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MASS SPECTRAL INVESTIGATIONS ON MICROORGANISMS

Bacterial cells undergo lysis readily, when suspended in mild aqueous acids, and release the cellular proteins along with other biomolecules. Molecular masses of the protein biomarkers released in-situ from individual intact bacterial cells could be directly measured by mass spectrometry. Limited sample clean up may be required at times, prior to mass spectral analysis, to remove any ionizable impurities such as salts, buffers and deergents. The marker proteins specific for individual genus, species and strains were determined by the comparison of the biomarkers measured for several closely related organisms. Even though there is a probability of over 4000 cellular proteins expressed in any single bacterial cell, only a small fraction of the projected marker proteins are identified consistently during the process. This could be due to the variation in the ionization properties of the proteins and the limited energy available to prompt their ionization. Variation in the sample processing and culture conditions had little effect in the marker proteins observed during the process. This experimental procedure enables the distinction of gram positive as well as gram negative cellular pathogens and their corresponding non-pathogenic counterparts. The identity of few bacterial cells present in unknown samples can be easily, rapidly and accurately established by adopting a procedure involving simple sample processing followed by direct mass spectral analysis and data processing. Thus, an uncomplicated approach has been developed to resolve a complex problem involving cellular pathogens. This method has enormous application potential in the rapid identification and subsequent prevention of any potential health hazard caused by the pathogenic bacteria, either under natural or induced conditions. There is a great potential for the total automation of the entire process in the future for simpler but more effective unattended operations in the laboratory as well as in the field.