currents
A bead-based kinase competition assay for chemical proteomics
GERARD DREWES
beads. They mixed their beads with a range of cell lysates and analyzed the bound proteins by MS. Kinobeads pulled down Nothing exemplifies the elegant subtlety of cell biology like 307 distinct protein kinases from 14 cell lines. protein kinases. Of the >500 kinases in the human genome, Once they had established that kinobeads bound a range each has a distinct role in keeping a cell functioning. Yet of kinases, the scientists wanted to test whether free kinase many have high structural homology, and they all essentially inhibitors could compete for binding with the immobilized incarry out the same reaction hibitors. They reasoned that (phosphorylation) on the the more the free inhibitor No compound 10 nM 100 nM 1 µM same substrate (another bound to its target, the less protein). With a few notable would be available to bind to exceptions, it has been very the bead. The investigators difficult to target an inhibitor mixed three ABL kinase into one kinase without afhibitors in varying concenfecting many of the others. trations with cell lysates and Traditional proteomic apthen exposed those lysates Bead Bead Bead Bead proaches for discovering to kinobeads. kinase inhibitors require that The researchers quanThe kinobeads assay. Lysates or cells are treated with an inhibitor over either the proteins or small titated bound kinases with a range of concentrations. At higher inhibitor concentrations, less target molecules be labeled or imisobaric tagging for relative protein is available for binding to kinobeads, whereas nontargets are unmobilized; this process can and absolute quantitation affected. introduce artifacts into the (known as iTRAQ) and MS measurements. analysis, then calculated Now Bernhard Kuster, Gerard Drewes, and colleagues at concentration for 50% inhibition in vitro (known as IC50 ) valCellzome (Germany and U.K.) have developed “kinobeads”, ues from their data. They found that all three inhibitors bound a tool to study the interaction of an inhibitor with many kinasto multiple kinases at significant levels. They also noted that es in solution simultaneously without the need for labels. To the method could be used early in the drug discovery process construct kinobeads, the scientists attached a panel of seven to help researchers better understand and fine-tune inhibitor nonselective ATP-competitive kinase inhibitors to sepharose selectivity. (Nat. Biotechnol. 2007, 25, 1035–1044)
Calcium phosphate precipitation and IMAC for phosphopeptide enrichment Although many methods for the enrichment of phosphopeptides exist, they are all far from perfect. Chemical derivatization techniques are selective, but because they include multiple steps, a lot of sample can be lost along the way when these methods are applied. In addition, side reactions can complicate the analysis. Chromatographic procedures such as immobilized metal-ion affinity chromatography (IMAC) are not specific for phosphorylations, and other negatively charged groups can bind. Therefore, Peter Roepstorff and colleagues at the University of Southern Denmark developed a new, highly selective method for phosphopeptide isolation that is compatible with LC/MS. The approach combines calcium precipitation with IMAC; this protocol greatly increases the number of phosphopeptides that can be isolated from complex proteomic mixtures compared with IMAC alone. © 2007 American Chemical Society
When tryptic digests of - and b-casein were analyzed by MS, few phosphopeptide signals were observed. However, after calcium precipitation of the digests, several new phosphopeptide signals were present in the spectrum. Next, - and b-casein were diluted 50-fold in a mixture that contained three other proteins. In this case, calcium phosphate precipitation did not greatly enhance the number of phosphopeptides observed by MS compared with the original sample. This result prompted Roepstorff and colleagues to combine the precipitation method with IMAC. When the diluted mixture was precipitated, desalted, and run on an IMAC microcolumn, phosphopeptides were significantly more enriched than with either precipitation or IMAC alone. Finally, the calcium precipitation and IMAC approach was applied successfully to the analysis of the rice embryo phosphoproteome. The researchers also demonstrated that even more phosphopeptides can be isolated when complex proteomes are analyzed
by performing successive rounds of IMAC after the calcium precipitation step. (Mol. Cell. Proteomics 2007, DOI 10.1074/mcp.M700278-MCP200)
A new protein PTM: pyrophosp horylation Protein pyrophosphorylation is a novel posttranslational modification (PTM) for proteins, according to Solomon Snyder and colleagues at the Johns Hopkins University School of Medicine, University College London, the University of Rhode Island, and the University of Utah. In previous work, the researchers demonstrated that diphosphoinositol pentakisphosphate (IP7), which is not a kinase, can donate a phosphate group to proteins that contain polyserine tracts. After further investigation, they now say that IP7 adds a second phosphate to amino acids that are phosphorylated already. In experiments with various yeast and mammalian substrates, prephosphorylation of residues (priming) was
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currents Toolbox NetworKIN Conventional biochemistry studies and high-throughput projects have helped researchers identify thousands of phosphorylation sites. The challenge, however, is to match up the phosphorylation sites to the responsible kinases. Sequence motifs help narrow the list of possible kinases, but this strategy typically does not allow researchers to pinpoint the specific kinase that phosphorylates a particular site in vivo. Therefore, Rune Linding, Peer Bork, Michael Yaffe, Tony Pawson, and colleagues at the European Molecular Biology Laboratory, the Max Delbrück Center for Molecular Medicine (both in Germany), Mount Sinai Hospital (Canada), the Massachusetts Institute of Technology, and Erasmus University (The Netherlands) developed NetworKIN. The new computational approach combines sequence motif data with contextual information, such as subcellular localization and temporal expression, to figure out which kinases phosphorylate certain substrates. In the first step, NetworKIN uses neural networks and scoring matrices to discard unlikely kinase families. In the next step, contextual information from the STRING database is analyzed for each substrate to create a probabilistic protein network. Kinases are identified by locating the one that is the closest to the substrate in the network. When researchers applied NetworKIN to 282 known phosphorylation sites, it had a higher prediction accuracy but a somewhat lower sensitivity than the motif method alone. (Cell 2007, 129, 1415–1426)
myProMS Patrick Poullet and colleagues at Institut Curie (France) have developed myProMS, a web server that helps biologists and chemists organize, mine, and share results from MS database search engines. The web server is connected to a MySQL or Oracle database so that multiple scientists can access the data. With myProMS, access is granted to all of the users only after a defined group of curators validates and approves the data. The server is available to academic researchers for free at http://bioinfo. curie.fr/myproms. (Proteomics 2007, 7, 2553–2556)
necessary for IP7 pyrophosphorylation. Phosphatase treatment of a phosphorylated peptide greatly reduced the ability of IP7 to modify the substrate. IP7 could not add a second phosphate, however, to peptides that were pyrophosphorylated already or that had methylated phosphate residues. IP7-phosphorylated peptides also had different properties than those phosphorylated by kinases and ATP. For example, IP7-pyrophosphor ylations were more acid-labile but more resistant to enzymatic dephosphorylation than kinase-mediated PTMs. In addition, two novel isomers of IP7 can pyrophosphorylate proteins. The bio-
Microfluidic 2D DIGE analyzer
logical significance of this new PTM is unknown, but the researchers propose that IP7-mediated pyrophosphorylation may be involved in cell signaling. (Proc. Natl. Acad. Sci. U.S.A. 2007, DOI 10.1073/ pnas.0707338104)
Proteomics with a PVDF membrane Scientists often transfer proteins onto polyvinylidene fluoride (PVDF) membranes when they prepare samples for western blotting, but Yu-Ju Chen and co-workers at Academica Sinica, National Taiwan University, and National Defense University (all in Taiwan) have
(a)
(b)
Compared with conventional macroscopic approaches, microfabricated devices could allow proteomics scientists to study rare proteins more effi(c) ciently. As a step toward con0 ducting proteomics on a chip, Richard Mathies and colleagues 1 at the University of California Berkeley and the U.S. Naval Re2 search Laboratory developed a cm microfluidic device for 2D difference gel electrophoresis (DIGE). The analyzer features an Tiny 2D DIGE. (a) Schematic of the micro-DIGE arced channel for IEF (the first analyzer. (b) MFIs connect the IEF and native gel dimension). Native gel electroelectrophoresis dimensions. (c) Electron microphoresis (the second dimengraph of an MFI. (Scale bar = 100 μm.) sion) is conducted in 20 longer channels that are perpendicular to the IEF arc. SDS is omitted from the ducted a 2D DIGE experiment. Timegel and buffer in the second dimension, course samples were obtained from an because it induces artifacts. Mathies E. coli strain that was induced to exand colleagues say that this tweak repress high levels of maltose-binding duces the orthogonality and peak caprotein and from an uninduced control pacity of the separation compared with strain. Initially, the current and elution traditional 2DE separations, but the depatterns were not reproducible. The revice is still a useful demonstration of producibility was improved, however, 2DE on a microfluidic analyzer. by adding urea to the second-dimenTo minimize the mixing of reagents sion separation matrix and by lowering between the dimensions, the researchthe electric field strength of that sepaers connected the dimensions with ration. In another experiment, samples much shallower and narrower channels were run only on the second dimencalled microfluidic interfaces (MFIs). sion for a 1D native PAGE separation. Although MFIs reduce fluid flow, they In each run, the protein contents of still allow proteins to be electrophoret440,000 cells were observed. Mathies ically transported from the first dimenand colleagues say that this amount of sion to the second. protein is the smallest to be analyzed To test the device for proteomics on-chip with electrophoresis methods. applications, the investigators con(Anal. Chem. 2007, 79, 7360–7366)
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currents discovered another application for these surfaces. In previous work, the researchers showed that proteins immobilized on PVDF membranes can be analyzed directly by MALDI TOFMS. Now, they show that the membranes can be used as affinity probes for protein quantitation and profiling. In this method, a sample of a bait protein (i.e., an antibody) is deposited directly onto a PVDF membrane and allowed to dry. The antibody sticks to the PVDF because of its affinity with the hydrophobic surface of the film. After the membrane is blocked, it is incubated with a solution containing the targeted protein. Nonspecific proteins are removed by washing the membrane. The antibody–antigen complexes are analyzed directly from the PVDF surface with MALDI TOFMS. The new assay has many benefits. Compared with bead-based immunoprecipitations, the PVDF method results
Metabonomics for toxicity monitoring
Bisphosphonates typically are prescribed to patients who suffer from bone conditions such as osteoporosis and metastatic bone disease. These drugs also can disturb kidney function in some patients, however. To test whether metabonomic methods can shed light on how these drugs work, Hans Senn and co-workers at F. Hoffmann–La Roche, Ltd., performed metabonomic analyses in addition to conventional toxicity assays. With the metabonomic approach, they discovered a novel endogenous metabolite in rat urine that could be a biomarker for monitoring the farnesyl pathway. Rats were injected with vehicle or with a low or high dose of ibandronate (Boniva) or zoledronate (Zometa). Most of the rats that were treated with zoledronate died prematurely between days 5 and 7. Conventional serum and urine analyses of surviving rats showed abnormal levels of several metabolites compared with control rats. In addition, microscopic and macroscopic defects were observed in the livers and kidneys of zoledronate-treated rats. In contrast, ibandronate-treated rats had normal kidneys and livers, and the level of only one metabolite was significantly abnormal.
in reduced sample loss, because it does not require multiple purification and elution steps. In addition, immobilization of the bait protein is much easier than with conventional protein arrays, which typically require the protein to be chemically modified. Chen and co-workers tested the assay by immobilizing antibodies against three proteins on separate membranes. The correct target proteins were isolated from simple mixtures and from human plasma. In addition, the investigators could determine the concentrations of the three proteins in plasma from controls and patients with gastric cancer, and their data correlated well with those that were obtained in previously reported studies. The researchers say that because the PVDF assay is so easy to perform, it could be applied to the rapid screening of clinical samples, especially if the sensitivity is improved. (Proteomics 2007, 7, 3038–3050)
In the metabonomics study, Senn and co-workers obtained NMR spectra from the urine samples and performed principal components analysis on the data. Slight differences were observed for the profiles of ibandronate-treated and control rats. However, the time trajectory data for rats that were given high doses of zoledronate were biphasic. Betaine levels were elevated in the first phase. In the second phase, betaine was still elevated, but the levels of other metabolites also were aberrant compared with controls. Only the second phase was observed for rats injected with a low dose of zoledronate. Therefore, the metabonomic analysis revealed new insights into the effects of bisphosphonates. The unbiased metabonomics approach also allowed the scientists to discover a novel endogenous metabolite called N-acetyl-felinine in rats. This metabolite was detected with NMR and LC/MS/MS only in rats given zoledronate. Surprisingly, N-acetyl-felinine previously was thought to be unique to cats. According to the researchers, N-acetyl-felinine synthesis could result from the inhibition of farnesyl diphosphate synthase. The inhibition of this enzyme probably inhibits bone resorption, they say. (Chem. Res. Toxicol. 2007, DOI 10.1021/tx700151t)
Toolbox Chaorder assesses experimental bias Can researchers really compare results from separate experiments performed in different labs with different machines? To find out, Amol Prakash and co-workers at the University of Washington, the Fred Hutchinson Cancer Research Center, the Institute for Systems Biology, Institut Pasteur (France), the Institute of Molecular Systems Biology (Switzerland), and Wesleyan University developed Chaorder. The tool measures the similarity of all raw MS1 peaks from two LC/MS experiments after retention-time alignment. The output is a graph; points on the graph that are close to each other indicate that experiments produced similar data, whereas those that are far apart indicate that the data are different. After Chaorder is applied to data sets, scientists can make more informed decisions about experimental designs. The researchers applied Chaorder to many experimental data sets to assess reproducibility. The model of mass spectrometer, the particular LC column, and the number of freeze/thaw cycles caused variations in the data. One of the most important parameters that affected reproducibility, however, was the run order; in most of the experiments, data tended to cluster according to the date or the order in which samples were run. In fact, the researchers caution that this effect can obscure the detection of real biomarkers. Chaorder is available by contacting Prakash at
[email protected]. (Mol. Cell. Proteomics 2007, DOI 10.1074/mcp. M600470-MCP200)
Glycan analysis Snigdha Banerjee and Ningombam Meitei at Devi Ahilya University (India) have developed a mathematical method for glycan fragmentation analysis. With this method, researchers can predict all of the possible first- and second-generation fragments that result from various cleavages. In addition, the masses of the fragments can be calculated. The researchers also introduced a new nomenclature for glycan structures. This method can be incorporated into an algorithm or applied manually. (Proteomics 2007, 7, 2530–2540)
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