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.

Inositol pyrophosphates are recognized components of cellular processes that regulate vesicle trafficking, telomere length, and apoptosis. We observed that pancreatic beta cells maintain high basal concentrations of the pyrophosphate diphosphoinositol pentakisphosphate (InsP7 or IP7). Inositol hexakisphosphate kinases (IP6Ks) that can generate IP7 were overexpressed. This overexpression stimulated exocytosis of insulin-containing granules from the readily releasable pool. Exogenously applied IP7 dose-dependently enhanced exocytosis at physiological concentrations. We determined that IP6K1 and IP6K2 were present in beta cells. RNA silencing of IP6K1, but not IP6K2, inhibited exocytosis, which suggests that IP6K1 is the critical endogenous kinase. Maintenance of high concentrations of IP7 in the pancreatic beta cell may enhance the immediate exocytotic capacity and consequently allow rapid adjustment of insulin secretion in response to increased demand.

We have characterized the positional specificity of the mammalian and yeast VIP/diphosphoinositol pentakisphosphate kinase (PPIP5K) family of inositol phosphate kinases. We deployed a microscale metal dye detection protocol coupled to a high performance liquid chromatography system that was calibrated with synthetic and biologically synthesized standards of inositol pyrophosphates. In addition, we have directly analyzed the structures of biological inositol pyrophosphates using two-dimensional 1 H-1 H and 1 H-31 P nuclear magnetic resonance spectroscopy. Using these tools, we have determined that the mammalian and yeast VIP/ PPIP5K family phosphorylates the 1/3-position of the inositol ring in vitro and in vivo. For example, the VIP/PPIP5K enzymes convert inositol hexakisphosphate to 1/3-diphosphoinositol pentakisphosphate. The latter compound has not previously been identified in any organism. We have also unequivocally determined that 1/3,5-(PP) 2 -IP 4 is the isomeric structure of the bis-diphosphoinositol tetrakisphosphate that is synthesized by yeasts and mammals, through a collaboration between the inositol hexakisphosphate kinase and VIP/PPIP5K enzymes. These data uncover phylogenetic variability within the crown taxa in the structures of inositol pyrophosphates. For example, in the Dictyostelids, the major bis-diphosphoinositol tetrakisphosphate is 5,6-(PP) 2 -IP 4 (Laussmann, T., Eujen, R., Weisshuhn, C. M., Thiel, U., Falck, J. R., and Vogel, G. (1996) Biochem. J. 315, 715-725). Our study brings us closer to the goal of understanding the structure/function relationships that control specificity in the synthesis and biological actions of inositol pyrophosphates.Signal transduction pathways frequently rely on a specific target protein recognizing a precise spatial arrangement of one or more phosphate groups on either another protein or a small metabolite. The six-carbon inositol ring offers what is arguably the most dramatic example of how even subtle modifications to phosphate topology can impart signaling specificity. The combinatorial manner in which phosphate groups can be arranged around the inositol skeleton creates a large family of phosphorylated molecules, many of which have individual, physiological roles (1). The inositol pyrophosphates, such as diphosphoinositol tetrakisphosphate (also known as PP-IP 4 ), PP-IP 5 2 (also known as IP 7 ) and (PP) 2 -IP 4 (also known as IP 8 ) (2, 3), are a specialized subgroup of the inositol-based signaling family that are distinguished by the presence of diphosphate groups. These particular molecules regulate a diverse range of cellular activities, including phosphate sensing, actin cytoskeleton dynamics, apoptosis, vesicle trafficking, transcription, and DNA repair (see Refs. 4 and 5 for reviews). The different isomers of inositol pyrophosphates can be distinguished by biological receptors (6, 7). Thus, there is great interest in understanding the structure/ function relationships of protein interactions with the inositol pyrophosphate ligands. .). Instrum...

The O-linked N-acetylglucosamine (O-GlcNAc) modification of serine/threonine residues is an abundant posttranslational modification present in cytosolic and nuclear proteins. The functions and subproteome of O-GlcNAc modification remain largely undefined. Here we report the application of the tagging-via-substrate (TAS) approach for global identification of O-GlcNAc-modified proteins. The TAS method utilizes an O-GlcNAc azide analogue for metabolic labeling of O-GlcNAc-modified proteins, which can be chemoselectively conjugated for detection and enrichment of the proteins for proteomics studies. Our study led to the identification of 199 putative O-GlcNAcmodified proteins from HeLa cells, among which 23 were confirmed using reciprocal immunoprecipitation. Functional classification shows that proteins with diverse functions are modified by O-GlcNAc, implying that OGlcNAc might be involved in the regulation of multiple cellular pathways.The modification of nuclear and cytoplasmic proteins at serine and threonine residues with O-linked N-acetylglucosamine (OGlcNAc) was first described over two decades ago. 1 The modification was found in various classes of proteins including enzymes, transcription factors, cytoskeletal proteins, signaling proteins, receptors, nuclear pore complex proteins, and kinases. 2 Similar to phosphorylation, the O-GlcNAc modification is dynamic with a turnover rate faster than that of the proteins it modifies. 3 The O-GlcNAc modification has been shown to affect protein-protein interactions, protein-DNA interactions, protein stability and activity, and cell signaling cascades. 4 Disregulation of the OGlcNAc modification has been implicated in the development of disease states including diabetes, cancer, and Alzheimer's. 2,4 Given the potentially broad regulatory influence of the O-GlcNAc modification, a more comprehensive understanding of the targets of O-GlcNAc transferase is needed to elucidate its functional consequences.In this report, we describe the global detection and proteomic analysis of O-GlcNAc-modified proteins in HeLa cells. An affinitytagged version of the O-GlcNAc modification is metabolically incorporated onto proteins using an azide-tagged analogue of N-acetylglucosamine. The azido-GlcNAc-modified proteins thus contain an azide handle for chemoselective conjugation using a biotinylated phosphine reagent. The resulting conjugates were affinity-purified with streptavidin beads and subsequently digested with trypsin and analyzed by nano-HPLC-MS/MS. Using this strategy, we identified 199 azido-GlcNAc-modified proteins in HeLa cells. We subsequently validated the presence of this modification among 10 previously reported and 13 newly identified O-GlcNAcmodified proteins using specific antibodies. Our results reveal that proteins with a wide range of functions are modified by O-GlcNAc, implying its diverse cellular functions.

Identification of proteins bearing a specific post-translational modification would imply functions of the modification. Proteomic analysis of post-translationally modified proteins is usually challenging due to high complexity and wide dynamic range, as well as unavailability of efficient methods to enrich the proteins of interest. Here, we report a strategy for the detection, isolation, and profiling of O-linked N-acetylglucosamine (O-GlcNAc) modified proteins, which involves three steps: metabolic labeling of cells with an unnatural GlcNAc analogue, peracetylated azido-GlcNAc; chemoselective conjugation of azido-GlcNAc modified proteins via the Staudinger ligation, which is specific between phosphine and azide, using a biotinylated phosphine capture reagent; and detection and affinity purification of the resulting conjugated O-GlcNAc modified proteins. Since the approach relies on a tag (azide) in the substrate, we designated it the tagging-via-substrate (TAS) strategy. A similar strategy was used previously for protein farnesylation, phosphorylation, and sumoylation. Using this approach, we were able to specifically label and subsequently detect azido-GlcNAc modified proteins from the cytosolic lysates of HeLa, 3T3, COS-1, and S2 cell lines, suggesting the azido-substrate could be tolerated by the enzymatic systems among these cells from diverse biological species. We isolated azido-GlcNAc modified proteins from the cytosolic extract of S2 cells and identified 10 previously reported and 41 putative O-GlcNAc modified proteins, by nano-HPLC-MS/MS. Our study demonstrates that the TAS approach is a useful tool for the detection and proteomic analysis of O-GlcNAc modified proteins.

(Z)-alpha-Fluoro-, (Z)-alpha-chloro-, and (Z)-alpha-bromoacrylates were obtained with unprecedented yield and stereocontrol (>99%) via addition of the corresponding commercial trihaloacetates to aldehydes at room temperature using stoichiometric Cr(II) salts or catalytic Cr(II) with a regeneration system. The intermediate 2,2-dihalo-3-hydroxy adducts could be isolated in good yields under conditions of limited reagent at 0 degrees C.

Dimethylthiocarbamates (DMTCs), prepared from the corresponding alcohols using commercial dimethylthiocarbamoyl chloride, are spectrally simple, achiral, and nonpolar. DMTCs are moderately to highly stable to a wide range of reagents and conditions including metal hydrides, hydroboration, ylides, NaOH, HCl, organolithiums, Grignards, DDQ, PCC, Swern, n-Bu(4)NF, CrCl(2), heat, and Lewis acids. They are readily removed by NaIO(4) or H(2)O(2) in the presence of other common alcohol protecting groups. [structure: see text]

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