Whole-cell analysis

Neher and coworkers, for example, have used the whole-cell analysis of currents... 

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... 

A final problem for bioinformatics and bioanalytical scientists is the characterization of engineered microorganisms. Whole-cell analysis by mass spectrometry has been used to confirm the introduction of therapeutic genes into adenovirus vectors,100 to confirm the expression of recombinant proteins in bacteria,101,102 and also in vaccinology.103 In the broader case, identification of... 

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. 

The analysis of individual cells, either by averaging analytical data from a number of individual cells ( whole-cell analysis ) or by analyzing only one cell at a time ( single-cell analysis ) often has enormous advantages over tissue analysis. 

Krishnamurthy, T. Ross, P. L. Rapid identification of bacteria by direct matrix-assisted laser desorption/ionization mass spectrometric analysis of whole cells. Rapid Commun. Mass Spectrom. 1996,10,1992-1996. 

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... 

Most often proteins are the bacterial biopolymers studied using MALDI MS either from fractions or whole cells. They are not the only isolated cellular biopolymers studied by MALDI, nor the first. Very soon after the introduction of MALDI there were a few reports of the analysis of bacterial RNA or DNA from bacterial fractions. One of the first applications of MALDI to bacteria fractions involved analysis of RNA isolated from E. coli,4 Other studies included analysis of PCR-amplified DNA,5 6 DNA related to repair mechanisms7 and posttranscriptional modification of bacterial RNA.8 While most MALDI studies involve the use of UV lasers, IR MALDI has been reported for the analysis of double stranded DNA from restriction enzyme digested DNA plasmids, also isolated from E. coli.9... 

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... 

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. 

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. 

Stump, M. J. Jones, J. J. Fleming, R. C. Lay, J. O., Jr. Wilkins, C. L. Use of double-depleted 13C and 15N culture media for analysis of whole cell bacteria by MALDI time-of-flight and Fourier transform mass spectrometry. J. Am. Soc. Mass Spectrom. 2003,14,1306-1314. 

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