Three Letters, One Molecule: How To Read A Peptide’s Sequence

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Three Letters, One Molecule: How To Read A Peptide’s Sequence

Most of us have learned to read a nutrition label or an ingredients list. Peptides come with a label of their own, and it looks far more forbidding: a string of capital letters such as KPV, or a run of abbreviations like Lys-Pro-Val. Once you know the code, though, that short string tells you almost everything about the molecule, down to what it should weigh.

This is a guide to reading it, using one of the smallest peptides there is as the example.

The alphabet of proteins

Peptides and proteins are chains of amino acids. Nature uses twenty common ones, and every peptide is simply a particular selection of them in a particular order. Short chains, up to roughly fifty links, are usually called peptides; longer ones are proteins.

Each amino acid has two standard abbreviations, agreed internationally so that chemists everywhere write them the same way:

• A three-letter code, which is usually the start of the name: Lys for lysine, Pro for proline, Val for valine.
• A one-letter code, which is more compact but less obvious. Proline and valine get P and V. Lysine gets K, because L was already taken by leucine and K was a free letter close to it.

So “KPV” and “Lys-Pro-Val” are the same molecule written two ways: a chain of lysine, then proline, then valine. Amidae’s library has a full table of amino acid codes and how to read a peptide sequence, including the letters that trip people up.

Order matters, and so does direction

A sequence is always written from one particular end of the chain to the other. By convention, the left-hand end has a free amino group (the N-terminus) and the right-hand end has a free acid group (the C-terminus). Chemists read it left to right, the same way the chain is built in cells.

That convention matters because the same three building blocks in a different order make a different molecule. KPV and VPK contain exactly the same atoms. They have the same formula and the same weight. But their shapes differ, and so does how they behave in an experiment. That is one reason a lab checking a peptide’s identity can’t rely on weight alone for every question; techniques that break the chain into fragments can confirm the order as well.

Adding up the weight

Reading a peptide sequence: lysine, proline and valine add up to 342.4 g/mol after two water molecules are lost

Here is where the code becomes genuinely useful. If you know the sequence, you can work out what the molecule should weigh with nothing more than a calculator.

Each amino acid has a known weight. When two of them join, the bond that forms between them (a peptide bond) releases one molecule of water. So the weight of a chain is the sum of its amino acids, minus one water for every bond.

That figure, 342.4, is the molecular weight a supplier should list for KPV, and the number a lab’s results should agree with. The formula follows the same logic: C16H30N4O4, which is the three amino acids’ atoms with two waters’ worth of hydrogen and oxygen taken away.

Two kinds of weight

If you ever see a lab report for a peptide, you may notice the mass doesn’t quite match the molecular weight. That is usually not a mistake.

The molecular weight above is an average. It accounts for the fact that a small share of carbon, nitrogen and other atoms in nature are slightly heavier versions, called isotopes. A mass spectrometer, the instrument that weighs molecules directly, sees each version separately, and the tallest signal is usually the one made only of the most common, lightest isotopes. That figure is called the monoisotopic mass. For KPV it is about 342.23, a fifth of a unit below the average.

A good lab report says which of the two it is quoting. A small difference between them is normal. A difference of tens of units is not.

Small changes at the ends

Sequences sometimes carry extra notation at either end. An “Ac-” at the start means the free amino end has been capped with an acetyl group. An “-NH2” at the end means the acid group has been turned into an amide. Both are common in research peptides, and both change the weight: the acetyl adds about 42, and the amide takes away about 1.

So “Ac-KPV-NH2” is not the same compound as KPV. It weighs about 383.5 instead of 342.4, and a lab report on one tells you nothing about the other. When you compare a product with its report, check that the ends match as well as the letters.

Putting it together: KPV

KPV is about as simple as a peptide gets: three amino acids, two bonds, one water lost per bond. That makes it a good test of whether a supplier’s information hangs together. The product listing for Amidae’s KPV, for example, gives the sequence as Lys-Pro-Val, the formula as C16H30N4O4, the molecular weight as 342.4 and the CAS registry number as 67727-97-3. Every one of those figures can be checked against the arithmetic above, and the mass found on any batch report should agree with them.

If a listing gives a sequence and a weight that don’t add up, or leaves out the sequence entirely, that is worth noticing before anything else.

The short version

• The letters are amino acids, written from the amino end to the acid end.
• The same letters in a different order make a different molecule with the same weight.
• The weight is the sum of the amino acids minus one water per bond.
• Extra notation at the ends, such as Ac- or -NH2, changes the compound and its weight.
• A lab report may give the monoisotopic mass, which sits slightly below the average.

With those five rules, a string like KPV stops being jargon and becomes something you can check for yourself.

References

1. IUPAC-IUB Joint Commission on Biochemical Nomenclature. Nomenclature and symbolism for amino acids and peptides. European Journal of Biochemistry. 1984;138(1):9–37.
2. CAS Registry Number 67727-97-3 (Lys-Pro-Val). CAS, a division of the American Chemical Society.