The stoichiometry of protein phosphorylation in adipocyte lipid droplets: analysis by N-terminal isotope tagging and enzymatic dephosphorylation.

Most phosphoproteomic studies to date have been limited to the identification of phosphoproteins and their phosphorylation sites, and have not assessed the stoichiometry of protein phosphorylation, a critical parameter reflecting the dynamic equilibrium between phosphorylated and non-phosphorylated pools of proteins. Here, we used a method for measuring phosphorylation stoichiometry through isotope tagging and enzymatic dephosphorylation of tryptic peptides. Using this method, protein digests are divided into two equal aliquots that are modified with either light or heavy isotope tags. One aliquot is dephosphorylated by alkaline phosphatase. Finally, the peptide mixtures are recombined and LC-MS/MS analysis is performed. With this method, we studied adipocytes of mice stimulated with CL316,243, a beta-3 adrenergic agonist known to induce lipolysis and marked phosphorylation changes in proteins of the lipid droplet surface. In lipid droplet preparations, CL316,243 administration increased phosphorylation of proteins related to regulation of signaling, metabolism and intracellular trafficking in white adipose tissue, including hormone-sensitive lipase which was 80% phosphorylated at the previously reported site, Ser-559, and the lipid surface protein perilipin, which was phosphorylated by approximately 60 and approximately 40% at previously unreported sites, Ser-410 and Ser-460.

Enzymatic activity of lysosomal carboxypeptidase (cathepsin) A is required for proper elastic fiber formation and inactivation of endothelin-1.

BACKGROUND: Lysosomal carboxypeptidase, cathepsin A (protective protein, CathA), is a component of the lysosomal multienzyme complex along with beta-galactosidase (GAL) and sialidase Neu1, where it activates Neu1 and protects GAL and Neu1 against the rapid proteolytic degradation. On the cell surface, CathA, Neu1, and the enzymatically inactive splice variant of GAL form the elastin-binding protein complex. In humans, genetic defects of CathA cause galactosialidosis, a metabolic disease characterized by combined deficiency of CathA, GAL, and Neu1 and a lysosomal storage of sialylated glycoconjugates. However, several phenotypic features of galactosialidosis patients, including hypertension and cardiomyopathies, cannot be explained by the lysosomal storage. These observations suggest that CathA may be involved in hemodynamic functions that go beyond its protective activity in the lysosome. METHODS AND RESULTS: We generated a gene-targeted mouse in which the active CathA was replaced with a mutant enzyme carrying a Ser190Ala substitution in the active site. These animals expressed physiological amounts of catalytically inactive CathA protein, capable of forming lysosomal multienzyme complex, and did not develop secondary deficiency of Neu1 and GAL. Conversely, the mice showed a reduced degradation rate of the vasoconstrictor peptide, endothelin-1, and significantly increased arterial blood pressure. CathA-deficient mice also displayed scarcity of elastic fibers in lungs, aortic adventitia, and skin. CONCLUSIONS: Our results provide the first evidence that CathA acts in vivo as an endothelin-1-inactivating enzyme and strongly confirm a crucial role of this enzyme in effective elastic fiber formation.

Global analysis of protein phosphorylation networks in insulin signaling by sequential enrichment of phosphoproteins and phosphopeptides.

Although the important role of protein phosphorylation in insulin signaling networks is well recognized, its analysis in vivo has not been pursued in a systematic fashion through proteome-wide studies. Here we undertake a global analysis of insulin-induced changes in the rat liver cytoplasmic and endosomal phosphoproteome by sequential enrichment of phosphoproteins and phosphopeptides. After subcellular fractionation proteins were denatured and loaded onto iminodiacetic acid-modified Sepharose with immobilized Al³âº ions (IMAC-Al resin). Retained phosphoproteins were eluted with 50 mM phosphate and proteolytically digested. The digest was then loaded onto an IMAC-Al resin and phosphopeptides were eluted with 50 mM phosphate, and resolved by 2-dimensional liquid chromatography, which combined offline weak anion exchange and online reverse phase separations. The peptides were identified by tandem mass spectrometry, which also detected the phosphorylation sites. Non-phosphorylated peptides found in the flow-through of the IMAC-Al columns were also analyzed providing complementary information for protein identification. In this study we enriched phosphopeptides to ~85% purity and identified 1456 phosphopeptides from 604 liver phosphoproteins. Eighty-nine phosphosites including 45 novel ones in 83 proteins involved in vesicular transport, metabolism, cell motility and structure, gene expression and various signaling pathways were changed in response to insulin treatment. Together these findings could provide potential new markers for evaluating insulin action and resistance in obesity and diabetes.

Quantitative analysis of a proteome by N-terminal stable-isotope labelling of tryptic peptides.

Covalent modification of peptides and proteins with compounds containing stable isotopes (isotope tagging) has become an essential tool to detect dynamic changes in the proteome following external or internal influence; however, using terminal amino groups for global isotope labelling of tryptic peptides is challenged by the similar reactivity of the amino groups of lysine residues. We describe a new quantitative method based on selective tagging of the terminal amino groups of tryptic peptides with pentafluorophenyl esters containing stable isotopes. The labelled peptides were resolved by two-dimensional nanoflow liquid chromatography on weak anion-exchange and reversed-phase columns and then identified and quantified by tandem mass spectrometry. The method was applied to compare the proteomes of plasma membranes from proliferating and differentiated human colorectal adenocarcinoma (Caco-2) cells and endosomes purified from the livers of rats stimulated with insulin and epidermal growth factor. The comparison of the results obtained by isotope tagging and biochemical assays demonstrate that global isotope tagging with pentafluorophenyl esters allows accurate quantification of complex protein samples.

Subcellular proteomics of cell differentiation: quantitative analysis of the plasma membrane proteome of Caco-2 cells.

Human colorectal carcinoma (Caco-2) cells undergo in culture spontaneous enterocytic differentiation, characterized by polarization and appearance of the functional apical brush border membrane. To provide insights into the biology of differentiation, we have performed a comparative proteomic analysis of the plasma membranes from proliferating cells (PCs) and the apical membranes from differentiated cells (DCs). Proteins were resolved by SDS-PAGE, in-gel digested and analyzed by RP-LC and MS/MS. Alternatively, proteins were digested in solution, and tryptic peptides were labeled with isotopic tags and analyzed by 2-D LC followed by MS/MS. Among the 1125 proteins identified in both proteomes, 76 were found to be significantly increased in the membranes of DCs and 61 were increased in PCs. Majority of the proteins increased in the apical membranes were metabolic enzymes, proteins involved in the maintenance of cellular structure, transmembrane transporters, and proteins regulating vesicular transport. In contrast, majority of the proteins increased in the membranes of PCs were involved in gene expression, protein synthesis, and folding. Both groups contained many novel proteins with yet to be identified functions, which could provide potential new markers of the intestinal cells or of colorectal cancer.