Maximizing immunopeptide identification and reproducibility from minute pediatric solid tumor biopsy samples

Introduction: Immunopeptidomics, a rapidly growing field of proteomics, plays a vital role in identifying and quantifying peptides presented by the major histocompatibility complex (MHC) to the immune system. Peptides presented by MHC molecules are typically present at very low levels especially in pediatric patient samples where MHC expression is also often low. Maximizing peptide yield, sensitivity, and intra- and inter-sample reproducibility with mass spectrometry is essential to discovering peptide-MHC (pMHC) targets for modern cancer immunotherapies. Here we compare sample preparation methodologies incorporating Sepharose bead columns and streptavidin magnetic beads combined with the ultra-high sensitivity mass spectrometry for the analysis of immunopeptides from pediatric solid malignancies.

Methods: HLA class-I peptide samples were prepared using two immunoprecipitation (IP) methods: CNBr-activated Sepharose 4B column-based capture and streptavidin-biotin based capture. Briefly, frozen tissues and frozen cell pellets were pulverized by liquid nitrogen cooled cryomilling and then resuspended in lysis buffer to perform IP with using pan-HLA class I monoclonal antibody W6/32. Peptides were eluted from HLA molecules and then purified with a 5 kDa MWCO filter and C18 resin. Resulting peptides were separated by HPLC (nanoElute 2, Bruker Daltonics) on a 250 mm x 75 µm, 1.7 µm column.60 min gradients were analyzed in parallel on a timsTOF Pro 2 and timsTOF Ultra (Bruker Daltonics) in PASEF (Parallel Accumulation and Serial Fragmentation) mode. Data were processed using PEAKS software.

Results: HLA class-I peptides from 1e8 cells of cancer cell lines yielded over 20,000 unique canonical peptides using CNBr-activated Sepharose 4B method, demonstrating the robustness of our large-scale IP approach. Osteosarcoma patient-derived xenograft (PDX) samples revealed an average of 4986 unique peptides (a range from 397 to 14,823 peptides), with variations linked to tumor input mass. In comparing small-scale IPs, the CNBr-activated Sepharose 4B method outperformed the streptavidin magnetic bead protocol, yielding 7710 and 4692 peptides, respectively, from 2e7 SKNAS neuroblastoma cells. While CNBr-activated Sepharose 4B offered higher overall peptide yields, it exhibited lower reproducibility compared to the streptavidin magnetic bead protocol, with approximately 40% and 50% overlap of peptide IDs between three technical replicates for the two sample preparation protocols run on the timsTOF Ultra. Moreover, the Bruker timsTOF Ultra identified > 40% more peptides compared to the timsTOF Pro 2. Importantly, an average of 13,098 unique HLA-I peptides, ranging from 3,879 to 29,261, were identified from pediatric glioma tissue samples of less than 200 mg using the timsTOF Ultra. These findings provide critical insights into optimizing reproducible immunopeptide identification in pediatric solid cancers, guiding future research and immunotherapy development.