Abstract
The combination of cross-linking/mass spectrometry (XL-MS) and ion mobility is still underexplored for conducting protein conformational and protein-protein interaction studies. We present a method for analyzing cross-linking mixtures on a timsTOF Pro mass spectrometer that allows separating ions based on their gas phase mobilities. Cross-linking was performed with three urea-based MS-cleavable cross-linkers that deliver distinct fragmentation patterns for cross-linked species upon collisional activation. The discrimination of cross-linked species from non-cross-linked peptides was readily performed based on their collisional cross sections. We demonstrate the general feasibility of our combined XL-MS/ion mobility approach for three protein systems of increasing complexity: (i) Bovine serum albumin, (ii) E. coli ribosome, and (iii) HEK293T cell nuclear lysates. We identified a total of 508 unique cross-linking sites for BSA, 868 for the E. coli ribosome, and 1,623 unique cross-links for nuclear lysates, corresponding to 1,088 intra- and 535 interprotein interactions and yielding 564 distinct protein-protein interactions. Our results underline the strength of combining XL-MS with ion mobility not only for deriving 3D-structures of single proteins, but also for performing system-wide protein interaction studies.
Competing Interest Statement
The authors have declared no competing interest.
Abbreviations
- ACN
- Acetonitrile
- BSA
- Bovine serum albumin
- Caps
- Collisional cross-section assisted precursor selection
- CCS
- Collisional cross section
- CSM
- Cross-link spectral match
- DSAU
- Disuccinimidyl diacetic urea
- DSBU
- Disuccinimidyl dibutyric urea
- DSPU
- Disuccinimidyl dipropionic urea
- DTT
- Dithiothreitol
- EM
- Electron microscopy
- FDR
- False discovery rate
- HEPES
- 2-[4-(2-Hydroxyethyl)-piperazine-1-yl]ethanesulfonic acid
- IM
- Ion mobility
- IMAC
- Immobilized metal ion chromatography
- LC
- Liquid chromatography
- MS
- Mass spectrometry
- MS/MS
- Tandem mass spectrometry
- NHS
- N-Hydroxysuccinimide
- PASEF
- Parallel accumulation serial fragmentation
- PBS
- Phosphate buffered saline
- RT
- Room temperature
- SAXS
- Small-angle X-ray scattering
- SDS
- Sodium dodecyl sulfate
- SEC
- Size exclusion chromatography
- TIMS
- Trapped ion mobility spectrometry
- TOF
- Time-of-flight
- XL
- Cross-linking
- XL-MS
- Cross-linking/mass spectrometry