No description available.

No description available.

No description available.

Peptide a-amidation is known as a signature of bioactivity due to the fact that half of the bioactive peptides found in the nervous and endocrine systems are a-amidated and that most known a-amidated peptides are bioactive. a-Amidated peptides are produced by the oxidative cleavage of glycine-extended precursors. Peptidylglycine a-amidating monooxygenase (PAM) is the only known enzyme responsible for catalyzing this reaction and its sole physiological function is to convert glycine extended prohormones to their a-amidated forms. High levels of PAM are found in certain tissues with no corresponding level of amidated products suggesting the presence of undiscovered a-amidated peptide hormones. Liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) has emerged as a powerful tool for peptide identification due to its advantages of speed, sensitivity and applicability to complex peptide mixtures. Normally, spectra are interpreted using database search engines. However, database searching is inefficient and ineffective for the identification of endogenous peptide with post-translational modifications (PTM) due to its low identification rate and high demand for computing power. There is a specific mass difference of 58.0055 units between an a-amidated peptide and its corresponding C-terminal glycine-extended precursor. The two peptides will have similar chromatographic retention time and MS/MS fragmentation patterns resulting from the identical amino acids sequences except for relatively the small differences at the C-termini. Based on this, a new LC-MS/MS based strategy for screening for a-amidated peptides was developed. This strategy depends on PAM inhibition and the mass accuracy of mass spectrometry (< 3 ppm). The coexistence of a-amidated peptides and their C-terminal glycine-extended precursors was insured by growing cells in the presence of a PAM inhibitor. After LC-MS/MS, masses and retention times of parent ions were extracted from raw data files and scanned by a script for peptide pairs with similar retention times and a mass difference around 58.0055. Resulting pairs were further validated by comparing their fragmentation patterns in MS/MS spectra. Only peptide pairs that met all three criteria were considered for further interpretation. This reduced the number of MS/MS spectra requiring interpretation by >99% and, thus, enable the manual inspection of MS/MS for the candidate peptide pairs. A total of 13 a-amidated peptides were successfully identified from cultured mouse pituitary AtT-20 cells using this method and a few of these newly identified a-amidated peptides exhibited bioactivity. The adaptability of this strategy to screening for other PTMs is also discussed. Peptidylglycine a-hydroxylating monooxygenase (PHM) is one of PAM domains which can be expressed separately. It is a copper dependent enzyme that catalyzes the first step of the two-step peptide amidation reaction. Removal of the copper ions results in the loss of enzyme catalytic activity. A PHM based a-amidated peptide enrichment method was developed. This method includes two steps. First, cells grown in culture were treated with a PAM inhibitor to effect the cellular accumulation of glycine-extended peptides. In the second step, copper-depleted PHM (apo-PHM) was used to selectively bind glycine-extended peptides present in the cell extract. All other unbound peptides were removed during wash runs. apo-PHM was then reinstated with copper to convert bound glycine-extended peptides to hydroxylated peptides and release them. Hydroxylated product can be converted to a-amidated peptide under basic conditions. Experiments carried out using model glycine extended peptides showed a 40 – 120-fold enrichment using HPLC-fluorometric assay or MALDI-TOF quantification. This method proved successful when working with complex samples like cell extracts. The relative intensity of a known a-amidated peptide mouse joining peptide (mJP) from an AtT-20 extract was dramatically increased after enrichment experiments.

Primary fatty acid amides (PFAMs) and N-acylglycines (NAGs) are important signaling molecules in the mammalian nervous system, binding to many drug receptors and demonstrating control over sleep, locomotor activity, angiogenesis, vasodilatation, gap junction communication, and many other processes. Oleamide is the best-studied of the PFAMs, while the in vivo activity of the others is largely unstudied. Even less is known about the NAGs, as their discovery as novel compounds is much more recent due to low endogenous levels. Herein is described extraction and quantification techniques for PFAMs and NAGs in cultured cells and media using solvent extraction combined with solid phase extraction (PFAM) or thin layer chromatography (NAG), followed by gas chromatography-mass spectroscopy to isolate and quantify these lipid metabolites. The assays were used to examine the endogenous amounts of a panel of PFAMs as well as the conversion of corresponding free fatty acids (FFAs) to PFAMs over time in several cell lines. The cell lines demonstrated the ability to convert all FFAs, including a non-natural FFA, and an ethanolamine to the corresponding PFAM. Different patterns of relative amounts of endogenous and FFA-derived PFAMs were observed in the cell lines tested. Essential to identifying therapeutic targets for the many disorders associated with PFAM signaling is understanding the mechanism(s) of PFAM and NAG biosynthesis. Enzyme expression studies were conducted to determine potential metabolic enzymes in the model cell lines in an attempt to understand the mechanism(s) of PFAM biosynthesis. It was found that two of the cell lines which show distinct metabolisms of PFAMs also demonstrate unique enzyme expression patterns, and candidate enzymes proposed to perform PFAM and NAG metabolism are described. RNAi knockdown studies revealed further information about the metabolism of PFAMs and calls into question the recently proposed involvement of cytochrome c. Isotopic labeling studies showed there are two pathways for PFAM formation. A novel enzyme is likely to be involved in formation of NAGs from acyl-CoA intermediates.