Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI ToF-MS) has been used to identify bacteria on the basis of its spectral nature. Identification of bacteria using MALDI ToF mass spectra depends on instrumental parameters and sample preparation protocol. Here we have optimized the sample preparation protocol for whole cell Rhizobium and evaluated the effect of its different growth phases to find out specific growth periods superior to the taxonomical approach. Sample preparation integrates the most important step influencing the quality and reproducibility of the spectra. The important factors in sample preparation include selection of matrix, solvent and acid concentration. A maximum numbers of peaks have been observed using 2,4-dihydroxy- phenylazobenzoic acid (HABA) rather than the commonly used α-cyano-4-hydroxy cinnamic acid (CHCA) or 3, 5-dimethoxy- 4-hydroxycinnamic acid (sinapinic acid) for whole cell Rhizobium identification. A protocol has been optimized after considering different experimental variables including various phases of bacterial growth and late log to stationary phase has been found to be best for identification of whole cell Rhizobium by this method.

Keywords: growth phase, Rhizobium sp., MALDI-ToF, matrix.