Fig. 1.

MHC-I enrichment is amenable to automation, reusable reagents, and dry storage of cartridges.A, overview of the stages in peptide processing and MHC-I presentation, enrichment, and mass-spectrometry-based analysis and quantification. In canonical presentation, intracellular proteins are polyubiquitinated within the cell and translocated to the proteasome for degradation. The resulting peptide fragments are transported into the endoplasmic reticulum via transporter associated with antigen processing (TAP) proteins. These fragments are then loaded into MHC-I complexes (consisting of an MHC-I heavy chain and ß2M), further trimmed by endoplasmic reticulum aminopeptidases (ERAP), and the stable MHC-I peptide complex is translocated to the cell surface. The display level of each unique peptide is determined by its abundance, degradation rate, and affinity for the MHC-I allele(s) in a given cell (26). Enrichment involves applying lysate containing MHC-I complexes to a solid support containing anti-MHC-I antibodies, washing away unbound contaminants, and then eluting the MHC-I proteins and associated peptides via acid treatment. Analysis involves peptide isolation and desalting, chromatographic separation, and analysis by mass spectrometry followed by data processing involving identification and quantification steps. B, table of small and large cartridge characteristics, including recombinant protein capacities, cellular abundance, and amount to be loaded on customized antibody cartridges. C, unique MHC-I peptides identified using a standard, single-use AssayMAP enrichment workflow, separated by species and effective cell count. D, antibody cartridge reuse scheme and circular enrichment workflow. Protein A cartridges are loaded with antibody and cross-linked, used to enrich MHC-I complexes, and washed before acid elution. Cartridges are then cleaned via priming with acid and TBS, stored at 4 °C, and reused in a similar manner. E, unique peptide count observed after nine consecutive uses of custom antibody cartridges, using GRANTA lysate (50 million cell equivalents each), on always-wet versus dried then rewetted cartridges.