Special Section: Mass Spectrometry for Proteomics Analysis

The scope of techniques covered by BioTechniques is extensive, ranging from permutations of PCR through microarray analysis to imaging of molecules in living cells. New methods that have been developed and applied in diverse areas of life science research populate every issue. Periodically, the editors select a particular technology or area of research and explore it in greater depth than is possible in the regular monthly issue, informing both experienced users of the technology and interested non-users alike. In this issue we are pleased to present a special section, Mass Spectrometry for Proteomics Analysis. Analysis of proteins by mass spectrometry encompasses a variety of approaches for the detection, characterization, and quantification of proteins and peptides. Review articles in this issue discuss ion/ion chemistry—a developing technology for protein sequence analysis (Good and Coon, p. 783), analysis of posttranslational modifications by tandem mass spectrometry (Larsen et al., p. 790), and strategies for sampling and bioinformatic analysis in biomarker discovery (Conrads et al., p. 799). John Fenn, 2002 Nobel laureate in Chemistry for his contributions to the development of electrospray ionization, steps away from the topic of mass spectrometry per se to share his thoughts on graduate education in a personal Perspective on page 780. The special section commences on p. 779 with an Introduction by John Yates of The Scripps Research Institute, which highlights the major milestones in this field of research and provides a brief overview of the areas treated in the Reviews.

Substitute Teacher

Despite the relative ease of genome manipulation in S. cerevisiae, researchers are always looking to learn still more convenient and rapid methods for substituting yeast promoters. Replacing a gene's native promoter with a heterolo-gous promoter of choice allows regulated expression and simplifies the task of discerning functional relevance. Although a host of clever chromosomal insertion strategies have been described over the years, the advent of the S. cerevisiae Genome Deletion Project provides an incredible resource for a further streamlined workflow. The strategy, explained by Liko et al. on p. 728 is appealingly simple. The genome deletion project resulted in a collection of strains in which a single ORF is replaced with a kanamycin resistance module. Although the purpose of the collection is to have a comprehensive resource of essentially all possible knockouts, the authors point out that for almost any given yeast promoter of interest there will be a strain in which the ORF immediately upstream has been replaced by kanamycin. Therefore, by PCR amplifying the kanamycin-promoter segment with amplification primers designed to be homologous to the targeted insertion site, researchers can generate a cassette suitable for promoter swapping. The authors illustrate the feasibility and ease of this approach by a variety of promoter substitutions. Their system should make promoter replacement a simple matter of a single PCR followed by standard yeast transformation.

Using the Force

The mention of “dark quenching” may conjure up images of light-saber battles between the forces of ruddy-faced Good and mellifluous-voiced Evil. In the molecular sciences, however, the scenario is usually far more benign and significantly more practical. The process of dark quenching, in which bringing two molecules together, a fluor and a quencher, results in the abrogation of a light signal, can be seen as being the flip-side of fluorescence resonance energy transfer (FRET). FRET assays, where the close proximity of two molecules creates fluorescence, have been used previously to monitor, in real-time, the process of homologous recombination in vitro. A challenge with this and other fluorescence assays is to obtain a sufficiently high signal over the background, an issue that commonly confounds many such experiments. Kaboev et al. on p. 736 of this issue therefore decided to go over to the dark side by using a dsDNA template carrying a FAM fluor as well as a DABCYL quencher molecule. The propinquity of the fluor and quencher mean that no signal is seen until recombination takes place, at which time one of the DNA strands is liberated and the inhibition is released, resulting in a fluorescent signal. Using this modification, as well as designing the probe with a triple repeat of the target sequence, the authors were able to demonstrate an impressive 5- to 10-fold improvement in the signal-to-background ratio.

Urchin Assay

Tubulin, in the form of multiple α and β dimers, makes up the dynamic, helix-like microtubules found in eukaryotic cells. These cylindrical structures are part of the cellular cytoskeleton and play an essential part in intracellular transport, as well as forming the mitotic spindle during cell division. Blocking tubulin polymerization affects microtubule assembly and perturbs cell division by disruption of the mitotic spindle and its role in sister chromatid separation. This effect is exploited for cancer treatment using the taxane class of drugs (such as paclitaxel and docetaxel), which target rapidly dividing tumor cells. Unfortunately, this treatment is a somewhat blunt instrument; numerous side effects and drug administration issues exist, provoking scientists to look for better drug candidates. However, the testing of new anti-proliferative molecules is currently a difficult and time-consuming affair. For this reason, Semenova et al. (p. 765) developed a simpler and more direct assay to assess antimitotics, utilizing sea urchin embryos at two different stages of development: fertilized eggs that were examined for mitotic arrest and free-swimming blastulae that were assessed for the effect of tubulin destabilization on cilia-dependent movement. The screening protocol developed by the authors was validated using known antimitotic molecules, providing evidence that it is a rapid, effective, and reproducible model for identification of potentially useful antiproliferative agents.

Living on the Edge

The appearance of ruffles, F-actin-rich membrane protrusions, has been linked with the establishment of cellular polarity. Despite knowledge of their existence both in vivo and in cultured cells, the biochemistry behind ruffle formation is still not fully resolved. Currently, evaluation of ruffles is performed manually by fluorescent or phase contrast microscopy, with the former using F-actin-specific fluorescently labeled phalloidin. This labor-intensive work is at best semiquantitative, as well as being tedious and frequently error prone. The availability of an automated means to evaluate ruffle formation is therefore sorely needed; this void has now been filled by a system described by Yi et al. on p. 745. The authors report on a new, fully-automated method that makes use of image processing and analysis of 2-D cell images. Following rhodamine-phalloidin staining of cells in culture, the image components were separated on the basis of differences in their intensity values, allowing for facile identification of the cell boundaries. Once this was achieved, open source ImageJ software was used to tease out the putative ruffle structures, and the results were classified and quantified using a piece of macro code written to work with ImageJ. The method is dramatically quicker by a factor of at least 10, while achieving very similar results to manual analysis. It can also potentially be used to analyze images captured by other forms of optical detection, including light microscopy.

Reduce, Reuse, Recycle

Epigenotyping has become increasingly prevalent as a result of the accumulating evidence linking aberrant DNA methylation with human disease. Specifically, methylation of CpG islands, which are present in the promoters and first exons of many genes, appears to be an early step in tumorigenesis. There are a plethora of techniques for standard genotyping; the same is true for epigenotyping methods. That said, one popular strategy is pyrosequencing of bisulfite-treated DNA. The well-established bisulfite strategy results in the conversion of cytosine, but not methylated cytosine, to uracil via deamination by bisulfite under acidic conditions. Pyrosequencing excels at direct quantitative sequencing, so its application to bisulfite-treated DNA can yield robust measurements of methylation. However, pyrosequencing is limited by sequence read length. As a result, only a few CpGs can be assessed in any one pyrosequencing reaction. This drawback can be expensive; more importantly, if the sample is a clinical specimen, the genomic DNA can be depleted before all the CpGs can be analyzed. On p. 721, Tost et al. describe how to circumvent this problem by stripping the sequencing primer off the template DNA in order to permit annealing of another primer and repetition of the sequencing process. The method uses the familiar combination of biotinylated primers and streptavidin-coated Sepharose™ beads, and allows up to seven repeated pyrosequencing runs without loss of accuracy.