Abstract
Abstract Rapid identification of microorganisms is essential for medical diagnostics, sanitary controls, and food safety. High‐throughput analytical platforms currently rely on whole‐cell MALDI‐TOF mass spectrometry to process hundreds of samples per day. Although this technology has become a reference method, it is unable to process most environmental isolates and opportunistic pathogens due to an incomplete experimental spectrum database. In most cases, its discriminating power is limited to the species taxonomical rank. By recording much more sequence information at the peptide level, proteotyping by tandem mass spectrometry is able to identify the taxonomic position of any microorganism in the tree of life and can be highly discriminating at the subspecies level. We propose here a methodology for ultra‐fast identification of microorganisms by tandem mass spectrometry based on direct sample infusion and a highly sensitive procedure for data processing and taxonomic identification. Results obtained on reference strains and hitherto uncharacterized bacterial isolates show identification to species level in 36 s of tandem mass spectrometry signal, 102 s when including the injection procedure. Flash proteotyping is highly discriminating, as it can provide information down to strain level. The methodology enables high throughput identification of isolates, opening up new prospects, particularly in culturomics, and diagnostics.