Changes in the Matrix Markedly Enhance the Resolution and Accurate Identification of Human Pathogens by MALDI-TOF MS

Abstract

The mass spectral profiles of three major/emerging Gram positive pathogens belonging to the genera Clostridium, Listeria and Propionibacterium were investigated using four MALDI-TOF (Matrix-Assisted Laser Desorption/ Ionization- Time of Flight) Mass Spectrometers from Waters, Shimadzu, Bruker and Ciphergen Biosystems. These instruments have been extensively used for developing a diagnostic platform for high throughput, low cost, near sample-free preparation for microbial identification. The results demonstrate the marked effect of spectral quality obtained by altering the matrix and the added value of simple preparative extraction procedures. While microbial spectral data are generally collected in the mass range 2 to 20 kDa for diagnostic signatures, most of the significant mass ions are <10 kDa. The change from the traditional use of CMBT (5-chloro-2-mercaptobenzothiazole) to DHB (2-(2, 5-DiHydroxyBenzoic acid) enabled successful identification of C. difficile whereas the substitution of CHCA (alpha-cyano-4-hydroxycinnamic acid) for DHB (2,5-dihydroxybenzoic acid) improved the identification of Listeria species. By contrast, the use of SELDI-TOF-MS (Surface Enhanced Laser Desorption/Ionization- Time of Flight- Mass Spectrometry), which employs affinity pre-capture of specific classes of molecules and analysed using sinapinic acid, showed a fivefold extension of the mass range of analytes, extending the potential for biomarker discovery and higher resolution strain typing of P. acnes.