Knowledge base
Peptide analysis glossary
46 terms used when reading certificates of analysis, chromatograms and mass spectra for research peptides, with links to the guides and compound pages where they matter.
A
Acetate salt
A peptide salt with acetate as the counter-ion, often prepared by exchange from the TFA salt. Each acetate adds about 60.05 Da per charged site.
Read more: Peptide Counter-Ions and Net Peptide Content: Why the Powder Weighs More Than the Peptide · GHRP-2
Adduct
An ion formed when a molecule picks up something other than a proton, most often sodium ([M+Na]+, +21.982 relative to [M+H]+) or potassium ([M+K]+, +37.956). Adducts are not impurities.
Read more: Common Peptide Degradation Products and the Mass Shifts That Reveal Them · Average, Monoisotopic and Most Abundant Mass: Reading Peptide Mass Spectrometry Values
Aib (α-aminoisobutyric acid)
A non-proteinogenic amino acid with two methyl groups on the α-carbon. It promotes helical structure and protects the peptide bond from enzymatic cleavage, which is why it appears in semaglutide, tirzepatide and ipamorelin.
Read more: Semaglutide · Tirzepatide · Ipamorelin
Amino acid analysis (AAA)
A method in which a peptide is hydrolysed to its amino acids, which are then quantified. It gives the amount of peptide in a sample and is the standard way to measure net peptide content.
Read more: Peptide Analytical Methods Compared: HPLC, LC-MS, Amino Acid Analysis, NMR and Karl Fischer · Peptide Counter-Ions and Net Peptide Content: Why the Powder Weighs More Than the Peptide
Amino acid residue
The part of an amino acid that remains in a peptide chain after it loses a molecule of water during bond formation. Peptide masses are calculated by adding residue masses and then the mass of the two chain ends.
Read more: Peptide Sequence Notation Explained: One-Letter Codes, D-Amino Acids and Terminal Groups · Average, Monoisotopic and Most Abundant Mass: Reading Peptide Mass Spectrometry Values
Area percent
The area of the main peak divided by the total area of all integrated peaks, times 100. It depends on the detection wavelength and on how the baseline and small peaks were integrated.
Read more: How to Read an HPLC Chromatogram for Peptide Purity · Peptide Purity vs Vial Content: Why the Numbers Differ
Average mass
The mass of a molecule calculated from the natural average atomic weight of each element. It is the value used for molecular weight and for large peptides, where the monoisotopic peak is too weak to see.
Read more: Average, Monoisotopic and Most Abundant Mass: Reading Peptide Mass Spectrometry Values
B
Bacteriostatic water
Sterile water that contains a preservative, usually 0.9 % benzyl alcohol, which inhibits bacterial growth in a multi-use vial. For laboratory use, benzyl alcohol can interfere with some assays and with cell cultures.
Read more: Laboratory Water and Diluents: Sterile Water, Bacteriostatic Water and Acetic Acid Solution
Batch (lot) number
An identifier that ties a vial, a COA and a manufacturing run together. A COA is only meaningful for the batch it names.
Read more: Batch-Specific COA Verification and Research Material Traceability
Batch traceability
The ability to follow a batch from supplier record through receiving, testing and use, using one stable identifier.
Read more: Batch-Specific COA Verification and Research Material Traceability · How to Build a Compound Identity File for Incoming Research Peptides
C
C-terminal amidation
Replacement of the C-terminal carboxylic acid by a carboxamide (-NH2). Many bioactive peptides, including ipamorelin, GHRP-2 and kisspeptin-10, are written with an amide and denoted with NH2 at the end of the sequence.
Read more: Peptide Sequence Notation Explained: One-Letter Codes, D-Amino Acids and Terminal Groups
C-terminus
The end of a peptide chain that carries a free carboxylic acid, or an amide if the peptide was synthesised with a C-terminal amide. The difference between an acid and an amide is 0.984 Da.
Read more: Peptide Sequence Notation Explained: One-Letter Codes, D-Amino Acids and Terminal Groups
Certificate of analysis (COA)
A document from the manufacturer or a testing laboratory that lists the tests performed on one batch, the methods used and the results obtained.
Read more: How to Read a Peptide Certificate of Analysis (COA) · How to Verify a Third-Party Peptide Test Report Without Altering It
Charge state
The number of protons attached to a peptide ion in electrospray ionisation, written as [M+nH]n+. Larger peptides carry more charges, so their peaks appear at lower m/z.
Read more: Average, Monoisotopic and Most Abundant Mass: Reading Peptide Mass Spectrometry Values · Mass Spectrometry for Peptide Identity: A Practical Guide
Counter-ion
The ion that balances the charge of a peptide in its solid form, usually trifluoroacetate (TFA) from synthesis and purification or acetate after exchange. The counter-ion is part of the powder mass but not of the peptide.
Read more: Peptide Counter-Ions and Net Peptide Content: Why the Powder Weighs More Than the Peptide
D
D-amino acid
The mirror-image (enantiomer) form of an amino acid. D-amino acids have the same mass as L-amino acids, so routine mass spectrometry does not tell them apart; chiral chromatography or special methods are needed.
Read more: Peptide Sequence Notation Explained: One-Letter Codes, D-Amino Acids and Terminal Groups
Deamidation
Conversion of asparagine or glutamine to aspartic or glutamic acid, which adds 0.984 Da. It is a major non-enzymatic degradation pathway, especially for asparagine followed by glycine or serine.
Read more: Common Peptide Degradation Products and the Mass Shifts That Reveal Them
Deconvolution
A calculation that converts a series of multiply charged ions into a single neutral mass. Reports for large peptides usually give the deconvoluted mass.
Read more: Average, Monoisotopic and Most Abundant Mass: Reading Peptide Mass Spectrometry Values
Disulfide bond
A covalent bond between the sulfur atoms of two cysteine residues. It lowers the mass by 2.016 Da compared with the two free thiols. Oxytocin, AOD9604, cagrilintide and IGF-1 LR3 contain disulfide bonds.
Read more: Oxytocin · AOD9604 · Common Peptide Degradation Products and the Mass Shifts That Reveal Them
E
Electrospray ionisation (ESI)
A soft ionisation method that produces multiply charged ions from peptides and proteins in solution. It is the usual way to couple HPLC to a mass spectrometer.
Read more: Mass Spectrometry for Peptide Identity: A Practical Guide · Average, Monoisotopic and Most Abundant Mass: Reading Peptide Mass Spectrometry Values
H
HPLC
High-performance liquid chromatography: a separation method that pushes a sample through a column under pressure. Peptide purity is normally measured by reversed-phase HPLC with UV detection at 214 nm.
Read more: How to Read an HPLC Chromatogram for Peptide Purity · Peptide Analytical Methods Compared: HPLC, LC-MS, Amino Acid Analysis, NMR and Karl Fischer
I
Identity test
A test that confirms that the material is the compound it is said to be, usually by mass spectrometry and, for peptides, by comparison with a reference standard.
Read more: Mass Spectrometry for Peptide Identity: A Practical Guide · How to Build a Compound Identity File for Incoming Research Peptides
Impurity profile
The list of related substances detected in a batch, with their approximate amounts and, where known, their identity. A good COA reports more than the main peak.
Read more: How to Read an HPLC Chromatogram for Peptide Purity · Peptide Analytical Methods Compared: HPLC, LC-MS, Amino Acid Analysis, NMR and Karl Fischer
K
Karl Fischer titration
A titration method that measures the water content of a solid sample. Lyophilized peptides can contain several percent of water.
Read more: Peptide Counter-Ions and Net Peptide Content: Why the Powder Weighs More Than the Peptide · Peptide Analytical Methods Compared: HPLC, LC-MS, Amino Acid Analysis, NMR and Karl Fischer
L
Lactam bridge
An amide bond formed between the side chains of an acidic residue (aspartic or glutamic acid) and a basic residue (lysine), which cyclises the peptide and removes a molecule of water (-18.011 Da). Melanotan II and PT-141 are closed this way.
Read more: Melanotan II · PT-141
Lipidation
Attachment of a fatty acid or fatty diacid to a peptide side chain, usually through a spacer, to promote binding to serum albumin. Semaglutide, liraglutide, tirzepatide and cagrilintide are lipidated.
Read more: Semaglutide · Liraglutide · Incretin-related and amylin peptides
Lyophilization
Freeze-drying: removal of water from a frozen sample under vacuum. Most research peptides are supplied as lyophilized powders because the dry form is more stable than a solution.
Read more: Lyophilized Peptides Explained: What Freeze-Drying Does and What It Does Not Guarantee
M
m/z
Mass divided by the number of charges. A peptide of mass 1000 Da carrying two protons appears at m/z 501.01.
Read more: Average, Monoisotopic and Most Abundant Mass: Reading Peptide Mass Spectrometry Values
Mass spectrometry (MS)
A technique that measures the mass-to-charge ratio (m/z) of ions. For peptides, it confirms identity by comparing the measured mass with the expected mass.
Read more: Mass Spectrometry for Peptide Identity: A Practical Guide · Average, Monoisotopic and Most Abundant Mass: Reading Peptide Mass Spectrometry Values
Methionine oxidation
Addition of one oxygen atom to the sulfur of methionine to give the sulfoxide (+15.995 Da). It is a common degradation product of peptides that contain methionine, such as Semax, MOTS-c and sermorelin.
Read more: Common Peptide Degradation Products and the Mass Shifts That Reveal Them · Semax · MOTS-c
Monoisotopic mass
The mass of a molecule calculated from the lightest stable isotope of each element (12C, 1H, 14N, 16O, 32S). It is the value used in high-resolution mass spectrometry of small and medium peptides.
Read more: Average, Monoisotopic and Most Abundant Mass: Reading Peptide Mass Spectrometry Values
N
N-terminal acetylation
Capping of the N-terminal amino group with an acetyl group (+42.011 Da). Thymosin alpha 1, thymosin beta-4 and melanotan peptides are acetylated, so they have no free N-terminal amine.
Read more: Peptide Sequence Notation Explained: One-Letter Codes, D-Amino Acids and Terminal Groups
N-terminus
The end of a peptide chain that carries a free amino group (or a capping group such as acetyl or pyroglutamate). Peptide sequences are written from the N-terminus to the C-terminus.
Read more: Peptide Sequence Notation Explained: One-Letter Codes, D-Amino Acids and Terminal Groups
Net peptide content
The percentage of the powder mass that is peptide, excluding water and counter-ions. It is always lower than 100 % and is measured by amino acid analysis or quantitative NMR, not by HPLC purity.
Read more: Peptide Counter-Ions and Net Peptide Content: Why the Powder Weighs More Than the Peptide · Peptide Purity vs Vial Content: Why the Numbers Differ
Non-proteinogenic amino acid
An amino acid that is not one of the 20 encoded by the standard genetic code, such as α-aminoisobutyric acid (Aib), norleucine (Nle), 2-naphthylalanine (2Nal) or 2',6'-dimethyltyrosine (Dmt).
Read more: Peptide Sequence Notation Explained: One-Letter Codes, D-Amino Acids and Terminal Groups
Nuclear magnetic resonance (NMR)
A technique that gives structural information about a molecule and, in its quantitative form, can measure content, including residual solvents and counter-ions such as TFA.
Read more: Peptide Analytical Methods Compared: HPLC, LC-MS, Amino Acid Analysis, NMR and Karl Fischer
P
Peptide
A chain of amino acids joined by peptide bonds. By convention, chains of up to about 50 residues are called peptides and longer chains are called proteins, although the boundary is not strict.
Read more: Peptide Sequence Notation Explained: One-Letter Codes, D-Amino Acids and Terminal Groups
Peptide purity
The proportion of the target peptide among all peptide-related species in a sample, usually reported as an HPLC area percentage. Purity says nothing about how much peptide is in the vial.
Read more: Peptide Purity vs Vial Content: Why the Numbers Differ · How to Read an HPLC Chromatogram for Peptide Purity
Pyroglutamate
A cyclic form of N-terminal glutamine or glutamic acid, written pGlu or pE. It has no free amine and is found in gonadorelin and ARA290. It lowers the mass by 17.027 Da compared with glutamine.
Read more: Gonadorelin · ARA290
R
Reference standard
A well-characterised sample of a compound that is used as a comparator in identity and purity tests.
Read more: How to Build a Compound Identity File for Incoming Research Peptides
Research use only (RUO)
A label meaning that a material is intended for laboratory research, not for use in humans or animals, in diagnostics, or as a medicine, supplement, cosmetic or food.
Read more: How to Read a Peptide Certificate of Analysis (COA)
Retention time
The time between injection and the maximum of a chromatographic peak. It identifies a compound only when it is compared with a reference run under the same conditions.
Read more: How to Read an HPLC Chromatogram for Peptide Purity
Reversed-phase HPLC (RP-HPLC)
HPLC with a non-polar stationary phase and a polar mobile phase whose organic content is increased during the run. Peptides elute in order of increasing hydrophobicity.
Read more: How to Read an HPLC Chromatogram for Peptide Purity
S
Stability-indicating method
An analytical method that can separate the intact compound from its degradation products, so a drop in purity can be detected.
Read more: Common Peptide Degradation Products and the Mass Shifts That Reveal Them
T
TFA salt
A peptide salt with trifluoroacetate as the counter-ion, the usual product of reversed-phase purification with a trifluoroacetic acid mobile phase. Residual TFA can affect cell assays and adds substantial mass.
Read more: Peptide Counter-Ions and Net Peptide Content: Why the Powder Weighs More Than the Peptide
Truncation and deletion sequences
Process-related impurities from solid-phase synthesis in which one or more amino acids are missing from the target peptide. They are the main impurities of long synthetic peptides.
Read more: How to Read an HPLC Chromatogram for Peptide Purity · Peptide Analytical Methods Compared: HPLC, LC-MS, Amino Acid Analysis, NMR and Karl Fischer