Presenter Bio: Karim Hayoun is a young talentuous PhD student developing tandem mass spectrometry proteotyping for fast identification of microorganisms. He is passionate by biochemistry, microbiology, and mass spectrometry.
Relevant Financial Disclosures
(within past 24 months)
No relevant financial relationship(s) to disclose.
Abstract
INTRODUCTION: Quick identification of pathogens is crucial for efficiently fighting against infectious diseases. Mass spectrometry is a powerful and discriminative tool for this. Whole-cell MALDI-TOF is successful for most pathogens but requires a pure sample usually obtained after cultivation. Tandem mass spectrometry proteotyping has been shown to be an interesting alternative for complex samples and could avoid the cultivation step.
OBJECTIVES: Here, we present an optimization of the sample preparation for MS/MS proteotyping of any type of microorganisms, as well as an optimization of the data treatment for a fast result.
METHODS: We proposed a bead-beating extraction of proteins and tested several protocols for fast trypsin proteolysis. A protocol based on magnetic beads was selected and further improved in order to achieve automatization of the sample preparation in 96-well plates. The bioinformatics pipeline for high-throughput MS/MS proteotyping was also optimized. The whole workflow is adapted for the identification of pathogens of clinical interest.
RESULTS: The optimized sample preparation method based on magnetic beads could be routinely performed in less than 1 h. The protocol was tested on an artificial mixture comprising Gram-positive and Gram-negative bacteria, as well as yeasts. We obtained perfect MS/MS proteotyping of several pathogenic strains. The optimized workflow enables to get an accurate identification of pathogen within 3 h after receiving the clinical sample, even if the sample is a mixture of microorganisms or in presence of MALDI-TOF incompatible matrix. Results show that even subtyping is possible with the same workflow. It is applicable to hundreds of samples treated in 96-well plates.
CONCLUSION: The results presented in this study illustrate the power of the approach, which addresses without a priori any kind of isolates, but also more complex samples such as mixtures of organisms.
= Discovery stage.
= Translation stage.
= Clinically available.
