Developing novel workflows for improved bottom-up, top-down and middle-down mass spectrometry analysis of proteins

Abstract

Mass spectrometry (MS) has been extensively used to analyse biological samples and has evolved into an essential tool for proteomics research. Recent tremendous technological advancements, including improving instrument accuracy, resolution and sensitivity, and more freely accessible protein databases, have led to the growing importance of this technique. This study contributes to this growing area of research by developing novel orthogonal workflows that push the boundary of mass spectrometry capabilities, and allowed greater insight into the protein environment. Specifically, this research describes new methodology for de novo characterisation of disulfide bonding connectivity and de novo protein sequencing.

Investigating protein 3D structure can provide essential information when addressing the issues in protein folding and function. One of the important structural features in proteins is the disulfide links between cysteine residue pairs, which play crucial roles in sustaining protein 3D structure. Although some conventional experimental techniques can provide information of the disulfide patterns within a specific proteoform, there has been little quantitative analysis due to cost and time limitations. Thus, there is a necessity for new approaches that increase confidence in disulfide mapping and maximise the protein sequence coverage obtained. Here, this study draws attention to the ability of a combination of pepsin and trypsin proteolysis and the usage of dual fragmentation of electron capture dissociation (ECD) and collision induced dissociation (CID) to assigning disulfide connectivity of proteins and maximise sequence coverage. This protease approach is based on the accurate mass measurement of proteins and high-resolution top-down fragmentation MS studies. Here we present our findings, which confirm that the developed method has significant advantages.

Another main challenge in mass spectrometry-based proteomics is de novo protein sequencing, especially for novel proteins such as monoclonal antibodies for which genome information is often limited or not available. However, due to limitations in peptide fragmentation coupled with coverage and ambiguities in spectra interpretation, complete de novo assembly of unknown protein sequences remains challenging. Thus, there is a drive for new strategies that increase fragment ion assignment efficiency in top-down mass spectra and maximise the protein sequence coverage obtained. Here, we use a strategy for selective chemical labelling of the protein N-terminus using reductive alkylation and utilise this chemistry to introduce a halogen-based mass defect tag to the N-terminus of Insulin B chain, Ubiquitin, Myoglobin and Rnase A. We outline the potential advantages of using this simple chemical derivatisation in top-down de novo protein sequencing.