Species-level discrimination of microorganisms by high-resolution paper spray – Ion mobility – Mass spectrometry

There is a projection of 10 million deaths per year by 2050 due to antimicrobial resistance, highlighting the need for rapid detection and accurate identification of microorganisms. Herein, we used the paper spray technique coupled with ion mobility separation and tandem mass spectrometry (PS-IM-MS/MS) to rapidly discriminate and identify five Bacillus species in 2 min of analysis time after only 4 h of incubation time. Bacterial cells were harvested by filtering their liquid cultures and ionized directly by PS. Numerical multivariate statistics (principal component analysis, followed by linear discriminant analysis) allowed species-level discrimination with a prediction rate of 92.4% and 97.6% utilizing the negative and positive ion information of PS-MS/MS, respectively. However, upon including the biomarkers' corresponding drift times, i.e., PS-IM-MS/MS, prediction rates of ca. 99.7% and 100% were obtained utilizing the negative and positive ion information, respectively. We attributed the enhancement in the prediction rates to the capability of IM separations in resolving isomers. Surfactins and phospholipids were the major ion populations detected in our experiments. They were found to have various diagnostic isomers for various Bacillus species. Therefore, improved prediction rates could be obtained upon including high-resolution IM separations in the workflow. These proof-of-concept studies described here suggest that PS-IM-MS/MS workflow would enable rapid discrimination of closely related bacteria species and strains.