Method MALDI-TOF

Dual analyses of ricin proteins are performed with MALDI-TOF-MS and LC-MS methods. MALDI-TOF-MS analysis is only able to determine an approximate molecular weight (MW) due to posttranslational modifications that cause ricin to be heavily glycosylated. Thus, LC-MS/MS is employed in concert with MALDI-TOF-MS analyses to identify peptides for accurate protein sequencing from a protein digest [80, 82-83]. Typical detection limits for the MALDI-TOF-MS analysis are between 50 ng/mL and 4 pg/mL. The proteomic... 

Two relatively new techniques, matrix assisted laser desorption ionization-lime of flight mass spectrometry (MALDI-TOF) and electrospray ionization (FS1), offer new possibilities for analysis of polymers with molecular weights in the tens of thousands. PS molecular weights as high as 1.5 million have been determined by MALDI-TOF. Recent reviews on the application of these techniques to synthetic polymers include those by Ilantoif54 and Nielen.555 The methods have been much used to provide evidence for initiation and termination mechanisms in various forms of living and controlled radical polymerization.550 Some examples of the application of MALDI-TOF and ESI in end group determination are provided in Table 3.12. The table is not intended to be a comprehensive survey. 

Hensley ei al.,n reported the only direct experimental observation of head-to-head linkages in PS by 2D INADEQUATE NMR on, 3C-enriched PS. The method did not enable these groups to be quantified with sufficient precision for evaluation of W tc- Zammit el a/.130 studied chain distribution of low molecular weight PS prepared with AIBN initiator by MALDI-TOF. Separate distributions of chains formed by combination and disproportionation were observed. They estimated kJkK. at 90 °C to be 0.057. 

During the past decade, MALDI-TOF MS has proven to be an effective tool for the analysis of oligo- and polymeric mannoglucans (for extensive reviews see [222,223]). SEC/MALDI mass spectrometry was employed in the analysis of hemicelluloses isolated by microwave heat-fractionation from spruce and aspen wood [94]. These methods allowed the separation and characterization of the oligo- and polysaccharide fractions derived from the xylan and mannan components of both woods [224]. 

However, because of the high price, MALDI-TOF mass spectrometers have not come into wide use. Vapor pressure osmometry (VPO), an old and traditional method for estimating molecular weight, is useful in the field of CPO chemistry. The experimental error of this measurement is approximately 10% however, the obtained data are sufficiently useful to estimate the number of porphyrins in a molecule. 

Current proteomics studies rely almost exclusively on 2D gel electrophoresis to resolve proteins before MALDI-TOF or ESI-MS/MS approaches. A drawback of the 2D gel approach is that it is relatively slow and work intensive. In addition, the in-gel proteolytic digestion of spots followed by mass spectrometry is a one-at-a-time method that is not well suited for high throughput studies. Therefore, considerable effort is being directed towards alternate methods for higher throughput protein characterization. 

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... 

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... 

It is now possible to evaluate the reproducibility and taxonomic utility of the MALDI-TOF MS method using spectra generated over a period of about eight years.3,61,62 Comparisons between MALDI-TOF MS and other standard methods have now been reported,23 and the method clearly can be used for rapid screening, if not yet for positive identification. 

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