Optimizing Immunopeptide Analysis Sample Preparation in Needle Biopsy Size Tissue Samples with AFA® Technology

Immunopeptides play an essential role in adaptive immunity by activating and ensuring T-cell specificity. Mass spectrometry (MS) is currently the only technology that can reliably measure and identify the immunopeptide profiles of biological samples at a large scale. However, quality of data in these assays is limited by sample input, extraction efficiency, and poor scalability. To address this issue, we optimized the native lysis of cells with AFA-based ultrasonication and immunoprecipitation while ensuring 96-well compatibility, scalability and reproducibility. Post MS data acquisition, the data was processed using SpectroMine for spectral library generation and Spectronaut for library-based data extraction. With the optimized workflow, we can now identify over 2,800 class-I immunopeptides with 2.5 mg healthy lung tissue and >11,000 class-I and >10,000 class-II immunopeptides with 15 mg fresh frozen tissue. Overall, we have established a scalable, efficient pipeline for cell line and tissue immunopeptidomics for both class-I and II immunopeptides. The workflow described in this presentation will demonstrate generation of high-quality identifications from minimal starting material and can be deployed to help shed light on immunopeptidomics heterogeneity through large-scale profiling of patients as it will be exemplified in the case of neoantigen identification in MSI-high colorectal cancer.

 

About AFA

Covaris Adaptive Focused Acoustics® (AFA®) is an advanced acoustic technology enabling the mechanical processing of samples through focused ultrasonication. With highly tunable energy, it gently disrupts the cell membranes of mammalian cells (e.g., GPCR assays), or abruptly disrupts the cell walls of bacteria (e.g., total protein extraction). AFA provides the flexibility to homogenize cells and efficiently extract proteins, RNA, DNA, Metabolites, or other targets in buffers optimized for downstream analytical methods.