Dead or Alive?

Activity-based protein profiling selectively identifies active enzymes by use of a probe that will only interact with a functional active site and not an inactive variant or zymogen. One means of quantifying catalytically active proteins is by reacting protein extracts with ABPP probes, performing a fluorescent labeling step that will light up the activity probe, and then running the output on an SDS gel. Converting to mass spectrometry-based quantification would be preferable to most proteomics researchers, but attempts to do this have not yet been fully satisfactory. Now, a report from Cravatt, Gygi, and colleagues promises an appealing solution they call CAPE, for catch-and-release activity profiling of enzymes. In this approach, the ABPP probe is a multipart molecule consisting of the reactive group, a cleavable linker, a biotin tag, and spacer arms. After the probe has reacted with the active site of a protein, the enzyme can be captured by biotin-avidin affinity chromatography. Following capture, the isolated protein can be released by cleavage, concentrated, and then subjected to mass spectrometry. Because the object is to obtain quantitative data, the cell cultures under analysis (the source of the ABPP-labeled proteins) will have been grown with amino acids containing heavy or light isotopes of carbon and nitrogen. Thus, cells from control and experimental cultures will have proteins that are distinguishable by MS. Since the extracts from the two conditions can be mixed and analyzed together, very accurate quantification of relative amounts is possible. The authors applied CAPE to analysis of serine hydrolases in two prostate cancer lines that display different propensities for metastasis when implanted into nude mice. Nine hydrolases were found to be present in significantly different levels in the two cell types. Significantly, a related stable isotope MS method that does not employ activity selection did not pick up three of these proteins, suggesting the importance of focusing on active proteins of a particular enzymatic class. Similarly, spectral counting MS, which is favored for label-free quantification, also failed to pick up three enzymes identified by CAPE. This approach is therefore likely to spur many informative experiments aimed at understanding the functional consequences of changes in protein activity.

-Everley et al. 2007. Assessing enzyme activities using stable isotope labeling and mass spectrometry. Molecular and Cellular Proteomics [Epub ahead of print, July 11, 2007].

Force Multiplier

Atomic force microscopy uses tiny cantilevers to measure the picoNewton-scale forces associated with biomolecular interactions. Ultrasensitive force measurements are, unfortunately, also ultra-susceptible to tiny variations in surface uniformity. Therefore, it makes to standardize and control the experimental conditions as much as possible reproducible, accurate quantification of molecular binding events. While mixed self-assembled monolayers (SAMs) have been widely employed, dendrons might provide significant advantages. A dendron is a molecule that can be polymerized into a defined, consistent shape. A group based in South Korea has previously shown that self-assembly of cone-shaped dendrons on surfaces can be used to generate a DNA microarray with enhanced hybridization efficiency and selectivity. This improved performance likely follows from the optimal spacing of the DNA probes, such that steric hindrance is minimized and hybridization efficiencies approach levels seen in solution. Now, a team composed of researchers from Korea and the United Kingdom has applied this mesospacing strategy to biomolecular force measurement via AFM. Probe and substrate surfaces were modified with conical dendrons bearing tethered oligonucleotides. Interactions between the DNA sequences was measured by recording attractive and unbinding forces. In work now appearing in JACS, the authors characterize their system and its potential for force-based DNA arrays. They find a length-dependent increase in attractive forces, showing that 10-bp differences in oligonucleotide size as well as single base mismatches can be easily distinguished. Their careful elaboration of the binding process suggests that dendron-based force detection strategies are likely to facilitate sensitive, efficient measurement of biomolecular interactions.

-Jung et al. 2007. Dendron arrays for the force-based detection of DNA hybridization events. JACS [Epub ahead of print, July 11, 2007].

Killing the Messenger

It's not always easy being an mRNA: one small sequence motif can make a transcript the target of miRNAs determined to chop it up or block its translation. A small consolation, perhaps, is that researchers are intensely interested in these unfortunate victims. miRNA-mediated processes are recognized as important regulators of gene expression, and much attention has been directed at which mRNAs are targeted by miRNA complexes. Although computational approaches have successfully predicted miRNA targets on the basis of mRNA-miRNA pairing rules, a team based at EMBL was convinced that a wet-lab approach would be a useful complement to in silico methods. Easow et al,. hypothesized that miRNPs that oversee translational inhibition would form stable complexes with their mRNA targets, and that this interaction could be used to isolate miRNP-associated mRNAs. Working in Drosophila cells, Easow et al,. constructed an HA-tagged version of Argonaute 1. The team immunopurified Ago1-containing complexes and then isolated the associated RNAs, which were then analyzed on Drosophila cDNA arrays. They found 89 RNAs that appeared to be enriched in the isolated miRNPs, suggesting that they might be targets of particular miRNAs. To provide supportive evidence for these putative hits, the authors turned to sequence analysis and looked for motifs complementary to known Drosophila miRNAs. This process returned a list of miRNAs whose target sequences were overrepresented in the isolated mRNAs. Additional confidence in these hits was obtained by characterizing luciferase expression constructs containing the motifs that were believed to target the transcripts for miRNP binding. Other experiments explored the effect of expressing an miRNA not normally present in this cell line, and suggested that it is possible to define targets of specific miRNAs of interest in this way. Although not all mRNAs that are known to be targeted by miRNPs were identified, this strategy did pick up new candidate targets, and thus represents an important way to supplement computational predication methods.

-Easow et al. 2007. Isolation of microRNA targets by miRNP immunopurification. RNA [Epub ahead of print, June 25, 2007].

Go With the Glow

Chemiluminescence features widely in protein analysis by Western blot, but with recent findings from Xiong et al,., this detection method may soon extend its reach even more broadly in the proteomics arena. In previous publications, the authors had described the successful chemiluminescent detection of metalloproteins by a metal ion-catalyzed reaction involving luminol and hydrogen peroxide. Since not all proteins contain an appropriate metal catalyst, the authors explored the feasibility of using a silver ion-based probe for global protein labeling following gel electrophoresis. The process is a little unusual compared to the steps familiar to those used to detection by Coomassie® blue or silver staining. After fixation, the gel is incubated first with a solution containing sodium acetate and sodium thiosulfate, and then in 0.4% silver nitrate. The gel slab is subsequently transferred to a glass plate and placed in a specially designed chamber into which saturated ammonia is pumped; this step produces the [Ag(NH3)2]+ probe. Once the gel is brought to a dark room, it is exposed to potassium persulfate and luminol, thus initiating the chemiluminescent reaction. The procedure takes less time than Coomassie staining/destaining, but it is more labor-intensive. However, the payoff is that chemiluminescent detection does not interfere with subsequent mass spectrometry. By contrast, Coomassie blue needs to be removed prior to trypsin digestion, while the formaldehyde necessary for silver staining can cause protein crosslinking and confound protein identification via MS. Although this new chemiluminescent method cannot compete with silver staining's sensitivity, it does have a larger dynamic range compared to traditional staining methods. Further improvements to sensitivity will likely be needed before chemiluminescence becomes a viable alternative for all in-gel protein detection needs, but the authors' detection approach is a worthy option for labs looking to maximize MS detection of electrophoretically separated protein samples.

-Xiong et al. 2007. A novel [Ag(NH(3))(2)](+) probe for chemiluminescent imaging detection of proteins after polyacrylamide gel electrophoresis. Proteomics [Epub ahead of print, July 3, 2007]