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

Abstract

Ion mobility separates molecules in the gas-phase based 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 500 Å2 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.

Deep learning the collisional cross sections of the peptide universe from a million experimental values

“…This highlights how our model, indeed, learned underlying principles. These could readily be extended to other peptide classes, such as modified 68 or cross-linked 69 peptides, using transfer learning 70 , with little additional experimental effort.…”

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

“…One goal of this study was to define general CCS characteristics of all cross-linked species that will later allow applying general selection procedures to separate cross-linked from non-cross-linked species. In the caps-PASEF approach described recently 19 , only intra- and interpeptide cross-links were selected for fragmentation, while “dead-end” cross-links were excluded. A specific region (polygon) of CCS values was defined in the distributions of monoisotopic mass versus CCS, in which the ions mainly correspond to cross-linked peptides that were then fragmented.…”

“…The timsTOF Pro mass spectrometer has been employed recently in a XL-MS study by the groups of Heck and Scheltema using the enrichable PhoX cross-linker 19 . In that study, an immobilized metal ion chromatography (IMAC) enrichment of cross-linker-containing peptides was performed before the MS analysis.…”

“…An optimized data analysis workflow for timsTOF Pro data using the software tools MSFragger and IonQuant has been reported, improving protein identification, sensitivity, quantification accuracy, and runtimes in proteomics experiments 18 . The timsTOF Pro mass spectrometer has been employed recently in a XL-MS study by the groups of Heck and Scheltema using the enrichable PhoX cross-linker 19 . In that study, an immobilized metal ion chromatography (IMAC) enrichment of cross-linker-containing peptides was performed before the MS analysis.…”

“…The various crossed species generated in a cross-linking experiment comprise the following three types: (i) intrapeptide cross-links (amino acids within one peptide are cross-linked), (ii) interpeptide cross-links (amino acids between two peptides are cross-linked), and (iii) peptides that are modified by a partially hydrolyzed cross-linker, which are commonly referred to as “dead-end” cross-links or “mono-links”. The major aim of that previous study 19 was to separate inter- and intrapeptide cross-links on one side from “dead-end” products on the other side as the latter ones do not yield direct distance information on interaction sites in proteins. The selection of cross-linked peptides was performed based on their characteristic CCS by applying a strategy termed “collisional cross-section assisted precursor selection (caps-PASEF)”.…”