space charge effects and RF power absorption) and that careful tuning prevents structural rearrangement, therefore making TIMS a suitable technology for nMS. 44 For the experiments performed in this manuscript, the amplitude of the RF-confining voltage in the TIMS funnel was set as low as possible, while still trapping a sufficient amount of ions, and trapping times in the TIMS analyzer were kept low to limit structural perturbation.…”

“…21,43,44 It has since been clarified that that ion heating likely occurs due to non-intrinsic properties of TIMS (space charge effects and RF power absorption) and that careful tuning prevents structural rearrangement, therefore making TIMS a suitable technology for nMS. 44 For the experiments performed in this manuscript, the amplitude of the RF-confining voltage in the TIMS funnel was set as low as possible, while still trapping a sufficient amount of ions, and trapping times in the TIMS analyzer were kept low to limit structural perturbation. 21 To confirm the results from Bleiholder and coworkers, TIMS-SID was performed using cholera toxin B (CTB), a 58 kDa pentamer, with bound GM1s ligands and compared to TIMS-CID of the same complex (TIMS parameters were identical for both TIMS-SID and TIMS-CID experiments).…”

Tandem‐trapped ion mobility spectrometry/mass spectrometry coupled with ultraviolet photodissociation

“…Further, we validate the feasibility of dissociating intact proteins at 2–3 mbar by irradiation with 213 nm UV light produced from an Nd:YAG laser. The construction of our novel, UVPD‐capable tandem‐TIMS (tTIMS/MS‐UVPD) instrument was based on experiences with our prior tandem‐TIMS instrument obtained through its application to the study of peptide oligomers and native‐like structures of proteins and their complexes 39–43 . The tTIMS/MS‐UVPD instrument described here combines the benefit of conformer selection of the precursor ion before UVPD with mobility separation of the fragment ions produced by UVPD.…”