Perspective: Mass Spec for the Masses

It would be difficult to imagine proteomics in the absence of mass spectrometry, but the advent of instrumentation capable of dealing with large proteins has only been a relatively recent development. A key technological innovation was electrospray ionization (ESI) and the consequent ability to interface liquid chromatographic separations with MS-based analysis. Richard D. Smith, one of the earliest entrants into this area, now provides a glimpse of the future of ESI-MS in a Perspective in this issue. Smith first focuses on measurement sensitivity as the limiting factor in proteomics. With the increasing interest in extracting comprehensive information from tiny samples (even, for instance, single cells), the ability to reproducibly detect vanishingly small quantities of analyte becomes an absolute requirement. Smith explains how flow rate and column inner diameter in the LC phase influence both sensitivity and quantitation and discusses improvements in ESI-MS instrumentation to make best use of nanoLC-based separations. This Perspective should provide valuable insight to anyone involved in the use of ESI-MS for biological samples or interested in the technology's suitability for clinical applications. -Page 147

Attacking the Flanks

Isolating novel gene variants from environmental metagenomes can reveal immense genetic diversity and may allow the isolation of genes with therapeutically or industrially relevant properties. One standard approach is to PCR amplify an environmental metagenome preparation using degenerate primers targeted to the conserved region of the gene of interest. Clearly, this approach will not allow the cloning of complete genes—therefore a genome walking strategy for capturing flanking regions is also required. Uchiyama and Watanabe had been disappointed with the performance of traditional inverse PCR methods for recovering flanking sequences so they developed a modified inverse PCR approach that is better suited to metagenome walking. Their approach hinges on affinity purification of the inverse PCR product from the background metagenome. After obtaining impressive selectivity in pilot experiments, the authors demonstrate real-world applicability by efficiently recovering flanking sequences from putative chitinase gene fragments derived from a groundwater metagenome. -Page 183

Connect the Dots

Spot-finding is an important objective in image analysis, and a number of segmentation algorithms have been discussed in the literature. What has been lacking thus far, however, is a freely available and customizable package for automatically detecting and quantifying spot-like objects in micrographs. Fortunately, a new report from Schiffman et al. describes just such a tool, which is made possible by the integration of the previously described FindSpots algorithm into the Open Microscopy Environment (OME). OME is an open-source image informatics platform that contains a number of visualization and analysis packages. Until the inclusion of FindSpots however, OME lacked a module allowing identification and measurement of objects in microscopy images or time-lapse movies. The authors not only show that FindSpots/OME performs equivalently to a commercial image analysis program, but also that the package can be used for complex analysis of protein dynamics. The power of FindSpots/OME should make it a natural fit with high-content screening studies. -Page 199

Rainbow Nucleus

AGT, or O⁶-alkylguanine-DNA alkyltransferase, is a small (21.6 kDa) DNA repair protein that is responsible for removing alkyl groups from guanine residues by transferring them to an active site cysteine residue, which permanently inactivates the enzyme. This system has been co-opted by scientists interested in labeling specific proteins via an alternative and more flexible pathway than using a fluorescent protein like GFP. AGT fusion protein constructs are engineered in tandem with fluorescent substrate analogs based on a common AGT inhibitor, O⁶-benzylguanine. By transfecting AGT fusion proteins into cells and then adding the customized fluorescent substrate—either to the media if membrane permeable, or by microinjecting nonpermeable substrates—the fusion protein can be rapidly covalently linked to the fluor. This strategy provides great flexibility for labeling, since an essentially limitless variety of substrate derivatives can be created, and those that best suit the particular experimental application can be chosen. Testing some new dyes, Keppler et al. demonstrate the labeling of histone 2B (H2B) using a mutant AGT that allows for the blocking of wild-type AGT activity. The new fluors are generally more photo-stable and provide broader emission spectra, extending into the far red and near-infrared end of the spectrum, where less damaging excitation wavelengths can be used. -Page 167

Why Be Normal

Gene expression profiling by real-time RT-PCR is a much more straight-forward way to quantitate transcript abundance than traditional end point RT-PCR assays. Despite this progress in instrumentation and speed for measuring transcript levels, the need for appropriate normalization remains. Most relative quantification strategies rely on normalization via a reference gene or genes. However, if no suitable housekeeping genes are available, or the use of several internal controls is undesirable, gene expression level may also be normalized to the input RNA amount. Unfortunately, this strategy fails to take into account any variation introduced by the RT step. As an alternative, the RNA-DNA hybrids can be measured after the RT reaction. This approach, however, is limited to experiments that use polyadenylated RNA as input, since with total RNA the fluorescent dye would also bind the double-stranded regions of rRNA. Libus and Štorchová directly address these limitations with a new measurement approach that uses post-RT RNase digestion and the single-stranded dye RiboGreen®. Under these conditions, the fluorescent signal is directly proportional to the amount of single-stranded cDNA, making this method a convenient and quick means of reliable quantitative RT-PCR normalization. -Page 156

The advent of fluorophores to follow intracellular proteins has revolutionized the field of microscopy. But it's not all good. The creation of reactive oxygen species (ROS) during laser illumination of cells can speed up the photobleaching effect and even cause cell death. To reduce photobleaching effects, special filters have been used, and more resistant fluors and antifade media have been developed. Another solution is to reduce the amount of time that the sample is illuminated. Using this concept, Nishigaki et al. describe a new option that profoundly extends the time that samples can be examined and dramatically reduces photobleaching through the use of a custom built stroboscopic light emitting diode (LED) illumination system. To demonstrate the utility of LED light, fresh sperm were used—a particularly appropriate model system due to their sensitivity and ability to provide continuous reporting of cell viability. Loaded with a fluorescent indicator, sperm illuminated with the LED remained viable and motile, while those under continuous illumination from a regular mercury lamp showed declining motility over the course of the experiment, demonstrating reduced phototoxicity with the LED system. Photobleaching was observed only after loss of motility, but was significantly more pronounced in samples under regular illumination. -Page 191