Peptide research glossary

The terms that appear on certificates of analysis, specification sheets and in our research notes, each defined once in plain language.

Analysis and testing

HPLC (High-performance liquid chromatography): A separation technique that pumps a dissolved sample through a packed column at high pressure. The components leave the column at different times and a detector records each as a peak. Peptides are usually analysed by reversed-phase HPLC on a C18 column, with UV detection around 214 to 220 nm, where the peptide bond absorbs. Reading an HPLC chromatogram.
Chromatogram: The detector trace from a chromatographic run: signal plotted against time. Each peak is a component that reached the detector, and the area under a peak is proportional to how much of that component the detector saw.
Retention time: How long a component takes to pass through the column to the detector. It helps tell peaks apart, but it depends on the column, solvents, gradient and flow rate, so retention times are only comparable between runs of the same method.
HPLC purity (Chromatographic purity, Area percent purity): The area of the main peak as a percentage of the total area of all detected peaks. It measures how much of what the detector saw is the target compound. It is not the same as net peptide content: a sample can be 99% pure by HPLC and still be well under 100% peptide by weight.
Mass spectrometry (MS): An analytical method that ionises molecules and measures their mass-to-charge ratio (m/z). For a peptide it confirms identity: the observed mass is compared with the mass calculated from the sequence.
LC-MS and LC-MS/MS (Liquid chromatography-mass spectrometry, Tandem mass spectrometry): Liquid chromatography coupled to a mass spectrometer, so each separated component is also weighed. LC-MS/MS goes a step further: selected ions are broken into fragments and the fragments are measured, which can confirm the sequence as well as the overall mass.
Identity testing: Confirming that a material is the compound it is labelled as, most often by matching its measured molecular mass to the theoretical mass of the stated sequence. Identity and purity are separate questions: a sample can be the right compound and still contain impurities.
Net peptide content (Peptide content): The share of a lyophilised powder's weight that is the peptide itself, as opposed to counter-ions, bound water and residual salts. It is measured separately from purity, typically by amino acid analysis or elemental nitrogen, and is commonly in the range of 60 to 90% for synthetic peptides even at high HPLC purity.
Endotoxin (Lipopolysaccharide, LPS): Lipopolysaccharide from the outer membrane of Gram-negative bacteria. It is measured with the LAL (Limulus amebocyte lysate) assay and reported in endotoxin units (EU). It matters most in cell-culture work, where even small amounts can trigger responses that confound a result.
Independent testing (Third-party testing): Analysis carried out by a laboratory with no commercial interest in the result, as opposed to testing by the manufacturer or the seller of the material. How independent purity testing works.

Chemistry and structure

Peptide: A short chain of amino acids joined by peptide (amide) bonds. By convention chains of up to about 50 residues are called peptides and longer ones proteins, though the boundary is not fixed. Peptide basics.
Amino acid residue: One amino acid unit within a peptide chain. It is called a residue because each amino acid loses the elements of a water molecule when it forms a peptide bond.
Sequence (Primary structure): The order of the amino acid residues in a peptide, written from the N-terminus to the C-terminus in one-letter or three-letter codes.
One-letter code: The standard single-letter abbreviations for the 20 proteinogenic amino acids, for example G for glycine, P for proline and K for lysine. A sequence written as GHK is glycine, histidine, lysine.
N-terminus and C-terminus: The two ends of a peptide chain: the free amino group at the N-terminus and the free carboxyl group at the C-terminus. Modifications at either end change the molecule's formula and mass.
C-terminal amidation: Replacement of the C-terminal carboxylic acid with an amide (written -NH2 after the sequence). It is common in naturally occurring peptide hormones and their synthetic analogues, and lowers the molecular mass by about 1 Da relative to the free acid.
N-terminal acetylation: Addition of an acetyl group to the N-terminal amine, as in the N-acetyl variants of some peptides. It adds 42.01 Da to the monoisotopic mass and removes the positive charge at that end.
Molecular formula: The count of each element in one molecule, written in Hill order (carbon, hydrogen, then the rest alphabetically). For a peptide it follows directly from the sequence and any end modifications.
Molecular weight (Average mass, Monoisotopic mass): The mass of one molecule. Average molecular weight, in g/mol, uses natural isotope abundances and is what specification sheets list. Monoisotopic mass uses the most abundant isotope of each element and is what a mass spectrometer's principal peak corresponds to for smaller peptides.
CAS number: A unique numeric identifier assigned by the Chemical Abstracts Service to a specific substance. It identifies a compound unambiguously across suppliers and databases. Not every research peptide has one.
Counter-ion (TFA salt, Acetate salt): Synthetic peptides are isolated as salts. Trifluoroacetate (TFA) remains from cleavage and HPLC purification; acetate is common after an ion-exchange step. The counter-ion adds to the weight of the powder but not to the peptide content.
Solid-phase peptide synthesis (SPPS): The standard way synthetic peptides are made. Amino acids are added one at a time to a growing chain anchored on a resin bead, then the finished chain is cleaved from the resin and purified, usually by preparative HPLC.
PEGylation: Covalent attachment of polyethylene glycol (PEG) chains to a molecule. Because PEG is a mixture of chain lengths, a PEGylated compound has a distribution of masses rather than one molecular weight.
DAC (Drug affinity complex): A reactive maleimide group attached to a peptide through an added lysine, as in CJC-1295 with DAC. It is designed to bond covalently to serum albumin. The same peptide without it is sold as the no-DAC form, and the two are different molecules.

Handling and storage

Lyophilisation (Freeze-drying): Removing water from a frozen sample under vacuum, so the ice passes straight to vapour. Peptides are supplied lyophilised because the dry powder is far more stable than a solution. Cold chain handling and storage.
Reconstitution: Dissolving a lyophilised powder in a measured volume of a suitable solvent to make a solution of known concentration. Concentration in mg/ml is the mass in the vial divided by the volume added. Reconstitution calculator.
Bacteriostatic water: Sterile water containing 0.9% benzyl alcohol, a preservative that inhibits bacterial growth, so that a vial can be entered more than once. Plain sterile water has no preservative and suits single-use preparation.
Dilute acetic acid: A weak acid solution, typically around 0.6%, used to dissolve peptides that are poorly soluble at neutral pH, most often basic peptides.
Aliquot: A measured portion taken from a larger volume. Dividing a stock solution into single-use aliquots means each portion is thawed once and the rest stays untouched.
Freeze-thaw cycle: One freezing and thawing of a solution. Repeated cycles can degrade peptides, through aggregation and concentration effects as ice forms, which is why stocks are aliquoted before freezing.
Hygroscopic: Readily absorbs moisture from the air. Lyophilised peptides are hygroscopic, so a cold vial should reach room temperature before it is opened, or condensation will form on the powder.
Oxidation and deamidation: Two common chemical degradation routes for peptides. Methionine and cysteine residues oxidise; asparagine and glutamine residues can deamidate. Cold, dry, dark storage slows both.

Documentation and regulation

Certificate of analysis (COA, CoA): A document reporting the results of analytical tests on one specific batch: what was tested, by which methods, the results, the laboratory and the date, tied to the batch's lot number. How to read a certificate of analysis.
Lot number (Batch number): The identifier of a single production batch, printed on each vial. It is what links a vial in hand to the certificate of analysis for the material inside it.
Research use only (RUO): Supplied for laboratory research, not for human or veterinary use, and not a medicine, food or cosmetic. In the UK, how a product is presented decides whether the MHRA regulates it as a medicine, which is why research-use material is described by what it is, not by what it might do. UK rules for research peptides.
In vitro and in vivo: In vitro means in cells, tissues or reactions outside a living organism; in vivo means in a living organism. A finding in one does not establish the same effect in the other.
Preclinical: Research done before any testing in people: in vitro work and animal studies. Much of the published literature on research peptides is preclinical, and it is reported here as such.

For laboratory research use only. Nothing here is dosing, administration, or medical guidance.