Optimization of MALDI-TOF MS for strain level differentiation of Arthrobacter isolates

Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been shown to be a rapid and sensitive method for characterization of bacteria, but it has not yet become a routine microbiological procedure. Currently there are no standardized protocols that would allow development of large libraries of reproducible protein profiles from a broad range of microorganisms to use for identification purposes. Important variables that may affect spectrum quality are MALDI matrices, solvents, cell growth condition, and culture age. In the present study our aim was to: (1) to determine optimal sample preparation and MALDI conditions for discrimination at the strain level; (2) to determine if changes in growth cycle correlated with MALDI spectrum changes; and (3) to compare level of isolate discrimination based on their MALDI spectra versus their 16S rRNA gene sequence. Using 16 strains of the Gram positive bacterium Arthrobacter, optimal spectra were obtained using two-layer sample application of intact cells grown on solid surface overlaid with a matrix consisting of sinapinic acid (SA) or alpha-cyano-hydroxy-cinnaminic acid (CHCA) in 50:50 acetonitrile:water solvent with 2% trifluoroacetic acid. Spectrum changes paralleled the coccus-rod-coccus growth cycle indicative of Arthrobacter. Strain differences based on their MALDI profiles (using Pearson coefficient and UPGMA) corresponded with their 16S rRNA gene phylogeny but it had greater discrimination.