The Monash Proteomics & Metabolomics Platform’s mission is to provide high quality proteomic, metabolomic, and lipidomic support to academic researchers, clinicians, and industry. We specialise in both collaborative and fee-for-service projects, offering standardised pipelines for diverse sample types. Our services range from general analyses (including discovery (DDA), quantification (TMT & DIA), crosslinking mass spectrometry, analysis of post-translational modification, N-terminal sequencing) to advanced workflows such as single-cell proteomics or immunopeptidomics. We offer comprehensive, end-to-end support through consultation, experimental design, sample processing, MS acquisition, and bioinformatic data analysis.
Our focus in this study is to refine our fee-for-service de novo antibody sequencing workflow to enhance data quality and thereby improving success rate and reduce both cost and turnaround time. Antibody sequence information is important for understanding antigen-binding mechanisms and for development of antibodies as therapeutic agents and research tools. Here, we present a method for direct de-novo sequencing of monoclonal IgG from purified antibody preparations. This involves using complementary proteases to generate peptides suitable for de novo sequencing via liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a bottom-up approach. We employ a dual-compensation voltage (CV); dual-fragmentation strategy that utilises high-energy collision dissociation (HCD) at one FAIMS CV and a combination of HCD and electron-transfer high-energy collision dissociation (EThcD) at the higher CV optimal for larger peptides. This method enhances acquisition and analysis efficiency, delivering complete sequence coverage of monoclonal antibody standards with less instrument time and significantly reduced manual data analysis, ultimately resulting in substantial time and cost savings.