AbstractThe closed nature of vendor file formats in mass spectrometry is a significant barrier to progress in developing robust bioinformatics software. In response, the community has developed the open mzML format, implemented in XML and based on controlled vocabularies. Widely adopted, mzML is an important step forward; however, it suffers from two challenges that are particularly apparent as the field moves to high-throughput proteomics: large increase in file size, and a largely sequential I/O access pattern. Described here is ‘toffee’, an open, random I/O format backed by HDF5, with lossless compression that gives file sizes similar to the original vendor format and can be reconverted back to mzML without penalty. It is shown that mzML and toffee are equivalent when processing data using OpenSWATH algorithms, in additional to novel applications that are enabled by new data access patterns. For instance, a peptide-centric deep-learning pipeline for peptide identification is proposed. Documentation and examples are available at https://toffee.readthedocs.io, and all code is MIT licensed at https://bitbucket.org/cmriprocan/toffee.