Label-Free Multiplex Proteotyping of Microbial Isolates.

To meet clinical diagnostic needs and for general microbiological screening, it is essential to be able to accurately and rapidly identify any microorganisms from complex microbiota. To gain insight into the individual components of microbiota, culturomics has been proposed as a means to systematically test hundreds of possible cultivation conditions and generate numerous microbial isolates with very distinct characteristics. High-throughput identification methods must now be developed to quickly screen these isolates. Currently, most multiplexing methods involve labeling, which comes at a cost. In this paper, we present an innovative label-free multiplexing method for the identification of microorganisms using tandem mass spectrometry. The method is based on offline reversed-phase fractionation of individual peptidomes. Multiplexing is achieved by mixing fractions of staged hydrophobicity; thus, each sample is mapped to specific elution times. In this proof-of-concept study, multiplexed samples were analyzed by tandem mass spectrometry in a single run and microorganisms present in the mixture were resolved by phylopeptidomics proteotyping. Using this methodology, up to 21 microorganisms could be identified in a single 60 min run performed with a Q-Exactive HF high-resolution mass spectrometer, resulting in a rate of one microorganism identified per 3 min of mass spectrometry, without any need for the use of labeling reagents. This approach opens new perspectives for the application of high-throughput proteotyping of bacteria using tandem mass spectrometry in large culturomics projects.