Intact cell MALDI

It is clear that both intact cell MALDI-TOF and PFGE have their limitations. PFGE analyses probes the chromosomal DNA of microorganisms for variations in the locations of specific restriction enzyme cleavage sites, while MALDI-TOF mass spectrometry of intact cells primarily examines abundant proteins such as ribosomal proteins35 and those associated with or near bacterial cell walls.58 In order for MALDI-TOF to detect a variation, a mutation must lead to noticeable changes in the expression of cell wall—associated... 

The results for bacterial whole-cell analysis described here establish the utility of MALDI-FTMS for mass spectral analysis of whole-cell bacteria and (potentially) more complex single-celled organisms. The use of MALDI-measured accurate mass values combined with mass defect plots is rapid, accurate, and simpler in sample preparation then conventional liquid chromatographic methods for bacterial lipid analysis. Intact cell MALDI-FTMS bacterial lipid characterization complements the use of proteomics profiling by mass spectrometry because it relies on accurate mass measurements of chemical species that are not subject to posttranslational modification or proteolytic degradation. 

Jones, J. J. Stump, M. J. Fleming, R. C. Lay, J. O. Wilkins, C. L. Strategies and data analysis techniques for lipid and phospholipid chemistry elucidation by intact cell MALDI-FTMS. /. Am. Soc. Mass Spectrom. 2004,15,1665-1674. 

Intact-cell MALDI-TOF analysis offers several attractive features for rapid screening of bacterial collections. Analysis is performed directly on the cells after minimal sample preparation, and data acquisition is complete in only a matter of minutes. Intact biomarkers are introduced into the MALDI-TOF instrument under these conditions. Whether the observed biomarker molecules are desorbed directly from the surface of the cell wall or are extracted from the cells and co-crystallized with the matrix is currently unresolved, but MALDI spectra of intact bacteria generally contain a large number of peaks in the mass range 1-20 kDa [31]. For bacterial cells, proteins are the most often observed biomarkers. While this approach samples only a small percentage of the total proteins produced in the cells, these profiles have been reported by many groups to be suitable for taxonomic identification, down to at least the strain level. The wide availability of the MALDI-TOF instrumentation and its relative ease of use, coupled with relatively simple sample preparation procedures, have been key features in the rapid advancement of this approach. 

To date, the vast majority of applications of intact cell MALDI-TOF analyses have been to bacteria. However, preliminary extensions of the approaches used in bacteria have been applied to fungi. From the standpoint of the discovery of novel chemotypes via natural products, this is obviously a desirable step, but it is also clearly in its infancy. Two groups reported [36,37] the observation of relatively simple mass spectral fingerprints from the intact fungal tissue they analyzed. This application might have the advantage of simplified spectral comparisons, relative to those for bacteria, but also means that such comparisons would have fewer data points to support them. 

Jackson, K.A., Edwards-Jones, V, Sutton, C.W., Fox, A.J. (2005) Optimisation of intact cell MALDI method for fingerprinting of methicillin-resistant Staphylococcus aureus. Journal of Microbiological Methods, 62, TIS-TBA. 

Hathout, Y., Ho, Y.R, Ryzhov, V., Demirev, R and Eenselau, C., Kurstakins A new class of lipopeptides isolated from Bacillus thuringieiisis. J. Nat. Prod., 63, 1492-1496 (2000). Pineda, R, Antoine, M., Demirev, R, Feldman, A., Jackman, J., Longenecker, M. and Lin, J., Microorganism identification by matrix-assisted laser/desorption ionization mass spectrometry and model-derived ribosomal protein biomarkers. Anal. Chem., 75, 3817-3822 (2003). Jackson, K.A., Edwards-Jones, V., Sutton, C.W. and Fox, A.J., Optimisation of intact cell MALDI method for fingerprinting of methicillin-resistant Staphylococcus aureus. J. Microbiol. Methods, 62, 273-284 (2005). 

The focus of this chapter is the development of a technique often called wholecell matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) or whole-cell MALDI-TOF MS. Some groups prefer to use terms such as intact or unprocessed rather than whole, but the intended meaning is the same regardless of which word is used. As noted in the first chapter of this book, there are many different methods for the analysis of bacteria. However, for the analysis of intact or unprocessed bacteria, whole-cell MALDI-TOF MS is the most commonly used approach. This method is very rapid. MALDI-TOF MS analysis of whole cells takes only minutes because the samples can be analyzed directly after collection from a bacterial culture suspension. Direct MALDI MS analysis of fungi or viruses is similar in approach1,2 but is not covered in this chapter. MALDI-TOF MS of whole cells was developed with very rapid identification or differentiation of bacteria in mind. The name (whole cell) should not be taken to imply that the cells are literally intact or whole. Rather, it should be taken to mean that the cells that have not been treated or processed in any way specifically for the removal or isolation of any cellular components from any others. In whole-cell analysis the cells have been manipulated only as necessary to... 

Another interesting comparison has recently been made between MALDI-TOF MS analysis of whole cells, and MALDI FTMS of the same organisms. This work is reported in greater detail in a dedicated chapter later in this book. It should be noted here that it appears to be much more difficult to obtain spectra from intact bacteria by MALDI FTMS than it is by MALDI-TOF MS. Thus far only a single research group has reported protein-like ions desorbed directly from intact cells by MALDI FTMS. 

Claydon, M. A. MALDI-ToF-MS, a new and novel technique for studies of intact cells. Anaerobe 2000, 6,133-134. 

Chemical characterization hypothesizes that certain molecules are unique and representative of individual microorganisms, and therefore the mass spectra can be characteristic of given species. A typical bacterial spectrum obtained by MALDI-TOF usually contains between 20 and 40 large peaks, mainly produced by proteins, phospholipids, and cyclic lipopeptides, in the MW range of 4—20 kDa.31 ESI is less frequently used samples containing intact cells can clog electrospray devices, and the formation of multiply charged species complicates the spectra. 

H., and Al Dahouk, S. (2013) Interlaboratory comparison of intact cell matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF MS) in the identification and differentiation of Brucella spp. J. Clin. Microbiol, in press. 

Sedo and Zdrahal provide in Chap. 7 specific examples of MALDITOF MS profiling for successful differentiation between strains of the Lactobacillus acidophilus group and sqXqcXcA Mycobacterium spp. In these two examples, careful optimization of the culture protocols contributed to the method robustness. In addition, strains within the Acinetobacter calcoaceticus-Acinetobacter baumannii complex. Staphylococcus aureus, and Bacillus subtilis ecotypes can be successfully typed by utilizing two alternative sample preparation protocols alternative MALDI matrix solution or microwave-assisted tiyptic digestion of the intact cells. 

This relationship between the final state of the sample and MS instramerrtation is mainly a consequence of the type of sample inlet and ionization technique used in a particular mass spectrometer. Referring to Fig. 2.1, the analysis of intact cells by matrix-assisted laser desorption/ionization-MS (MALDI-MS), (JaskoUa and Karas 2011) one of the simplest approaches for microorganism analysis by MS, (Holland et al. 1996) requires the isolation of a pure microbial colony, which is then deposited directly onto the MALDl plate. The subsequerrt mass spectral profiles. 

Fig. 2.2 Structures and scanning electron microscopy (SEM) photographs of different MALDI matrices deposited onto a stainless steel plate. Lower SEM photographs show E. coli cells cocrystallized with different MALDI matriees (matrix applied with a spray deposition technique). Arrows point to intact cells within the erystalline matrix. (Adapted from Toh-Boyo et al. 2012, eopyright American Chemical Society)...

In addition to gel electrophoresis, proteins can also be characterized using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Developed by Karas etal. (1987), this system uses a laser to irradiate intact cells, which ionizes surface cell proteins that are then characterized. The patterns of proteins yield a profile unique to the microorganism and are therefore used for identification purposes. MALDI-TOF-MS offers the advantage of rapid results with minimal sample preparation and reagents. However, the cost of equipment makes these methods impractical for wineries. 

Over the past 10 years, methods have been optimized for the direct analysis of individual cells, groups of cells, and small tissue sections [8-27]. Peptide profiling of complex mixtures by MALDI TOP MS has been performed without previous molecular separation to show differences between cell types and physiological conditions, identify novel peptides, assess post-translational processing, and demonstrate peptide localization within the tissues. Additional experiments have demonstrated sub-cellular protein localization within intact cells [23,28]. Efforts have also been made to characterize bacterial strains based on intact peptide and protein profiles [12,15,18]. Most of this early work involved analysis of peptides and low-molecular-weight proteins, typically from established cell lines. 

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