Monoclonal antibody (mAb) therapeutics are vital to many areas of human health ranging from vaccines to immunotherapy treatments for various forms of cancer. As new pathogens emerge and immuno-oncology targets are discovered, a rapid, sensitive, and accurate method for interrogation of the polyclonal antibody (pAb) response and discovery of new potent mAbs is crucial. Identification of an antibody involves sequencing the amino acids in each of its subunits (a heavy chain and a light chain), as well as pairing of those subunits: determining which heavy chain (HC) pairs with which light chain (LC) in the higher order structure. Although widely accepted strategies for pAb identification such as single-B-cell cloning and hybridoma methods supply subunit sequence and pairing, they are severely limited in their representation of patient serum antibody diversity/abundance. Recently introduced proteome-centric methods demonstrate the concept of determining pAb subunit sequences directly but show limitations in de novo sequencing accuracy/sensitivity and fail to provide heavy/light chain (HC/LC) pairing for higher-order structure. The objective of this project is to develop a new method for streamlined and accurate serum pAb sequencing and HC/LC pairing based on top-down and middle-down proteomics. Technical innovations of this proposal are centered around novel MS/MS instrumentation and methods for electron capture dissociation and ultraviolet photodissociation which offer new capabilities including (A) improved MS/MS sequence coverage of intact mAbs and large peptides and (B) in-instrument cleavage of disulfide bonds for HC/LC pairing that scales to complex pAb samples. The primary contributions of this proposal will be a multi-dimensional proteomics platform ready for biological applications as well as novel MS/MS technologies and informatics tools ready for broad dissemination.