A new phosphoproteomics strategy from Xue et al. in Molecular and Cellular Proteomics takes the KALIP (kinase assay–linked phosphoproteomics) workflow “pro”—to the protein level, that is. The new method, called proKALIP, permits identification of kinase substrates that need to be intact proteins in order to be phosphorylated. Like the original KALIP approach, in vitro and in vivo phosphorylation assays are run side-by-side to look for substrates that show up in both. In first-generation KALIP, cells are treated with a phosphatase inhibitor, proteins are extracted and digested, and phosphopeptides are immunoaffinity purified, thus narrowing the substrate screen to factors that are legitimately phosphorylated in the cellular environment. These peptides are incubated with phosphatase (subsequently heat inactivated) and the sample is then split in two for treatment with and without the kinase of interest. Phosphopeptide purification and mass spectrometry for substrate identification follow. The advantage of using peptide substrates is that endogenous kinases have been digested away and thus can't muddy the results. The catch is that KALIP may miss legitimate substrates. proKALIP skips the digestion step, but, after affinity purification, the beads are incubated with an irreversible kinase inhibitor to block interference by endogenous kinases. Another change is isotopic labeling to control for incomplete phosphate removal during the phosphatase step. Accordingly, a “heavy” extract incubated with exogenous kinase is mixed with a “light” control sample, and vice versa, so that mass spectrometry can detect peptides that are differentially phosphorylated. At the same time, as with KALIP, extracts from cells treated with and without kinase inhibitor are analyzed by mass spectrometry to reveal the kinase-dependent phosphoproteome. Limitations include the requrement for an efficient antibody for immunoprecipitation of the potential substrate pool and the need for a specific kinase inhibitor (though potent knockdown may be a viable alternative). The authors’ used proKALIP to identify 21 new candidate substrates for SYK, a tyrosine kinase implicated in tumorigenesis, showing the value of combining in vitro kinase screening with endogenous phosphorylation profiling.
L. Xue et al. Identification of direct tyrosine kinase substrates based on protein kinase assay-linked phosphoproteomics. Mol Cell Proteomics. [Epub ahead of print, June 22, 2013; doi:10.1074/mcp.O113.027722].Stick together
The poorly matched ex who clings on to a relationship, the cad who callously dumps his intended—these are soap opera plot descriptions but could also be applied to the drama of nucleic acid hybridization. The tension comes from a fundamental trade-off between affinity and selectivity: the longer the probe, the greater its affinity for targets, whether they are perfectly complementary or mismatched. To date, this problem has been addressed by emphasizing selectivity over affinity—an unstructured probe is hybridized under stringent conditions, or a structured probe's internal base pairing competes with probe/target hybridization. When harsh temperature and buffer conditions aren't appropriate, structured probes seem optimal. However, these probes can't withstand the washes involved in procedures such as in situ hybridization. An alternative is to covalently link an unstructured probe to its complement. However, since the crosslinker is exposed in the unstructured oligonucleotide, linkage to nontarget sequences can occur during transient off-target hybridizations. In an article in JACS, Vieregg et al. step back from the zero-sum struggle between affinity and selectivity and describe a new class of probe, the shielded covalent (SC) probe. The oligonucleotide consists of a hairpin with a photoactivatable crosslinker in the stem. A single-stranded toehold binds the target sequence, and by competitive branch migration, the hairpin unravels until there is full probe/target hybridization. A blast of 365 nm light crosslinks a nucleoside analogue to the target, securing the pair for stringent washes or other harsh conditions. Because the probe is structured, selective hybridization is possible without high temperature. Moreover, the structure means that the crosslinker is unlikely to participate in off-target reactions. The authors tested DNA and RNA probes with both types of oligonucleotide targets, showing near-quantitative capture of perfectly matched targets, with discrimination ratios of two to three orders of magnitude against sequences with two mismatches. Single-nucleotide mismatches with DNA target and probes gave a median discrimination ratio of 90, which is within the range of other probes designed for this level of resolution. The new probes selectively bound their targets in a solution containing an excess of single or double mismatched sequences, maintaining yield and selectivity at target sequence concentrations down to the 1 nM detection limit of the assay. Conveniently, the crosslink is nearly fully reversible after UV irradiation at 311 nm. While further work is needed to validate these probes in real-world detection, their attributes suggest that they may be particularly suited to visualizing gene expression in situ or regulating gene expression in photoaccessible tissues.
J.R. Vieregg et al. 2013. Selective nucleic acid capture with shielded covalent probes. J Am Chem Soc. 135:9691-9.