Effect of humidity during sample preparation on bacterial identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a highly reliable and efficient technology for the identification of microbial pathogens. We previously found that 40% humidity was the optimal condition for the preparation of samples (co-crystallization of the sample and matrix) for serum peptidomic analysis via MALDI-TOF MS profiling. This optimum temperature was applied to obtain the highest reproducibility and throughput and greatest number of peaks. We therefore hypothesized that humidity control was also essential for MALDI-TOF MS bacterial identification. In this study, we constructed a simple sample preparation device that enables humidity control and used it for co-crystallization of the sample and matrix. Identification scores for five Gram-negative bacteria and six Gram-positive bacteria were determined using the MALDI BioTyper® system at three humidity ranges (10–20%, 30–40%, and 50–60%). As a result, higher identification scores were obtained at 30–40% humidity than at 10–20% or 50–60% humidity. At 30–40% humidity, 517/550 (94.0%) isolates scored greater than 2.0, indicating the success of species-level identification. Similarly, 537/550 (97.6%) isolates scored greater than 1.7, indicating the success of genus-level identification. Thus, 30–40% humidity generated optimal MALDI-TOF MS identification scores and the highest percentage of correct identifications. These results could lead to further improvements in the accuracy of MALDI-TOF MS bacterial identification.