Comparative analysis of protein extraction methods for the identification of seafood-borne pathogenic and spoilage bacteria by MALDI-TOF mass spectrometry

Abstract

Species differentiation of food pathogenic and spoilage bacteria is important to ensure food quality and safety. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been applied to species identification of microorganisms, proving to be a rapid and cost-effective technique and allowing species differentiation due to the highly specific spectral profiles obtained. In this work, bacterial strains from our laboratory intern collection of seafood-borne pathogenic and spoilage species were studied by MALDI-TOF MS. Different sample preparation protocols were applied and compared to each other. Two methods were based on the analysis of whole bacterial cells that were suspended in an organic solvent or applied directly to the sample target. In a different sample preparation technique, cell extracts were obtained from intact bacterial cells by a dissolution/centrifugation step. The protocol applied for the study of cell extracts was shown to be very fast and simple, allowing the standardization of sample preparation. Furthermore, the analysis of cell extracts had several advantages with respect to the analysis of suspensions of whole bacterial cells. Thus, spectral profiles obtained from cell extracts showed less noise and more reproducible peaks as compared to spectra obtained from intact cells. The analysis of cell extracts by MALDI-TOF MS was also applied to create a mass spectral library of the main pathogenic and spoilage bacteria potentially present in seafood and was demonstrated to be a rapid and accurate method for microbial species differentiation, as well as for the classification of unknown strains isolated from seafood.