Optimization of sample preparation method, and evaluation of formic acid protein extracts for MALDI-typing of Staphylococcus aureus. (c2013)

Abstract

Matrix Assisted Laser Desorption Ionization mass spectrometry (MALDI-MS) has been recently introduced in to the field of microbiology. The use of this technique overcomes some of the limitations of current phenotypic and genotypic methods. MALDI-typing of microorganisms is an approach based on the differentiation of MALDI-acquired protein fingerprints. Employed in clinical settings, it allows rapid identification of microorganisms down to the strain level. Leading to high morbidity and mortality rates, antibiotic resistant strains of Staphylococcus aureus have become a worldwide concern. Herein, we carried out a comparative study of 20 variations of an acid/alcohol bacterial protein extraction method using a clinical isolate of S. aureus. Protein fingerprints of these extracts acquired in linear mode (800-20,000 Da) were used for assessment of information content (number of peaks) and identity (size of proteins/peptides, m/z). Two methods were shown to be most efficient for sample preparation, i.e. formic acid/methanol and trifluoroacetic acid/ethanol, yielding 28 peaks each. Proteins obtained by the classic formic acid/ethanol extraction were separated using 2-dimensional electrophoresis (2-DE) yielding more than 50 protein spots. Nine proteins were successfully identified using peptide mass fingerprinting (PMF), two of which are virulence related proteins, SpoVG and endonuclease IV. These proteins are considered prospects for MALDI-typing.