Whole cell MALDI

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... 

As noted above, whole-cell MALDI-TOF MS was intended for rapid taxonomic identification of bacteria. Neither the analysis of specific targeted bacterial proteins, nor the discovery of new proteins, was envisioned as a routine application for which whole cells would be used. An unknown or target protein might not have the abundance or proton affinity to facilitate its detection from such a complex mixture containing literally thousands of other proteins. Thus, for many applications, the analysis of proteins from chromatographically separated fractions remains a more productive approach. From a historical perspective, whole-cell MALDI is a logical extension of MALDI analysis of isolated cellular proteins. After all, purified proteins can be obtained from bacteria after different levels of purification. Differences in method often reflect how much purification is done prior to analysis. With whole-cell MALDI the answer is literally none. Some methods attempt to combine the benefits of the rapid whole cell approach with a minimal level of sample preparation, often based on the analysis of crude fractions rather... 

TAXONOMY FROM ISOLATES TO WHOLE-CELL MALDI... 

Holland et al. proposed a simpler and faster approach using the protein profile obtained directly from whole cells rather than from cellular extracts or affinity beads.17 This was the first report of the whole-cell MALDI-TOF MS... 

Figure 6.1 Sections (mlz 5000-10,000) from unpublished spectra—standard (a) and blind-coded (b)—obtained from Enterobacter cloaca from work reported in Holland et al.17 showing the very low mass resolution, additional peaks, and three consistent signals used to identify this organism in the first reported whole-cell MALDI experiments in 1996.

The direct whole-cell method of Holland et al. was extremely rapid, even in comparison to Lubman s MALDI analysis of fractions collected after bacterial sonnication. With the whole-cell approach bacteria were simply sampled from colonies on an agar plate, mixed with the matrix, air-dried, and introduced in batches into the mass spectrometer for analysis. In all of the spectra obtained in these and later experiments, each bacterial strain showed a few characteristic high-mass ions that were attributed to bacterial proteins. Studies demonstrating the whole cell methodology for strain-level differentiation were reported independently by Claydon et al. at almost the same time.18 Shortly thereafter a third study on whole-cell MALDI included bacteria from pathogenic and nonpathogenic strains appeared.19... 

Figure 6.2 Whole-cell MALDI-TOF spectra of two Vibrio species taken using a modern TOF mass spectrometer. Even in the linear mode V. parahaemolyticus can be easily differentiated from V. vulnificus.

BIOLOGY-BASED CHANGES IN WHOLE-CELL MALDI SPECTRA... 

Despite the large number whole-cell MALDI protocols that have been tested a single approach has not yet been widely adopted. There remain different and sometimes conflicting reports in the literature regarding optimum methodologies.3 In addition to the ferulic acid matrix mentioned above sinapinic acid, a-yano-4-hydroxycinnamic acid, 2,4-hydroxyphenyl benzoic... 

An interesting variation on the whole-cell MALDI approach was recently reported in a study aimed more at FTMS than TOF MS, but the results are nevertheless interesting and important to users of both methods for analysis of bacteria 40. Wilkins s group showed both MALDI-TOF and MALDI-FTMS spectra of whole bacteria grown on isotopic media depleted in C13 and N14. Because most bacterial identification protocols involve a culture step prior to analysis, it is possible to manipulate the sample based on control of the growth media. For mass spectral analysis manipulation of the isotope profile... 

The most common criticism of whole-cell MALDI is that the method requires a relatively large number of cells, usually obtained directly from culture media. In principle, an analysis of even a few unknown bacteria (a colony-forming unit) is possible after a culture step. More important is the number of bacteria needed in a sample or on the sample probe for successful analysis. Detection of a very small number of bacteria could eliminate the need for a preliminary culture step. This would be a considerable asset for environmental analysis (unless to many bacteria were detected) and for the detection of a bioterrorism-related release of bacteria. 

FUNDAMENTALS OF COMPLEX BIOLOGICAL ANALYSIS 13.3.1 Whole-Cell MALDI-FTMS Analysis... 

Approximately 90% to 95% of whole-cell MALDI MS profiles are representative of the ribosomal proteins abundant in rapid growth whole cells. Although the identification of proteins, from whole bacterial cells, by MALDI-TOF MS analysis is ambiguous, at best, due to the low-mass accuracy and resolving power, several researchers realized that many of the observed... 

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