VIP: The 28-Residue Vasoactive Intestinal Peptide and What a Certificate Shows

Vasoactive intestinal peptide, VIP, is one of the older entries in any peptide catalogue. Said and Mutt isolated it from porcine small intestine and reported it in Science in 1970; Mutt and Said published its 28-residue sequence in 1974. It is a basic peptide with a C-terminal amide, a member of the same structural family as secretin and glucagon, and it carries a single methionine that decides most of its handling. VIP is listed in the catalogue at 5 mg, from $84.

This guide covers the discovery, the family relationship, the sequence and mass as the catalogue records them, the degradation routes that follow from the sequence, and what a certificate of analysis for a 28-mer with these particular residues should show.

Published work is described by model throughout. The material is supplied for laboratory research only.

Isolation and sequence

The 1970 Science paper by Said and Mutt reports a polypeptide isolated from porcine small intestine with activity on blood vessels in their animal preparations, hence the name. Mutt and Said's 1974 paper in the European Journal of Biochemistry gives the full amino acid sequence of the porcine peptide, 28 residues, determined with the help of the enzyme kallikrein to generate fragments. The authors also noted the resemblance to secretin and glucagon, which placed the peptide in a family.

The human peptide turned out to be the same. Itoh and colleagues reported the human precursor sequence in Nature in 1983, from a complementary DNA clone, and the VIP it encodes matches the porcine sequence; the same precursor also carries a second related peptide, PHM-27. For a buyer this means that the catalogue sequence, the porcine sequence from 1974 and the human sequence are one and the same thing.

The secretin and glucagon family

VIP belongs to a family of peptides that share a common ancestry and a common shape: a run of roughly 27 to 44 residues, an N-terminal histidine in most members, and a tendency to form an alpha-helix when bound to a receptor. Sherwood, Krueckl and McRory's 2000 review in Endocrine Reviews traces the family's origin and lists its members. The receptors are class B G protein-coupled receptors, and the 2012 IUPHAR review by Harmar and colleagues sets out the three that bind VIP and its closest relative PACAP: VPAC1, VPAC2 and PAC1.

The family relationship is why VIP is worth reading alongside GLP-1 and the incretin peptides. The incretins and the growth hormone-releasing hormone analogues in the catalogue are VIP's structural cousins, and the handling habits built for one carry over to the others: all are medium-length, helix-forming peptides with an N-terminal region that enzymes attack and a C-terminus that is often amidated.

Sequence and mass as the catalogue records them

The catalogue records the sequence His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2, 28 residues, the formula C147H238N44O42S, a molecular weight of 3326.83 g/mol and CAS number 37221-79-7. The NH2 at the end denotes the C-terminal amide: the final asparagine ends in a primary amide rather than a free carboxylic acid.

Counting along the sequence: two arginines, three lysines and the N-terminal histidine on the basic side, against two aspartates on the acidic side. The peptide is therefore net positive at neutral pH, which is what "basic" means on a certificate. There are two tyrosines, one methionine, no cysteine and no tryptophan. The one sulfur in the formula is the methionine at position 17.

PubChem's entry under the VIP CAS number, CID 53314964, records the formula C147H237N43O43S at 3326.8 g/mol, which is the free-acid form of the same sequence; the catalogue formula is the amide. The two differ by the exchange of OH for NH2, which is a difference of about one dalton in mass, and the certificate's observed mass will not reliably distinguish the two on a 3300 Da peptide. The amide is confirmed by the sequence and the synthesis, not by the intact mass.

Instability: the methionine and the asparagines

VIP's sequence contains the residue most responsible for chemical instability in stored peptides: methionine. Its thioether oxidises readily to the sulfoxide, adding 16 Da, and under harsher conditions to the sulfone, adding 32 Da. Li, Schöneich and Borchardt's 1995 review sets out the mechanisms, and Manning and colleagues' 2010 review of protein stability covers methionine oxidation alongside the other routes. Oxidation needs only dissolved oxygen, trace metal and time, and it is faster in solution than in the lyophilised solid.

The second route is deamidation. VIP has three asparagines, at positions 9, 24 and 28, and one glutamine. Asparagine followed by serine, as at positions 24 and 25, is among the most deamidation-prone motifs, and the C-terminal asparagine amide is a second site. Deamidation adds about one dalton and introduces a negative charge, so it shifts the HPLC retention time slightly and is often seen as a shoulder on the main peak before it is seen in the mass. How peptides degrade maps these routes to the residues that enable them.

The handling that follows is the familiar one for methionine peptides: keep the solid at -20°C, exclude moisture and light, minimise time in solution, and avoid oxidising conditions and headspace in reconstituted vials. Methionine oxidation is the reason a VIP lot stored for a long time in solution can show a growing +16 peak on re-analysis even when the vial was never opened.

What a certificate should show

A certificate for VIP should carry the lot number, a reversed-phase HPLC purity with its method, an intact mass, and ideally a sequence confirmation. HPLC purity explained covers how to read the trace. For this sequence, the specific things to look for are:

• A single main peak with no shoulder, since a shoulder on a methionine- and asparagine-rich peptide is more likely to be an oxidised or deamidated form than a synthetic impurity.
• An observed mass reconciling with 3326.83 g/mol on the average basis, or about two daltons lower if the certificate reports a monoisotopic figure, which is the normal gap for a peptide of this size.
• No peak at +16 Da above the main mass. Its presence means the lot was already partly oxidised when tested.
• The sequence written out with the C-terminal NH2, since the amide is not something the intact mass confirms.
• A test date close to the ship date, because the degradation routes above run with time.

Deletion sequences, the main synthetic impurity class, are resolved easily on a 3327 Da peptide: the smallest, a missing alanine, is 71 Da away. The certificate's difficulty with VIP is not the synthesis impurities; it is the one-dalton degradation products.

What the published work describes

Beyond the original vascular preparations, the VIP literature is largely a literature of receptor pharmacology and immunology in cells and rodents. The Harmar review covers receptor distribution and signalling through cyclic AMP. Delgado and Ganea's 2013 review in Amino Acids summarises work on the peptide's actions on immune cells, principally in cultured mouse and human cells and in mouse models. The catalogue record describes VIP as studied for its immunomodulatory and vasodilatory signalling, which is an accurate account of what those bodies of work examine in their models. This guide makes no statement about effects in people.

What to establish before ordering

• That the sequence on the certificate matches the catalogue sequence, including the C-terminal amide.
• The observed mass against 3326.83 g/mol and whether the figure is average or monoisotopic.
• The absence of a +16 peak and of any shoulder on the main HPLC peak.
• The test date, and how the lot has been stored since.
• Net peptide content, since a peptide with six basic residues is isolated as a salt and the gravimetric 5 mg is not 5 mg of peptide.