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

 
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Award ID(s):
1654608
NSF-PAR ID:
10367514
Author(s) / Creator(s):
 ;  ;  ;  ;  ;  ;  ;  ;  
Publisher / Repository:
Wiley Blackwell (John Wiley & Sons)
Date Published:
Journal Name:
Rapid Communications in Mass Spectrometry
Volume:
35
Issue:
22
ISSN:
0951-4198
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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