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

Abstract

Rationale Tandem‐ion mobility spectrometry/mass spectrometry methods have recently gained traction for the structural characterization of proteins and protein complexes. However, ion activation techniques currently coupled with tandem‐ion mobility spectrometry/mass spectrometry methods are limited in their ability to characterize structures of proteins and protein complexes. Methods Here, we describe the coupling of the separation capabilities of tandem‐trapped ion mobility spectrometry/mass spectrometry (tTIMS/MS) with the dissociation capabilities of ultraviolet photodissociation (UVPD) for protein structure analysis. Results We establish the feasibility of dissociating intact proteins by UV irradiation at 213 nm between the two TIMS devices in tTIMS/MS and at pressure conditions compatible with ion mobility spectrometry (2–3 mbar). We validate that the fragments produced by UVPD under these conditions result from a radical‐based mechanism in accordance with prior literature on UVPD. The data suggest stabilization of fragment ions produced from UVPD by collisional cooling due to the elevated pressures used here (“UVnoD2”), which otherwise do not survive to detection. The data account for a sequence coverage for the protein ubiquitin comparable to recent reports, demonstrating the analytical utility of our instrument in mobility‐separating fragment ions produced from UVPD. Conclusions The data demonstrate that UVPD carried out at elevated pressures of 2–3 mbar yields extensive fragment ions rich in information about the protein and that their exhaustive analysis requires IMS separation post‐UVPD. Therefore, because UVPD and tTIMS/MS each have been shown to be valuable techniques on their own merit in proteomics, our contribution here underscores the potential of combining tTIMS/MS with UVPD for structural proteomics.

Tandem-trapped ion mobility spectrometry/mass spectrometry (tTIMS/MS): a promising analytical method for investigating heterogenous samples

“…1,3 Top-down protein analysis in tTIMS/MS is currently supported by collision-induced dissociation (CID) 1,3 and UV photodissociation (UVPD) conducted in-between the TIMS-1 and TIMS-2 devices at 2-3 mbar of nitrogen gas (discussed below). 6…”

“…1,2,4,52,[94][95][96][97] Nevertheless, also the commercial version in parallel accumulation mode enables native MS applications as described. 5,6 Fig. 1B and C further show schematics of the two tTIMS/MS instruments which are the focus of this review.…”

“…The second, more recent tTIMS/MS was constructed from a commercial timsTOF Pro instrument (Bruker Daltonics, Billerica, MA). 5,6 This setup couples two commercial TIMS devices with 96 mm tunnels in an orthogonal manner ("orthogonal tTIMS/ MS", Fig. 1C).…”

“…To this end, we showcase the various operational modes tTIMS/ MS offers to the analyst and discuss case studies ranging from peptide assemblies 2 to native protein systems [3][4][5] and top-down analysis of intact protein systems. 1,3,6 We exclude a detailed discussion of ion mobility spectrometry (IMS) and trapped ion mobility spectrometry (TIMS), on which several excellent reviews are available. [7][8][9][10][11][12] The single exception we make here is that of ion heating in tTIMS: the topic of ion heating is of such paramount significance to any ion mobility study that our review would not be complete without its discussion.…”

“…3,4 More recently, to improve sensitivity and sequence coverage for top-down analysis of larger protein systems, we constructed an orthogonal tTIMS/MS instrument based on a commercial timsTOF Pro instrument (Bruker Daltonics, MA) 5 and coupled it with UV photodissociation. 6 This orthogonal tTIMS/MS instrument was designed to enable native complex-top-down studies using automated TIMS 2 -MS 2 workflows by performing parallel-accumulation serial fragmentation (PASEF) on fragment ions generated from UVPD. 63 We realize that an instrumental method enabling multiple ion activation, IMS separation, and ion selection steps prior to Melvin A.…”