MS-based immunopeptidomics is maturing into an automatized and high-throughput technology, producing small- to large-scale datasets of clinically relevant major histocompatibility complex (MHC) class I-associated and class II-associated peptides. Consequently, the development of quality control (QC) and quality assurance systems capable of detecting sample and/or measurement issues is important for instrument operators and scientists in charge of downstream data interpretation. Here, we created MhcVizPipe (MVP), a semiautomated QC software tool that enables rapid and simultaneous assessment of multiple MHC class I and II immunopeptidomic datasets generated by MS, including datasets generated from large sample cohorts. In essence, MVP provides a rapid and consolidated view of sample quality, composition, and MHC specificity to greatly accelerate the “pass–fail” QC decision-making process toward data interpretation. MVP parallelizes the use of well-established immunopeptidomic algorithms (NetMHCpan, NetMHCIIpan, and GibbsCluster) and rapidly generates organized and easy-to-understand reports in HTML format. The reports are fully portable and can be viewed on any computer with a modern web browser. MVP is intuitive to use and will find utility in any specialized immunopeptidomic laboratory and proteomics core facility that provides immunopeptidomic services to the community.

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Understanding the molecular principles that govern the composition of the mammalian MHC-I immunopeptidome (MHC-Ii) across different primary tissues is fundamentally important to predict how T cell respond in different contexts in vivo. Here, we performed a global analysis of the mammalian MHC-Ii from 29 and 19 primary human and mouse tissues, respectively. First, we observed that different HLA-A, -B and -C allotypes do not contribute evenly to the global composition of the MHC-Ii across multiple human tissues. Second, we found that peptides that are presented in a tissue-dependent and -independent manner share very distinct properties. Third, we discovered that proteins that were evolutionarily hyperconserved represent the primary source of the MHC-Ii at the organism-wide scale. Finally, we uncovered a remarkable antigen processing and presentation network that may drive the high level of heterogeneity of the MHC-Ii across different tissues in mammals. This study opens up new avenues toward a system-wide understanding of antigen presentation in vivo and may serve as ground work to understand tissue-dependent T cell responses in autoimmunity, infectious diseases and cancer.

Immunopeptidomics refers to the science of investigating the composition and dynamics of peptides presented by major histocompatibility complex (MHC) class I and class II molecules using mass spectrometry (MS). Here, we aim to provide a technical report to any non-expert in the field wishing to establish and/or optimize an immunopeptidomic workflow with relatively limited computational knowledge and resources. To this end, we thoroughly describe step-by-step instructions to isolate MHC class I and II-associated peptides from various biological sources, including mouse and human biospecimens. Most notably, we created MhcVizPipe (MVP) (https://github.com/CaronLab/MhcVizPipe), a new and easy-to-use open-source software tool to rapidly assess the quality and the specific enrichment of immunopeptidomic datasets upon the establishment of new workflows. In fact, MVP enables intuitive visualization of multiple immunopeptidomic datasets upon testing sample preparation protocols and new antibodies for the isolation of MHC class I and II peptides. In addition, MVP enables the identification of unexpected binding motifs and facilitates the analysis of non-canonical MHC peptides. We anticipate that the experimental and bioinformatic resources provided herein will represent a great starting point for any non-expert and will therefore foster the accessibility and expansion of the field to ultimately boost its maturity and impact.

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