Mobility-assisted psuedo-MS3 sequencing of protein ions

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.