The peptide hormone ghrelin is the only known protein modified with an O-linked octanoyl side group, which occurs on its third serine residue. This modification is crucial for ghrelin's physiological effects including regulation of feeding, adiposity, and insulin secretion. Despite the crucial role for octanoylation in the physiology of ghrelin, the lipid transferase that mediates this novel modification has remained unknown. Here we report the identification and characterization of human GOAT, the ghrelin O-acyl transferase. GOAT is a conserved orphan membrane-bound O-acyl transferase (MBOAT) that specifically octanoylates serine-3 of the ghrelin peptide. Transcripts for both GOAT and ghrelin occur predominantly in stomach and pancreas. GOAT is conserved across vertebrates, and genetic disruption of the GOAT gene in mice leads to complete absence of acylated ghrelin in circulation. The occurrence of ghrelin and GOAT in stomach and pancreas tissues demonstrates the relevance of GOAT in the acylation of ghrelin and further implicates acylated ghrelin in pancreatic function.G hrelin is a 28-aa peptide hormone produced principally by stomach tissue with an unusual acyl modification on its critical serine-3 residue. Ghrelin is the endogenous ligand for the growth hormone secretagogue receptor 1a (GHSR1a), and its acyl modification is critical for the activation of the GHSR1a (1). In addition to stimulating growth hormone release from pituitary, ghrelin also promotes food intake, carbohydrate utilization, and adiposity (2-5). Accordingly, ghrelin levels are modulated by changes in nutritional status such as feeding and fasting or exposure to high-fat diets (5, 6). Importantly, ghrelin is the only peptide hormone of peripheral tissue origin that increases food intake (2).More recent studies have identified roles for acylated ghrelin in regulating insulin secretion and blood glucose. Acylated ghrelin occurs in pancreas tissues, and GHSR1a receptor blockade with specific antagonists or treatments with antiserum against acylated ghrelin enhance glucose-induced increases in insulin release whereas acylated ghrelin decreases insulin release (5, 7-10). These observations have further implicated acylated ghrelin in the regulation of metabolism.In stomach tissue and in circulation, acylated forms of ghrelin are modified via an ester linkage with n-octanoic acid and to a lesser extent with decanoyl and decenoyl fatty acids (1, 3). Importantly, the acyl modification in ghrelin is essential for function, with octanoyl and decanoyl fatty acids being optimal (11). Ghrelin is highly conserved in vertebrates, and the third serine residue, which is uniquely modified by the ester-linked acyl group, occurs in all mammal, avian, and fish species (3).The enzyme(s) responsible for acylation of ghrelin has remained unknown. Work by Takada et al. (12) described that porcupine, an enzyme with structural similarities to membrane-bound O-acyl transferases (MBOAT), is required for serine-209 acylation with palmitoleic acid and for transport of...

Proteolytic cleavage of FGF21, evaluated in human plasma, confirmed three known proline hydrolysis sites. Although DPP-IV participates in consecutive cleavages from the N-terminus, fibroblast activation protein was implicated as the enzyme responsible for the C-terminal cleavage that inactivates FGF21.

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Immunoprecipitation (IP) combined with matrix-assisted laser desorption ionization (MALDI) time of flight (Tof) mass spectrometry has been used to develop quantitative assays for amyloid-beta (Abeta) peptides in cerebrospinal fluid (CSF). Inclusion of (15)N labelled standard peptides allows for absolute quantification of multiple Abeta isoforms in individual samples. Characterization of variability associated with all steps of the assay indicated that the IP step is the single largest contributor to overall variability. Optimization of the assay resulted in overall coefficient of variation

Heart fatty acid binding protein (Fabp3) is a cytosolic protein expressed primarily in heart, and to a lesser extent in skeletal muscle, brain, and kidney. During myocardial injury, the Fabp3 level in serum is elevated rapidly, making it an ideal early marker for myocardial infarction. In this study, an MS-based selected reaction monitoring method (LC-SRM) was developed for quantifying Fabp3 in rat serum. Fabp3 was enriched first through an immobilized antibody, and the protein was digested on beads directly. A marker peptide of Fabp3 was quantified using LC-SRM with a stable isotope-labeled peptide standard. For six quality control samples with Fabp3 ranging from 0.256 to 25 ng, the average recovery following the procedure was about 73%, and the precision (%CV) between replicates was less than 7%. The Fabp3 concentrations in rat serum peaked 1 h after isoproterenol treatment, and returned to baseline levels 24 h after the dose. Elevated Fabp3 levels were also detected in rats administered with a PPAR α/δ agonist, which has shown to cause skeletal muscle necrosis. Fabp3 can be used as a biomarker for both cardiac and skeletal necroses. The cross-validation of the LC-SRM method with an existing ELISA method is described.