Boronic acid: A versatile handle for the enrichment of cross-linked peptides

Abstract

Virtually all cellular processes in living organisms rely on protein machinery of complex three-dimensional structures and protein-protein interactions (PPIs). Therefore, significant effort has been devoted for elucidating protein structures and mapping PPI networks to understand relevant biological processes. Cross-linking mass spectrometry (XL-MS) is a robust technique that can provide conformational restraints within proteins and protein complexes for modelling protein structures and analysing PPI networks on a whole proteome scale both in vitro and in vivo. In XL-MS, protein samples are chemically cross-linked, digested by proteases, and analysed by LC-MS/MS to identify cross-linked peptides. However, one challenge associated with XL-MS is the sample complexity. Because of the low abundance of cross-linked peptides in the mixture, it is challenging to identify cross-linked peptides. The aim of this thesis is to develop a robust affinity purification (AP) protocol to enrich cross-linked peptides prior to LC-MS/MS analysis. Boronic acids (BAs) can form reversible covalent bonds with diols and they are highly versatile as building blocks of many useful chemical transformations. In addition, they have low toxicity as several BA containing drugs have been approved by FDA. Therefore, we investigated the use of boronic acid as a handle for enriching cross-linked peptides. We have synthesized two BA-containing cross-linkers and a diol-containing resin for AP. Using model proteins, protein complexes and cell lysates, we found that BA-based enrichment is highly sensitive and specific. The BA cross-linkers can also be used for live-cell cross-linking due to its cell permeability. Therefore, we believe this new enrichment handle will further expand the power of XL-MS in the analysis of complex samples.