Benefits of Collisional Cross Section Assisted Precursor Selection (caps-PASEF) for Cross-linking Mass Spectrometry

Abstract

Ion mobility separates molecules in the gas-phase on their physico-chemical properties, providing information about their size as collisional cross-sections. The timsTOF Pro combines trapped ion mobility with a quadrupole, collision cell and a time-of-flight mass analyzer, to probe ions at high speeds with on-the-fly fragmentation. Here, we show that on this platform ion mobility is beneficial for cross-linking mass spectrometry (XL-MS). Cross-linking reagents covalently link amino acids in close proximity, resulting in peptide pairs after proteolytic digestion. These cross-linked peptides are typically present at low abundance in the background of normal peptides, which can partially be resolved by using enrichable cross-linking reagents. Even with a very efficient enrichable cross-linking reagent, like PhoX, the analysis of cross-linked peptides is still hampered by the co-enrichment of peptides connected to a partially hydrolyzed reagent -termed mono-linked peptides. For experiments aiming to uncover protein-protein interactions these are unwanted byproducts. Here, we demonstrate that gas-phase separation by ion mobility enables the separation of mono-linked peptides from cross-linked peptide pairs. A clear partition between these two classes is observed at a CCS of 500Å 2 and a monoisotopic mass of 2 kDa, which can be used for targeted precursor selection. A total of 50 -70% of the mono-linked peptides are prevented from sequencing, allowing the analysis to focus on sequencing the relevant cross-linked peptide pairs. In applications to both simple proteins and protein mixtures and a complete highly complex lysate this approach provides a substantial increase in detected cross-linked peptides.

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 .…”

“…This maximizes the benefit of the PASEF principle and allows, for example, to exclude singly-charged species by their relative ion mobility. Steigenberger et al 67 extended this logic to include precursors of interest based on their collisional cross section in a method termed caps-PASEF. The authors made use of the shifted distribution of mono-and cross-linked peptides in the mass vs. CCS space to bias their acquisition towards the more informative crosslinked peptides.…”

Deep learning the collisional cross sections of the peptide universe from a million training samples

“…This highlights how our model indeed learned underlying principles. These could readily be extended to other peptide classes, such as modified or cross-linked 56 peptides, using transfer learning 57 , with little additional experimental effort.…”

Evaluation of Acquisition Modes for the Quantitative Analysis of Cross-Linked Peptides by Targeted and Untargeted Mass Spectrometry

“…These observations are consistent with previous observations of IM adding an extra dimension of separation, and the ability of IM to improve analysis of cross-linked peptides. 25,32 It is also hypothesized that the packaging of a continuous ion beam by ion mobility, problematic at high concentrations, may have a positive effect at the low concentrations of cross-linked peptides by enhancing detectability.…”

“…[20][21][22][23] However, despite its reported improvements in sensitivity and signal to noise, IM-MS has been only been exploited for the study of crosslinked peptides in a handful of cases to date. 24,25 By reviewing these acquisition modes for quantitative XL-MS, it is hoped that the field of cross-linking can be expanded for the comparative analysis of an increased number and variety of biological samples.…”