Apelin-13 and the Pyroglutamate It Becomes
Apelin-13 has a problem that almost no other peptide of its size has: it converts into a different, stable molecule on its own, and the converted form is seventeen daltons lighter. Anyone reading a certificate for this peptide needs to know which of the two it describes.
Sequence and mass
Apelin-13 is Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe, the thirteen C-terminal residues of the apelin precursor protein. As the free peptide it has a monoisotopic mass of 1549.83 Da and an average mass of 1550.85 Da. CAS 217082-60-5.
The pyroglutamate form, written Pyr-1 apelin-13 or [Pyr1]apelin-13, has a monoisotopic mass of 1532.80 Da and an average of 1533.81 Da. It is the same thirteen residues with the N-terminal glutamine cyclised.
What cyclisation does
An N-terminal glutamine can attack its own backbone nitrogen, forming a five-membered ring and releasing ammonia. The result is pyroglutamate, and the reaction costs the molecule 17.03 daltons.
It happens spontaneously in aqueous solution, faster at neutral to slightly acidic pH and faster still at elevated temperature. It is not a synthesis defect and it is not degradation in the usual sense; it is a chemical property of any peptide that begins with glutamine, and apelin-13 is the textbook example.
The pyroglutamate form is the more stable of the two, because the cyclised N-terminus has no free amine to react further. Material that begins as the free peptide will drift toward the cyclised form on storage in solution.
Reading a certificate for this peptide
Three things have to line up. The name should say whether it is apelin-13 or [Pyr1]apelin-13. The mass should be near 1550.9 or near 1533.8 respectively. And the chromatogram should be consistent with the stated form.
Where they disagree, the mass is usually right and the name is usually the thing that was copied from a previous document. A stated mass of 1533.8 under a heading reading apelin-13 describes the cyclised peptide whatever the heading says.
A chromatogram showing two peaks separated by a small retention difference, with masses seventeen daltons apart, is showing partial conversion. That is a real and common finding, not an artefact.
Methionine at position eleven
The sequence carries a methionine, which brings the same oxidation liability that any methionine does: a plus-sixteen sulfoxide that elutes earlier and can form during handling rather than during synthesis.
On a peptide that is already being watched for a minus-seventeen conversion, this means two mass shifts are in play at once, and a species at 1549.8 minus 17 plus 16 sits only one dalton away from the unmodified parent. Resolving that requires either a high-resolution measurement or, more practically, chromatography that separates the species before they reach the detector.
A strongly basic peptide
Two arginines, a lysine and a histidine against no acidic residues other than the C-terminal carboxyl give apelin-13 a substantial net positive charge at working pH. Tailing on silica-based columns is the predictable consequence, and trifluoroacetic acid is the usual ion-pairing remedy.
The same basicity makes it ionise efficiently in positive-mode electrospray. The doubly protonated ion near m/z 776.4 and the triply protonated ion near m/z 517.9 are usually the dominant species, and the seventeen dalton conversion appears as a shift of 8.5 and 5.7 m/z units respectively in those charge states, which is why deconvolution to a neutral mass makes the comparison easier to read.
Apelin-17 and apelin-36 are different molecules
The apelin precursor yields several fragments of different lengths, and the number in the name is the residue count. Apelin-17 and apelin-36 extend further toward the N-terminus and have correspondingly larger masses. They are not interchangeable with apelin-13 and their certificates are not comparable.
The C-terminal phenylalanine is shared across the family, which means a C-terminal fragment observed in a mass spectrum does not by itself distinguish one family member from another.
What confirms a lot
A stated form, free peptide or pyroglutamate, with a deconvoluted neutral mass that matches it; a chromatogram showing whether both forms are present and in what proportion; and awareness that the proportion is not fixed. A lot analysed as the free peptide and stored in solution will not stay that way, which is an argument for lyophilised storage rather than for distrusting the certificate.
Related reading
Terminal cyclisation is one of several modifications that change a peptide chemically without changing its sequence. The broader set is discussed in half-life and analog modifications, and the mass arithmetic in monoisotopic versus average mass.
