Investigating cross‐linked peptides in bovine aortic lysyl oxidase (LB129)

Abstract

Lysyl oxidase (LOX) isolated from bovine aorta contains five disulfide bonds and the lysyl tyrosyl quinone (LTQ) cross‐link. In attempting to locate the inhibitor‐modified amino acid residues resulting from addition of various suicide inhibitors, which may be in cross‐linked regions of the protein, we have been optimizing procedures for mass spectrometric identification of cross‐linked peptides produced during proteolysis of the enzyme. To covert the reactive quinone group of the LTQ to an invariant structure, LOX was reacted with phenylhydrazine before proteolysis. Recombinant human and purified bovine lysyl oxidase were first subjected to various proteolyses and mass spectrometry acquisition methods to determine optimal experimental conditions. Proteolytic procedures must produce peptides of manageable size and with termini that can be at least partially sequenced; chymotrypsin proved to be more suitable than trypsin. Optimizing conditions significantly increased the sequence coverage of LOX from 60% to 95%. Targeted MS/MS was used to identify cross‐link containing peptides using calculated m/z values and by utilizing the software packages xQuest/xProphet (ETH Zürich) and pLink (CAS) to find the cross‐link containing peptides.Grant Funding Source: Supported in part by Nuclea Biotechnologies