GLP-3 molecular characteristics and structure

Composition at a glance

Sequence

Highlighted positions carry non-standard residues or modifications. Lys* at position 17 carries the lipid side chain. The C-terminal Ser³⁹ is amidated.

Peptide composition

Counts from the sequence above. Ser, Gly and Pro account for 13 of the 39 residues, concentrated in the C-terminal segment (positions 29–39).

Non-standard residues

α-Aminoisobutyric acid (Aib), positions 2 and 20

Aib is a non-proteinogenic amino acid with two methyl groups on its α-carbon instead of one hydrogen and one side chain.¹ The extra methyl group restricts the rotation of the peptide backbone around that residue. Aib is widely used in peptide design to stabilize helical structure and to make specific bonds less accessible to proteolytic enzymes.

α-Methyl-leucine, position 13

This is leucine with an added methyl group on the α-carbon. It keeps leucine's hydrophobic side chain while adding the same backbone constraint as Aib.

The lipid side chain at Lys¹⁷

The ε-amino group of lysine 17 carries a side chain built from a hydrophilic AEEA spacer (8-amino-3,6-dioxaoctanoic acid), a γ-glutamic acid and a 20-carbon fatty diacid. This side chain accounts for a substantial share of the molecule's mass and much of its hydrophobic character.

Where it shows up in our data: on reversed-phase HPLC, more hydrophobic peptides are retained longer on the column.² On the current certificates the main peak elutes late in the run, at roughly 14.4 to 14.9 minutes, which is consistent with a large lipidated peptide. Retention time depends on the column, gradient and solvent system, so it is only comparable between runs using the same method.

Ionizable groups and mass spectra

The molecule carries several acidic groups (Asp⁹, Asp¹⁵, Glu²⁴, Glu²⁸, the linker γGlu and the terminal diacid carboxyl) and basic sites including the N-terminal amine and Lys¹⁶. In positive-mode electrospray, a peptide of this size picks up several protons and appears as multiply charged ions. That is why the certificates show signals near m/z 1184 (4+) and 1578 (3+) rather than at 4731.³ The calculation is shown in Mass spectrometry and peptide identity.

Identity considerations

Several features of the structure matter when confirming identity:

• Molecular weight is the primary check. At 4731.33 g/mol, a missing residue (57–186 Da), an incomplete lipid side chain or a missing amide would each shift the mass far beyond the precision of LC-MS. That is why mass confirmation catches most synthesis errors. See LC-MS analysis of GLP-3.
• Non-standard residues are invisible to composition alone. Aib and α-methyl-leucine differ from alanine and leucine by one methyl group (14 Da each), so they are confirmed through total mass rather than by amino acid analysis.
• Sequence order needs more than mass. A peptide with the same residues in a different order has the same mass. Confirming order requires fragmentation (tandem MS) or sequencing.
• Hydrophobicity supports, but does not prove, identity. The lipid side chain produces the late HPLC retention seen on every lot, which is consistent with the structure but not specific to it.

Residues absent from the sequence

GLP-3 RT contains no methionine, cysteine, tryptophan, histidine or asparagine. Those residues are associated with several common degradation pathways in peptides, such as oxidation, disulfide scrambling and rapid deamidation. Their absence is relevant to storage, discussed in Lyophilized peptide storage and stability.