A mass spectrometry-based method to screen for α-amidated peptides

Abstract

Amidation is a post-translational modification found at the C-terminus of ~50% of all neuropeptide hormones. Cleavage of the C(α)-N bond of a C-terminal glycine yields the α-amidated peptide in a reaction catalyzed by peptidylglycine α-amidating monooxygenase (PAM). The mass of an α-amidated peptide decreases by 58 Da relative to its precursor. The amino acid sequences of an α-amidated peptide and its precursor differ only by the C-terminal glycine meaning that the peptides exhibit similar RP-HPLC properties and tandem mass spectral (MS/MS) fragmentation patterns. Growth of cultured cells in the presence of a PAM inhibitor ensured the coexistence of α-amidated peptides and their precursors. A strategy was developed for precursor and α-amidated peptide pairing (PAPP): LC-MS/MS data of peptide extracts were scanned for peptide pairs that differed by 58 Da in mass, but had similar RP-HPLC retention times. The resulting peptide pairs were validated by checking for similar fragmentation patterns in their MS/MS data prior to identification by database searching or manual interpretation. This approach significantly reduced the number of spectra requiring interpretation, decreasing the computing time required for database searching and enabling manual interpretation of unidentified spectra. Reported here are the α-amidated peptides identified from AtT-20 cells using the PAPP method.

Figure 1. Detection of α-Amidated Peptides by Mass Spectrometry

A, Peptide amidation reaction catalyzed by peptidylglycine α-amidating monooxygenase (PAM). In this reaction, the precursor peptide is cleaved, removing the C-terminal glycine and creating a new amidated C-terminus during PAM catalysis, resulting in a total difference of 58.0055 mass units (C2H2O2) between the glycine-extended and amidated forms. B, MS/MS spectra of mouse joining peptide (mJP) and its precursor. The two MS/MS spectra have similar fragment patterns. Corresponding b ions (connected with solid lines) have same mass values while y ions (connected with dashed elbow connectors) have a constant mass difference of 58 resulting from the cleavage of C₂H₂O₂ group from the C-terminus by PAM.

Figure 2. MS/MS Spectra of Two mJP Related Peptide Pairs

These spectra have same the y ions as mJP and its precursor.

Figure 3. De novo Identification of an α-Amidated Peptide

A, Spectral pair found by PAPP. Two spectra have peaks with a 58 mass difference (tentative y ions, marked with dashed elbow connectors) and peaks with the same m/z value (tentative b ions, marked with solid lines). B, Fragment ions found in the spectrum of the larger peptide and the corresponding possible amino acid residues. C, Mass errors of all possible sequences. D, MS/MS spectrum of the synthetic peptide ELEGERPL-NH₂.