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.

Peptide hormones are responsible for cellular functions critical to the survival of an organism. Approximately 50% of all known peptide hormones are post-translationally modified at the C-terminus. Enzymatic oxidative conversion of C-terminal glycine extended peptide precursors results in an a-amidated peptide and glyoxylate. Peptidylglycine a-amidating monooxygenase (PAM) is the single known enzyme responsible for catalyzing this reaction. PAM is an O2, Cu(II), and Zn(II) dependent bifunctional enzyme. Initially, PAM hydroxylates the glycyl a-carbon followed by dealkylation of the hydroxylated intermediate to an a-amidated product and glyoxylate. PAM is also responsible for the conversion of glycine extended fatty acids to fatty acid amides and glyoxylate. PAM catalyzes the activation of all glycine-extended prohormones including biomolecules ranging from neuro to physio-homeostatic hormones. Identification of a-amidated hormones from a biological source has been severely hindered by the lack of a specific assay for this distinctive class of biological hormones, indicating that numerous a-amidated hormones remain undiscovered. Based on the selective in situ chemistry of PAM, a novel and specific assay was developed for the discovery of a-amidated hormones. The identification of novel a-amidated hormones will lead to an increased understanding of post-translational modifications and will pioneer a new understanding of a-amidated hormone biosynthesis, regulation, and bioactivity. Discovery of novel a-amidated biomolecules could also lead to their use as pharmaceuticals as there are several currently marketed a-amidated peptide based pharmaceuticals.Inhibition of PAM in cell culture leads to the accumulation of glycine-extended hormones in the conditioned medium. The medium was fractionated by chromatographic techniques and each specific fraction was then assayed by the newly developed platform technology for the presence of a-amidated hormones. For every a-amidated hormone synthesized by PAM, glyoxylate is also formed. Based on this 1:1 molar ratio, several novel spectrophotometric, fluorescent, and chemi-luminescent enzyme linked assays for glyoxylate were developed, which when utilized on cell culture fractions proved positive for the identification of a-amidated hormones. Each novel spectroscopic assay was independently verified by a variety of known methodologies. Moreover the assay was utilized to identify two known a-amidated hormones accumulated from cell culture, which were further verified by Mass Spectral analysis.