Application of MALDI Biotyper to cyanobacterial profiling.

RATIONALE: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has been used for bacterial profiling. A few reports have shown MALDI-MS profiling of isolated/cultured cyanobacteria; however, these applications have been limited. In this study, we have investigated whether rapid profiling and differentiation of cyanobacteria including harmful genera Microcystis and Anabaena (Dolichospermum) can be performed by MALDI Biotyper analysis of intact cells. METHODS: Twenty-two cyanobacterial strains including 12 Microcystis, 7 Anabaena, 1 Pseudanabaena, 1 Planktothrix, and 1 Synechocystis were cultured. Also, natural pond water containing cyanobacteria was collected. Intact cyanobacterial cells were deposited on a target plate, and analyzed using an Autoflex Speed MALDI-TOF mass spectrometer with Biotyper software. Mass spectra obtained from m/z 2000 to 20000 were used for clustering and spectral library searching of cyanobacterial strains. RESULTS: MALDI-MS analysis of cultured cyanobacterial cells showed clear ion signals under optimized conditions. Hierarchical clustering of mass spectra using Biotyper resulted in a tight cluster of Microcystis strains which was clearly differentiated from a cluster of Anabaena strains. Spectral library searching was able to identify Microcystis aeruginosa NIES-298 and Synechocystis sp. PCC 6803 even when these two cells were mixed. Furthermore, cyanobacterial cells in the pond water were successfully classified as Anabaena. CONCLUSIONS: We have demonstrated that MALDI-MS in combination with Biotyper analysis is applicable to cyanobacterial profiling. Increasing the size of the spectral library may facilitate monitoring of cyanobacteria in crude cyanobacterial blooms. Copyright © 2016 John Wiley & Sons, Ltd.

Cyanobacterial Classification with the Toxicity Using MALDI Biotyper.

An abnormal growth of cyanobacteria in eutrophicated freshwaters can cause various environmental problems. In particular, Microcystis producing hepatotoxic cyclic heptapeptides microcystins (MCs) has been globally observed. Recent studies have demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) offers a rapid classification of cyanobacteria; however, they have not fully considered the toxicity yet. In this study, we have performed MALDI-TOF MS for intact cyanobacterial cells using Biotyper software and optimized their conditions to achieve cyanobacterial classification with the toxicity. The detection mass range used for Biotyper was extended to cover small molecules, but their intense ions were suppressed as a function of the used instrument Autoflex Speed, which enabled simultaneous observations of large molecular fingerprints and small MCs with comparable ion intensity. Hierarchical clustering of mass spectra obtained under the optimized conditions differentiated toxic and non-toxic clusters of Microcystis strains and furthermore formed a tight cluster of non-toxic strains possessing the MC biosynthesis gene mcyG. Spectral libraries were expanded to >30 genera (>80 strains) under the default and optimized conditions to improve the confidence of cyanobacterial classification. Consequently, spectral library searching allowed for characterization of cyanobacteria from a field sample as mixed toxic and non-toxic Microcystis cells, without isolating those cells.