Cross-linking/Mass Spectrometry Combined with Ion Mobility on a timsTOF Pro Instrument for Structural Proteomics

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.

Trapped Ion Mobility Spectrometry and Parallel Accumulation–Serial Fragmentation in Proteomics

“…TIMS and PASEF provide efficient access to large-scale CCS measurements, allowing to characterize the multi-dimensional data space in unprecedented detail 93 . As CCS values are determined by the amino acid sequence, it will be interesting to extend these studies to further peptide classes, for example cross-linked or modified peptides, in particular given the prospect J o u r n a l P r e -p r o o f of separating positional isomers in the gas phase 67,[112][113][114] . We believe that emerging deep learning techniques will propel this development to make full use of the additional information 115 .…”

Mimicked synthetic ribosomal protein complex for benchmarking crosslinking mass spectrometry workflows

“…Aiming to dig deeper in the interactome of complex samples, reagents bearing an affinity tag for selective enrichment of cross-linked peptides were further developed. [14][15][16] The optimization of cross-linker specific acquisition strategies 17 and most recently the implementation of ion-mobility 18,19 or FAIMS filtering 20 as additional separation technique further boosted the number of possible crosslink (XL) identifications. The broad spectrum of applications, linker types and acquisition strategies 4 led to the development of lots of specialized data analysis tools 21 which makes it challenging, especially for newcomers, to decide for an appropriate analysis workflow.…”

“…Aiming to dig deeper in the interactome of complex samples, reagents bearing an affinity tag for selective enrichment of cross-linked peptides were further developed. 14–16 The optimization of cross-linker specific acquisition strategies 17 and most recently the implementation of ion-mobility 18,19 or FAIMS filtering 20 as additional separation technique further boosted the number of possible crosslink (XL) identifications.…”