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Mobility-assisted psuedo-MS3 sequencing of protein ions

Cite this dataset

Graham, Katherine; Grisolia, Vincent; Borotto, Nicholas (2024). Mobility-assisted psuedo-MS3 sequencing of protein ions [Dataset]. Dryad.


The sequencing of intact proteins within a mass spectrometer has many benefits but is frequently limited by the fact that tandem mass spectrometry (MS/MS) techniques often generate poor sequence coverages when applied to protein ions. To overcome this limitation exotic MS/MS techniques that rely on lasers and radical chemistry have been developed. These techniques generate high sequence coverages, but they require specialized instrumentation, create products through multiple dissociation mechanisms, and often require long acquisition times. Recently, we demonstrated that protein ions can be dissociated in a trapped ion mobility spectrometry (TIMS) device prior to mobility separation in a commercial timsTOF. All generated product ions were distributed throughout the mobility dimension and this separation enabled deconvolution of complex tandem mass spectra and could enable facile pseudo-MS3 interrogation of generated product ions with the downstream quadrupole and collision cell. A second activation step improves sequence coverage because the most labile bonds have been depleted during the first dissociation and subsequent dissociation events are more evenly distributed throughout the product ion backbone. In this work, we explore the potential of this mobility-assisted pseudo-MS3 (MAP) method on a commercial timsTOF and timsTOF Pro 2. We demonstrate that while MAP only generates 92% of the sequence coverage of the most effective MS/MS technique, it accomplished this feat in 1.5 mins and could be facilely integrated with liquid chromatographic separations.

README: Mobility-Assisted Psuedo-MS3 Sequencing of Protein Ions

In this deposit are the raw Bruker datafiles that were used to generate all figures found within the manuscript with the same title. The protein analyzed and the instrument utilized is explicitly indicated in the title of each data file. We recommend utilizing the software Bruker DataAnalysis to view both mass and mobility spectra, however, any software package capable of analyzing timsTOF data can be employed. The methods section in the accompanying manuscript describe in detail how the instrumental parameters are altered over the time course of the measurement. 


Compressed Bruker data files with all mass spectra.


National Institute of General Medical Sciences, Award: R35GM151104