Amino acids, residues and the peptide bond
Amino acids share a common backbone: an amino group, a carboxyl group and a variable side chain. When two amino acids join, the carboxyl group of one condenses with the amino group of the next, releasing a molecule of water and forming an amide linkage known as the peptide bond.
Because water is lost at every linkage, the mass of a peptide is not the sum of its constituent amino acid masses. It is the sum of the residue masses plus one water molecule for the intact chain. This is the single most common source of arithmetic confusion when checking a reported molecular weight against a sequence.
Each unit within the assembled chain is called a residue rather than an amino acid, which is why a peptide is described as being a certain number of residues long.
Where peptides end and proteins begin
There is no universally enforced boundary between a peptide and a protein. In common usage, chains up to roughly fifty residues are described as peptides and longer chains as proteins, but the line is a convention rather than a rule.
A more useful distinction in practice is structural. Short peptides frequently have no stable folded conformation in solution, while proteins generally adopt defined three-dimensional structures. That difference has direct analytical consequences: unstructured peptides tend to behave more predictably in reversed-phase chromatography, because there is no folded structure to unfold on the column.
Reading a sequence
Peptide sequences are written from the N-terminus, the end carrying the free amino group, to the C-terminus, the end carrying the free carboxyl group. This direction is a convention and is always assumed unless stated otherwise.
Sequences appear in one-letter code, such as GEPPPGKPADDAGLV, or three-letter code, such as Gly-Glu-Pro-Pro-Pro-Gly-Lys. The two describe the same molecule.
One-letter code only works for the twenty standard proteinogenic amino acids. Peptides containing non-standard residues, D-amino acids or synthetic building blocks cannot be fully written in one-letter code, which is why some compounds are described by name and structure rather than by a sequence string.
Terminal and side-chain modifications
Synthetic peptides are frequently modified at their ends. C-terminal amidation replaces the terminal carboxylic acid with an amide; N-terminal acetylation caps the free amino group. Both are common, both change the molecular weight, and both must be stated for a sequence to be unambiguous.
The mass consequences are small but diagnostic. Amidation reduces the mass by approximately 0.98 Da relative to the free acid, and acetylation adds approximately 42 Da. A mass spectrum that appears to be off by one of these increments usually indicates a modification that was present but not recorded, rather than a different peptide.
How synthetic peptides are produced
Most research peptides are produced by solid-phase peptide synthesis. The chain is assembled one residue at a time on an insoluble resin support, with each cycle consisting of a deprotection step followed by a coupling step. Because the growing chain stays anchored to the resin, excess reagents and by-products can simply be washed away between cycles.
No coupling step is perfectly efficient. Small failures accumulate, producing deletion sequences that are missing one or more residues. These related impurities are chemically similar to the target peptide, which is precisely why they are the hardest ones to separate and the main reason chromatographic purification is required.
After cleavage from the resin, the crude peptide is typically purified by preparative reversed-phase chromatography and then lyophilized to a dry solid.
Salt form, net peptide content and counter-ions
Purified synthetic peptides are usually isolated as salts. Trifluoroacetic acid is widely used in purification, so peptides are frequently supplied as TFA salts, and acetate salts are also common.
This matters for anyone weighing material. The mass in a vial includes counter-ions and residual water, so it is not identical to the mass of peptide. Net peptide content, sometimes called peptide content by amino acid analysis, is the figure that expresses how much of the vial is actually peptide, and it is a different measurement from chromatographic purity.
A vial can be 99% pure by HPLC and still be well below 99% peptide by mass, because purity describes the composition of the peptide fraction while net peptide content describes the composition of the solid.
- Chromatographic purity — the proportion of total peak area attributable to the target peptide.
- Net peptide content — the proportion of the supplied solid that is peptide rather than counter-ion, water or salt.
- Identity confirmation — evidence that the material present is the intended sequence, typically from mass spectrometry.
Frequently asked questions
- What is the difference between a peptide and a protein?
- The distinction is conventional rather than strict. Chains up to roughly fifty residues are usually called peptides and longer chains proteins. A more practical difference is that short peptides often have no stable folded structure in solution, while proteins generally do.
- Why is a peptide's molecular weight not the sum of its amino acids?
- Because a molecule of water is lost at every peptide bond formed. The mass of the chain equals the sum of the residue masses plus one water molecule for the intact peptide.
- What does it mean when a peptide is supplied as a TFA salt?
- It means trifluoroacetate counter-ions are associated with the peptide, usually as a consequence of the purification process. The counter-ions contribute to the mass in the vial, so the weighed mass is greater than the mass of peptide alone.
Sources and further reference
Compounds this applies to
The considerations above are not abstract. Each of these compounds is listed in the Peptide Tactics catalog for research use in India, with its reference data, analytical notes and documentation status set out in full.
BPC-157
BPC-157 is a synthetic pentadecapeptide — a chain of fifteen amino acids — corresponding to a partial sequence of the gastric peptide referred to in the literature as body protection compound. It is listed here as a research catalog item with the compound reference data recorded in public chemical databases.
View listing and pricingMOTS-c
MOTS-c is a 16-residue mitochondrial-derived peptide. It is unusual among the peptides in this catalog because it is encoded within the mitochondrial genome rather than the nuclear genome, in an alternative open reading frame inside the 12S ribosomal RNA gene.
View listing and pricingGHK-Cu
GHK-Cu is the copper(II) complex of the tripeptide glycyl-L-histidyl-L-lysine. Unlike the other entries in this catalog it is a metal-peptide complex rather than a peptide alone, which changes how it is described, handled and analysed.
View listing and pricingSee the full research peptide catalog, the India sourcing guide or how to request documentation.
Continue reading
- Peptide purity explained — What a peptide purity percentage actually measures, why it differs from net peptide content, and which method details make a purity claim readable.
- Lyophilized peptides explained — What freeze-drying does to a peptide, why research peptides are supplied as dry solids, how to read a cake, and what residual moisture means.
- Understanding peptide certificates of analysis — How to read a peptide certificate of analysis: which fields carry real information, how to spot a template, and what a COA cannot tell you.