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 Al³⁺ ions (IMAC-Al resin).

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.

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.

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.

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.