A recently developed proteomics strategy, designated tagging-viasubstrate (TAS) approach, is described for the detection and proteomic analysis of farnesylated proteins. TAS technology involves metabolic incorporation of a synthetic azido-farnesyl analog and chemoselective derivatization of azido-farnesyl-modified proteins by an elegant version of Staudinger reaction, pioneered by the Bertozzi group, using a biotinylated phosphine capture reagent. The resulting protein conjugates can be specifically detected and͞or affinity-purified by streptavidin-linked horseradish peroxidase or agarose beads, respectively. Thus, the technology enables global profiling of farnesylated proteins by enriching farnesylated proteins and reducing the complexity of farnesylation subproteome. Azido-farnesylated proteins maintain the properties of protein farnesylation, including promoting membrane association, Ras-dependent mitogen-activated protein kinase kinase activation, and inhibition of lovastatin-induced apoptosis. A proteomic analysis of farnesylated proteins by TAS technology revealed 18 farnesylated proteins, including those with potentially novel farnesylation motifs, suggesting that future use of this method is likely to yield novel insight into protein farnesylation. TAS technology can be extended to other posttranslational modifications, such as geranylgeranylation and myristoylation, thus providing powerful tools for detection, quantification, and proteomic analysis of posttranslationally modified proteins.

The natural product salicylihalamide is a potent inhibitor of the Vacuolar ATPase (V-ATPase), a potential target for antitumor chemotherapy. We generated salicylihalamide-resistant tumor cell lines typified by an overexpansion of lysosomal organelles. We also found that many tumor cell lines upregulate tissue-specific plasmalemmal V-ATPases, and hypothesize that tumors that derive their energy from glycolysis rely on these isoforms to maintain a neutral cytosolic pH. To further validate the potential of V-ATPase inhibitors as leads for cancer chemotherapy, we developed a multigram synthesis of the potent salicylihalamide analog saliphenylhalamide.As products of evolution, natural products are selected for interaction with living systems. As such, an unbiased quest to study their function will inevitably lead to discoveries in biology, potentially with therapeutic implications. 1 By accessing congeners for mode-of-action studies, optimization of potency and pharmacokinetic, toxicological, and metabolic properties, synthesis takes center stage as an enabling tool to execute a natural product-based discovery and development program. 2 Herein, we report our efforts to validate inhibition of the Vacuolar ATPase (V-ATPase), the target of salicylihalamide, as a strategy for cancer chemotherapeutic intervention. This program led to the selection and multigram synthesis of a salicylihalamide analog saliphenylhalamide (2, SaliPhe).The marine metabolite salicylihalamide A (1), 3 the first member of a family of marine and terrestrial metabolites characterized by a signature N-acyl-enamine appended macrocyclic salicylate, has elicited a great deal of interest from the synthetic community 4 -certainly due in part because of their growth-inhibitory activities against cultured human tumor cells and oncogene-transformed cell lines through mechanisms distinct from standard clinical antitumor agents. 5 The cellular target of SaliA remained elusive until after our first total synthesis, 3b when Boyd and coworkers reported that SaliA and other related benzolactone enamides inhibit V-ATPase activity in membrane preparations of mammalian cells, but not V-ATPases from Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript yeast and other fungi -an observation that distinguishes them from previously identified VATPase inhibitors. 6 Our biochemical studies utilizing a reconstituted, fully purified bovine brain V-ATPase confirmed this activity and demonstrated that SaliA binds irreversibly to the trans-mem...

A chemoselective alkylation method is described for the isolation and subsequent identification of thiophosphorylated peptides/proteins. The method involves thiophosphorylation of proteins using adenosine 5-O-(thiotriphosphate) (ATP␥S) followed by selective in situ alkylation of the newly thiophosphorylated proteins resulting in a stable covalent bond. The chemoselective alkylation exploits the relatively high nucleophilicity at low pH of the sulfur in thiophosphate residues, whereas the nucleophilicities of phosphates, amines, and other functionality of amino acids are negligible or significantly suppressed. Modified alkylation reagents linked to biotin or solid supports (e.g. glass or Sepharose beads) with or without a photocleavable linker facilitate the isolation of the thiophosphorylated peptide/proteins. This approach is demonstrated through the localization of phosphorylation sites on myosin regulatory light chain. We anticipate that this technique will be useful for isolation and subsequent identification of newly thiophosphorylated proteins, produced either in vivo or in vitro, thus facilitating the dissection of protein phosphorylation networks. Molecular & Cellular Proteomics 2:242-247, 2003.Protein phosphorylation can profoundly affect the function of proteins and is often associated with cellular regulation, inter alia, such as transcription, replication, apoptosis, and signal transduction (1). Analysis of the human genome reveals that ϳ2% of human genes code for protein kinases or protein phosphatases (2). While many kinases are known to be involved in various cellular functions and disease progression, their substrates and phosphorylation sites remain largely unknown. Since characterization of protein phosphorylation networks requires a detailed knowledge of kinase/phosphatase substrates and their specificities as well as localization of phosphorylation sites, efficient techniques for the isolation of newly phosphorylated peptides/proteins would expedite progress in these areas.A variety of methods have been described for the isolation of phosphate-containing peptides/proteins, including metal ion affinity purification (3), tagging phosphorylated residues through chemical reactions (4, 5), and affinity purification by immobilized anti-phosphoryl-residue antibodies (6). Many of the preceding methods have been used for the identification of protein phosphorylation sites, for the isolation of phosphorylated signal proteins, and for global surveys of protein phosphorylations (3-6). While powerful, these methods are not able to distinguish newly phosphorylated proteins from steady-state phosphorylated proteins. Since one-third of proteins in a cell are estimated to be phosphorylated (7,8), isolation of newly phosphorylated proteins from unphosphorylated and/or steady-state phosphorylated proteins would simplify the identification of newly phosphorylated proteins responding to diverse signal stimulations. Herein we report a novel method for the isolation of thiophosphorylated peptides/proteins. The meth...