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

Barbara Steigenberger,Henk W.P Van Den Toorn,Emiel Bijl,Jean-François Greisch,Oliver Räther,Markus Lubeck,Roland J Pieters,Albert J.R Heck,Richard A Scheltema

doi:10.1074/mcp.ra120.002094

Barbara Steigenberger, Henk W.P Van Den Toorn + Show 7 more

Open Access

https://doi.org/10.1074/mcp.ra120.002094

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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 TOF 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 MS (XL-MS). Cross-linking reagents covalently link amino acids in 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.

Highlights

Development of a novel data acquisition routine that a-priori distinguishes cross-linked from monolinked peptides called caps-PASEF
Even though the study of protein structure is dominated by techniques like NMR, crystallography and cryo-EM, structural proteomics techniques driven by MS have an increasingly important, integrative role to uncover new details not achievable by the conventional techniques
Collisional Energy Optimization—As cross-linked peptides are different from unmodified peptides previously optimized settings potentially do not apply and we attempted to optimize the fragmentation conditions for the identification of cross-linked peptides [25, 26]

Summary

Introduction

Development of a novel data acquisition routine that a-priori distinguishes cross-linked from monolinked peptides called caps-PASEF. Cross-linking reagents covalently link amino acids in proximity, resulting in peptide pairs after proteolytic digestion. 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. A total of 5070% 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.

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Conclusion