Although matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) can be employed to rapidly characterize pathogenic bacteria, bacterial cultures are generally required to obtain sufficient quantities of the bacterial cells prior to MALDI MS analysis. If this time-consuming step could be eliminated, the length of time required for identification of bacterial strains would be greatly reduced. In this paper, we propose an effective means of rapidly identifying bacteria - one that does not require bacterial culturing - using functional nanoparticle-based proteomic strategies that are characterized by extremely short analysis time. In this approach, we used titania-coated magnetic iron oxide nanoparticles (Fe3O 4@TiO2 NPs) as affinity probes to concentrate the target bacteria. The magnetic properties of the Fe3O4@TiO 2 NPs allow the conjugated target species to be rapidly isolated from the sample solutions under a magnetic field. Taking advantage of the absorption of the magnetic Fe3O4 NPs in the microwave region of the electromagnetic spectrum, we performed the tryptic digestion of the captured bacteria under microwave heating for only 1-1.5 min prior to MALDI MS analysis. We identified the resulting biomarker ions by combining their MS/MS analysis results with protein database searches. Using this technique, we identified potential biomarker ions representing five Gram-negative bacteria: Escherichia coli O157:H7, uropathogenic E. coli, Shigella sonnei, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Finally, we demonstrated the practical feasibility of using this approach to rapidly characterize bacteria in clinical samples.