Progress and challenges in mass spectrometry-based analysis of antibody repertoires

Humoral immunity is divided into the cellular B cell and protein-level antibody responses. High-throughput sequencing has advanced our understanding of both these fundamental aspects of B cell immunology as well as aspects pertaining to vaccine and therapeutics biotechnology. Although the protein-level serum and mucosal antibody repertoire make major contributions to humoral protection, the sequence composition and dynamics of antibody repertoires remain underexplored. This limits insight into important immunological and biotechnological parameters such as the number of antigen-specific antibodies, which are for example, relevant for pathogen neutralization, microbiota regulation, severity of autoimmunity, and therapeutic efficacy. High-resolution mass spectrometry (MS) has allowed initial insights into the antibody repertoire. We outline current challenges in MS-based sequence analysis of antibody repertoires and propose strategies for their resolution. Recent advances in high-resolution mass spectrometry (MS) instruments and liquid chromatography (LC)-MS/MS data analysis software have enabled novel insights into the serum and mucosal antibody repertoire.There is a lack of standardization and benchmarking of antibody repertoire proteomics (Ab-seq) in both experimental and analytical pipelines.Knowledge of the sequence composition and dynamics of the antibody repertoire remains limited due to the complexity of antibody biology as well as Ab-seq workflow-related technological, experimental, and computational challenges that hinder the development of vaccines, antibody therapeutics, and immunodiagnostics.Newly developed strategies can improve Ab-seq workflows and technology.