Optimizing High-Throughput Shotgun Immunoproteomics for Antigen Identification

ABSTRACT: Identification of individual proteins (i.e., antigens) which initiate and/or perpetuate adaptive immune responses has potential to greatly impact pre-clinical and clinical work across numerous fields. To date, however, the methodologies available to identify antigens responsible for driving adaptive immune responses have been plagued by numerous issues which have drastically limited their widespread adoption. Therefore, in this study, we sought to optimize a shotgun immunoproteomics approach to alleviate these persistent issues and create a high-throughput, quantitative methodology for antigen identification. Three individual components of a previously published approach, namely the protein extraction, antigen elution, and LC-MS/MS analysis steps, were optimized in a systematic manner. These studies determined that preparation of protein extracts using a one-step tissue disruption method in immunoprecipitation (IP) buffer, eluting antigens from affinity chromatography columns with 1% trifluoroacetic acid (TFA), and TMT-labeling & multiplexing equal volumes of eluted samples for LC-MS/MS analysis, resulted in quantitative longitudinal antigen identification, with reduced variability between replicates and increased total number of antigens identified. This optimized pipeline provides a multiplexed, highly reproducible, and fully quantitative approach to antigen identification which is broadly applicable to determine the role of antigenic proteins in inciting (i.e. primary antigens) and perpetuating (i.e., secondary antigens) a wide range of diseases.

INSTRUMENT(S): Orbitrap Eclipse, Orbitrap Fusion Lumos

ORGANISM(S): Bos Taurus (bovine)

TISSUE(S): Pericardium

SUBMITTER: Akhilesh Pandey  

LAB HEAD: Akhilesh Pandey

PROVIDER: PXD038761 | Pride | 2023-04-18

REPOSITORIES: Pride