TR&D 3

Hybrid SID, IM, & UVPD Methods for Complex-Down MS of Protein Complexes — Lead: Sophie Harvey

Improved SID devices coupled with different IM devices in different platforms to enable SID-IM and/or IM-SID. This makes available information on the complex interfaces (from SID) and conformations (from IM) in a single experiment. In addition, the coupling of SID and UVPD will allow us to directly dissociate the intact complex first to folded subcomplexes by SID and then further dissociate by UVPD the subcomplexes and monomers released in the first activation, providing information such as proteoform sequence. CID/UVPD will be performed when fragmentation of unfolded complexes is desired.

Aim 1

Couple Q Exactive Orbitrap SID with IM or UVPD
  • Couple higher energy SID to high-resolution IM in (Q) Exactive EMR Orbitrap and SID with UVPD in the Q Exactive UHMR for proteoform characterization

Aim 2

SID on prototype Q-cyclic IM-TOF; SID-IM-UVPD
  • Test linear IM and Waters prototype cyclic multi-pass IM
  • Add SID
  • Combine SID with IM and UVPD for subcomplex formation (SID), IM selection, and covalent dissociation of subcomplexes (UVPD)

Aim 3

TIMS-SID and TIMS-SID-UVPD in 15T FT-ICR
  • Test Bruker TIMS on the ICR
  • Couple TIMS with SID on the ICR for SID interrogation of shape separated and selected species
  • Combine TIMS-SID- UVPD for proteoform characterization