Culture conditions and sample preparation methods affect spectrum quality and reproducibility during profiling of Staphylococcus aureus with matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) has emerged as a promising tool to rapidly characterize Staphylococcus aureus. Different protocols have been employed, but effects of experimental factors, such as culture condition and sample preparation, on spectrum quality and reproducibility have not been rigorously examined. We applied MALDI‐TOF MS to characterize a model system consisting of five methicillin‐sensitive (MSSA) and five methicillin‐resistant S. aureus isolates (MRSA) under two culture conditions (agar and broth) and using two sample preparation methods [intact cell method and protein extraction method (PEM)]. The effects of these treatments on spectrum quality and reproducibility were quantified. PEM facilitated increases in the number of peaks and mass range width. Broth cultures further improved spectrum quality in terms of increasing the number of peaks. In addition, PEM increased reproducibility in samples prepared using identical culture conditions. MALDI imaging data suggested that the improvement in reproducibility may result from a more homogeneous distribution of sample associated with the broth/PEM treatment. Broth/PEM treatment also yielded the highest rate (96%) of correct classification for MRSA. Taken together, these results suggest that broth/PEM maximizes the performance of MALDI‐TOF MS to characterize S. aureus. Significance and Impact of the Study Two culture conditions (agar or broth) and two sample preparation methods (intact cell or protein extraction) were evaluated for their effects on profiling of Staphylococcus aureus using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). Results indicated that MALDI‐enabled profiling of S. aureus is most effective when cultures are grown in broth and processed using a protein extraction‐based approach. These findings should enhance future efforts to maximize the performance of this approach to characterize strains of S. aureus. Significance and Impact of the Study: Two culture conditions (agar or broth) and two sample preparation methods (intact cell or protein extraction) were evaluated for their effects on profiling of Staphylococcus aureus using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). Results indicated that MALDI‐enabled profiling of S. aureus is most effective when cultures are grown in broth and processed using a protein extraction‐based