Whole cell sampling, with

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... 

In the whole cell sample, doublet 1 has the same parameters as observed in purified pMMO. The 8.0 T spectrum of Figure 2.21b shows that the iron of doublet 1 resides in a diamagnetic complex, and thus this material most likely represents the same species in both samples. At this juncture, it will be useful to compare the amount of doublet 1 iron with catalytic activity. The purified sample of Figure 2.20a contained 240 flM iron associated with doublet 1 taking into account the pMMO protein concentration and our conclusion that doublet 1 represents a dinuclear cluster, we... 

One hundred and twenty-three individual phytoplankton clones representing twelve algal classes were examined for total DMS(DMSP) production. DMS was measured in whole cultures, cell and filtrate fractions (as outlined in Figure 1). DMS measurements for whole cultures (cells and filtrate) are given in Table I. Values for DMS levels in all fractions, whole culture, filtered cells and filtrate are not reported here but comparisons of the whole culture sample with the filtered cells and accompanying filtrate revealed that, for the most part, amounts of DMS in the two fractions were equivalent to the amount in the whole culture. Differences can largely be attributed to filtering problems. Even... 

FIGURE 14.1 Recently developed whole cell sampling schemes, (a) Continuous cell introduction. (From Chen, S. andLillard, S.J., AnaZ. Chem., 73, 111, 2001. With permission.) (b) Laser cavitation. (From Sims, C.E. et al.. Anal. Chem., 70, 4570, 1998. With permission.) (c) Fast electrical lysis. (From Han, R, et al.. Anal. Chem., 75, 3688, 2003. With permission.)... 

Heat protein in SDS sample buffer for 10 min at 37°C before loading on an SDS gel. SATBl can be detected in 5 to 10 fig whole-cell extract with the wild-type (25)s probe. Load 5 fil prestained marker or 1 / biotinylated marker (BioRad). 

Changes in the reference electrode junction potential result from differences in the composition of die sample and standard solutions (e.g., upon switching from whole blood samples to aqueous calibrants). One approach to alleviate this problem is to use an intermediate salt bridge, with a solution (in the bridge) of ions of nearly equal mobility (e.g., concentrated KC1). Standard solutions with an electrolyte composition similar to that of the sample are also desirable. These precautions, however, will not eliminate the problem completely. Other approaches to address this and other changes in the cell constant have been reviewed (13). 

Whole cell OPH activity was measured by following the increase in absorbancy of p-nitrophenol from the hydrolysis of substrate (0.1 mM Paraoxon) at 400 nm (sm = 17,000 M cm ). Samples of culture (1 ml) were centrifuged at 10,000 g and 4 C for 5 min. The cells were washed, resuspended with distilled water, and 100 pi was added to an assay mixture containing 400 pi 250 mM CHES [2-(N-cyclohexylamino)ethane-sulfonic acid] buffer, pH 9.0, 100 pi 1 mM Paraoxon, and 400 pi distilled water. One unit of OPH activity was defined as pmoles Paraoxon hydrolyzed per min. Each value and error bar represents the mean of two independent experiments and its standard deviation. 

Qmntitatlvz detznmivuition oi HzmogtobZn Ai by micAjochAo-matogAaphy - By means of this simple method (29, 30), Hb-A2 may be rapidly and accurately determined In blood samples from subjects with and without Hb-S or Hb-D The procedure makes use of DEAE-Cellulose, Inexpensive laboratory glassware, and allows one technician to determine the level of Hb-A2 In 50 samples a day Whole blood samples, red cell hemolysates, and blood... 

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... 

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... 

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. 

Thus far we tacitly assumed that our EPR tubes were filled with pure, or at least to some extent purified, biomolecular preparations. However, what will we find when we try to measure, for example, whole cells In other words, how complex can a sample actually be without us losing track of all the overlaying signals What would be the dynamic range of signal amplitudes that we can resolve from a single sample ... 

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