Laboratory reference

Peptide laboratory glossary

73 short definitions of terms used when handling, analysing and preparing solutions of synthetic peptides. Definitions are about the chemistry only. Each entry links to the guide that covers it in more depth.

73 terms

A

Acetate salt#: A peptide salt in which acetate (from acetic acid) is the counter-ion. It is made by exchanging the counter-ion after purification, and it gives a less acidic solution in unbuffered water than a trifluoroacetate salt does. Guide: Counter-Ions in Synthetic Peptides: TFA Salts and Acetate Salts See also: Counter-ion See also: TFA salt
Acetylation (N-terminal)#: The addition of an acetyl group to the free amino terminus of a peptide. It removes the positive charge of that amine and adds 42.011 Da (monoisotopic) to the mass. Guide: Solid-Phase Peptide Synthesis: An Overview of the Chemistry See also: N-terminus See also: Amidation (C-terminal)
Adduct#: In mass spectrometry, an ion formed when a molecule picks up an extra ion other than a proton, most often sodium (+21.982 over the protonated ion) or potassium (+37.956). Guide: Mass Spectrometry for Peptide Identity See also: Mass spectrometry See also: m/z (mass-to-charge ratio)
Adsorption#: The sticking of molecules to a surface, such as the wall of a vial. It can remove a large share of a peptide from a very dilute solution without any chemical change. Guide: Peptide Solubility and Aggregation: Laboratory Basics See also: Aggregation See also: Aliquot
Aggregation#: The clumping of individual molecules into larger assemblies. Concentration, temperature, pH, salts and interfaces all influence it, and it can show up as haze, particles or loss of signal in chromatography. Guide: Peptide Solubility and Aggregation: Laboratory Basics Guide: Peptide Storage Stability and Degradation Pathways See also: Solubility See also: Isoelectric point (pI)
Aliquot#: A measured portion taken from a larger sample or solution, or the act of dividing a stock into portions that are stored separately so that each is thawed once. Guide: Peptide Solubility and Aggregation: Laboratory Basics See also: Stock solution See also: Freeze–thaw cycle
Amidation (C-terminal)#: A peptide whose C-terminus ends in an amide (–CONH2) rather than a free carboxylic acid. It removes the negative charge of the terminus and changes the mass by −0.984 Da compared with the free acid. Guide: Solid-Phase Peptide Synthesis: An Overview of the Chemistry See also: C-terminus See also: Acetylation (N-terminal)
Amino acid analysis#: A method that breaks a peptide into its amino acids by hydrolysis, then separates and quantifies them. Because the composition is known, the result can be used to work out how much peptide a sample contains. Guide: Peptide Purity Versus Net Peptide Content See also: Net peptide content See also: Hydrolysis
Amino acid sequence#: The order of amino acid residues in a peptide, written from the N-terminus to the C-terminus, usually with one- or three-letter codes. Guide: Mass Spectrometry for Peptide Identity See also: Residue See also: N-terminus See also: C-terminus
Area percent#: The area of one chromatographic peak divided by the total area of all integrated peaks, times 100. It is the usual way a chromatographic purity is reported. Guide: How to Read an HPLC Chromatogram and a Purity Value See also: Purity See also: Chromatogram
Aspartimide (succinimide)#: A five-membered ring that can form from an aspartic acid or asparagine residue. It is an intermediate in deamidation and isomerization and a side product in peptide synthesis. Guide: Peptide Storage Stability and Degradation Pathways Guide: Solid-Phase Peptide Synthesis: An Overview of the Chemistry See also: Deamidation See also: Racemization
Average mass#: The mass of a molecule calculated with the average atomic weights of its elements, weighted by natural isotope abundance. It sits near the centre of an isotope cluster and is the figure usually quoted as molecular weight. Guide: Mass Spectrometry for Peptide Identity See also: Monoisotopic mass See also: Molecular weight

B

Buffer#: A solution containing a weak acid and its conjugate base, which resists changes in pH when small amounts of acid or base are added. Guide: Peptide Solubility and Aggregation: Laboratory Basics See also: pH

C

Cake#: The dry, porous solid left in a vial after freeze-drying. Its appearance reflects the freezing and drying conditions but is not a measure of content or purity. Guide: Lyophilization: Why Research Peptides Are Freeze-Dried See also: Lyophilized See also: Lyophilization
Charge state#: The number of charges carried by an ion in mass spectrometry. Electrospray of a peptide typically produces several charge states, each at its own m/z. Guide: Mass Spectrometry for Peptide Identity See also: m/z (mass-to-charge ratio)See also: Electrospray ionization (ESI)
Chromatogram#: A plot of detector signal against time from a chromatographic run. Each peak represents a component, or group of components, leaving the column together. Guide: How to Read an HPLC Chromatogram and a Purity Value See also: HPLC See also: Retention time
Cleavage#: In solid-phase peptide synthesis, the step that detaches the finished peptide from the resin and removes side-chain protecting groups, usually with strong acid. Guide: Solid-Phase Peptide Synthesis: An Overview of the Chemistry See also: Resin See also: Crude peptide
Concentration#: The amount of a substance per unit volume of a solution, for example mg/mL (mass concentration) or mM (molar concentration). Guide: Peptide Purity Versus Net Peptide Content See also: Molarity See also: Stock solution
Cosolvent#: A second, water-miscible solvent added in a small proportion to help dissolve a poorly soluble substance. Guide: Peptide Solubility and Aggregation: Laboratory Basics See also: Solvent See also: Solubility
Counter-ion#: The ion that balances the charge of another ion. In synthetic peptides, positively charged amino groups are balanced by anions such as trifluoroacetate, acetate or chloride. Guide: Counter-Ions in Synthetic Peptides: TFA Salts and Acetate Salts See also: TFA salt See also: Acetate salt See also: Hydrochloride salt
Coupling#: The step in peptide synthesis in which an activated amino acid reacts with the free amine on the growing chain to form a new peptide bond. Guide: Solid-Phase Peptide Synthesis: An Overview of the Chemistry See also: Deprotection See also: Solid-phase peptide synthesis (SPPS)
Crude peptide#: The material recovered after cleavage from the resin and before purification. It contains the target peptide together with synthesis by-products. Guide: Solid-Phase Peptide Synthesis: An Overview of the Chemistry See also: Cleavage See also: Deletion sequence
C-terminus#: The end of a peptide chain that carries a free carboxyl group (or an amide, if the peptide is amidated). By convention a sequence is written ending at the C-terminus. See also: N-terminus See also: Amidation (C-terminal)

D

Deamidation#: The conversion of an asparagine or glutamine side chain into the corresponding carboxylic acid. For asparagine it usually runs through a succinimide ring and adds 0.984 Da to the mass. Guide: Peptide Storage Stability and Degradation Pathways See also: Aspartimide (succinimide)See also: Hydrolysis
Deletion sequence#: An impurity in a synthetic peptide that lacks one residue because a coupling did not go to completion. Its mass is lower than the target by that residue’s mass. Guide: Solid-Phase Peptide Synthesis: An Overview of the Chemistry See also: Crude peptide See also: Coupling
Deprotection#: Removal of a temporary protecting group, for example the Fmoc group from the amino terminus of the growing chain, so that the next coupling can take place. Guide: Solid-Phase Peptide Synthesis: An Overview of the Chemistry See also: Fmoc See also: Coupling
Desiccant#: A drying agent, such as silica gel, used to keep air in a container dry. Guide: Lyophilization: Why Research Peptides Are Freeze-Dried See also: Hygroscopic See also: Residual moisture
Diluent#: The liquid used to dissolve or dilute a sample. In laboratory work the term is used interchangeably with solvent when a solid is dissolved. See also: Solvent
Dilution#: Lowering the concentration of a solution by adding more solvent. The relation C1 × V1 = C2 × V2 links the concentrations and volumes before and after. See also: Serial dilution See also: Stock solution
Disulfide bond#: A covalent S–S bond between the side chains of two cysteine residues. Each disulfide lowers the mass by about 2.016 Da compared with the two free thiols. Guide: Solid-Phase Peptide Synthesis: An Overview of the Chemistry See also: Oxidation See also: Residue

E

Electrospray ionization (ESI)#: A mass spectrometry ionization method in which a solution is sprayed through a charged needle. Peptides typically appear with several charges, so one molecule gives a series of peaks. Guide: Mass Spectrometry for Peptide Identity See also: MALDI See also: Charge state
Extinction coefficient#: A constant relating how much light a substance absorbs at a given wavelength to its concentration (Beer–Lambert law). For peptides it can be calculated from the amino acid composition. Guide: Peptide Purity Versus Net Peptide Content See also: Net peptide content

F

Fmoc#: Fluorenylmethyloxycarbonyl, a base-removable protecting group for amino groups. Fmoc chemistry is the most widely used strategy for solid-phase peptide synthesis. Guide: Solid-Phase Peptide Synthesis: An Overview of the Chemistry See also: Deprotection See also: Solid-phase peptide synthesis (SPPS)
Freeze–thaw cycle#: One complete freezing and thawing of a sample. Repeated cycles expose a solution to ice surfaces and concentration changes, which is why stocks are often divided into portions. Guide: Peptide Storage Stability and Degradation Pathways See also: Aliquot See also: Aggregation

G

Gradient#: In HPLC, a planned change in the composition of the mobile phase over time, for example from water-rich to richer in acetonitrile. Guide: How to Read an HPLC Chromatogram and a Purity Value See also: Mobile phase See also: RP-HPLC
Gross weight#: The total mass of a powder on the balance, including peptide, counter-ions, water and any other solids. It is larger than the mass of peptide. Guide: Peptide Purity Versus Net Peptide Content See also: Net peptide content

H

HPLC#: High-performance liquid chromatography: a technique that separates the components of a mixture by pumping it through a packed column, then detects them as they leave. Guide: How to Read an HPLC Chromatogram and a Purity Value See also: RP-HPLC See also: Chromatogram
Hydrochloride salt#: A salt in which chloride (from hydrochloric acid) is the counter-ion. Peptide hydrochlorides are made by exchange, often by freeze-drying from dilute hydrochloric acid. Guide: Counter-Ions in Synthetic Peptides: TFA Salts and Acetate Salts See also: Counter-ion See also: Acetate salt
Hydrolysis#: The breaking of a chemical bond by reaction with water. In peptides it can cleave the backbone amide bond or convert side-chain amides. Guide: Peptide Storage Stability and Degradation Pathways See also: Deamidation See also: Amino acid analysis
Hygroscopic#: Tending to take up water from the surrounding air. Many freeze-dried cakes are hygroscopic, which is why containers are kept sealed and vials are warmed to room temperature before opening. Guide: Lyophilization: Why Research Peptides Are Freeze-Dried See also: Residual moisture See also: Desiccant

I

Ion-pairing reagent#: An acid or salt added to the HPLC mobile phase whose ion pairs with charged groups on the analyte, improving retention and peak shape. Trifluoroacetic acid is the usual choice for peptides. Guide: How to Read an HPLC Chromatogram and a Purity Value See also: TFA (trifluoroacetic acid)See also: RP-HPLC
Isoelectric point (pI)#: The pH at which a molecule carries no net charge. Peptide solubility is typically lowest near the isoelectric point. Guide: Peptide Solubility and Aggregation: Laboratory Basics See also: Solubility See also: pH

L

Lyophilization#: Freeze-drying: removal of water from a frozen sample by sublimation under reduced pressure, leaving a dry solid. Guide: Lyophilization: Why Research Peptides Are Freeze-Dried See also: Lyophilized See also: Lyophilization cycle
Lyophilization cycle#: The programme of shelf temperature and chamber pressure over time used in freeze-drying, consisting of freezing, primary drying (ice sublimation) and secondary drying (removal of bound water). Guide: Lyophilization: Why Research Peptides Are Freeze-Dried See also: Lyophilization See also: Cake
Lyophilized#: Describes a material that has been freeze-dried. It names a physical form and says nothing about the purity, content or identity of the material. Guide: Lyophilization: Why Research Peptides Are Freeze-Dried See also: Lyophilization See also: Cake

M

MALDI#: Matrix-assisted laser desorption ionization: a mass spectrometry method in which the sample is mixed with a light-absorbing matrix and ionized by a laser pulse, mostly giving singly charged ions. Guide: Mass Spectrometry for Peptide Identity See also: Electrospray ionization (ESI)See also: m/z (mass-to-charge ratio)
Mass spectrometry#: An analytical technique that ionizes molecules and measures their mass-to-charge ratios, used to check the molecular weight and sequence of peptides. Guide: Mass Spectrometry for Peptide Identity See also: m/z (mass-to-charge ratio)See also: Tandem mass spectrometry (MS/MS)
Mobile phase#: The liquid pumped through an HPLC column to carry the sample along. In reversed-phase peptide work it is a water and acetonitrile mixture with an acid additive. Guide: How to Read an HPLC Chromatogram and a Purity Value See also: Gradient See also: Ion-pairing reagent
Molarity#: Concentration expressed as moles of solute per litre of solution (mol/L, or mM for millimoles per litre). See also: Concentration See also: Molecular weight
Molecular weight#: The mass of one mole of a substance in g/mol (numerically equal to the mass of one molecule in daltons). Catalogue values are usually average masses. Guide: Mass Spectrometry for Peptide Identity See also: Average mass See also: Monoisotopic mass
Monoisotopic mass#: The mass of a molecule calculated using the most abundant isotope of each element (carbon-12, hydrogen-1, nitrogen-14, oxygen-16, sulfur-32). It corresponds to the first peak of an isotope cluster. Guide: Mass Spectrometry for Peptide Identity See also: Average mass See also: Molecular weight
m/z (mass-to-charge ratio)#: The quantity a mass spectrometer reports: the ion’s mass divided by its number of charges. A singly charged ion has m/z equal to its mass. Guide: Mass Spectrometry for Peptide Identity See also: Charge state See also: Mass spectrometry

N

Net peptide content#: The percentage of a powder’s mass that is peptide. The remainder is water, counter-ions and other solids. It is a different quantity from purity. Guide: Peptide Purity Versus Net Peptide Content See also: Purity See also: Gross weight
N-terminus#: The end of a peptide chain that carries a free amino group (unless it is acetylated). By convention a sequence is written starting at the N-terminus. See also: C-terminus See also: Acetylation (N-terminal)

O

Oxidation#: A reaction in which a molecule gains oxygen or loses electrons. In peptides it mainly affects methionine (to the sulfoxide, +15.995 Da), cysteine, tryptophan, tyrosine and histidine. Guide: Peptide Storage Stability and Degradation Pathways See also: Disulfide bond See also: Residue

P

pH#: A logarithmic measure of the hydrogen-ion activity of a solution. pH 7 is neutral; lower values are acidic and higher values basic. Guide: Peptide Solubility and Aggregation: Laboratory Basics See also: Buffer See also: Isoelectric point (pI)
Purity#: The share of a sample, by some stated measure, that is the intended substance. For peptides it is usually the area percent of the main HPLC peak, which is not the same as net peptide content. Guide: How to Read an HPLC Chromatogram and a Purity Value Guide: Peptide Purity Versus Net Peptide Content See also: Area percent See also: Net peptide content

R

Racemization#: Conversion of an amino acid residue from one mirror-image form (L) to the other (D). The mass does not change, so it is detected by chromatography rather than mass alone. Guide: Peptide Storage Stability and Degradation Pathways Guide: Solid-Phase Peptide Synthesis: An Overview of the Chemistry See also: Aspartimide (succinimide)
Residual moisture#: Water remaining in a freeze-dried solid, expressed as a percentage of its weight, commonly measured by Karl Fischer titration. Guide: Lyophilization: Why Research Peptides Are Freeze-Dried See also: Hygroscopic See also: Net peptide content
Residue#: An amino acid as it appears inside a peptide chain, after losing a molecule of water in forming the peptide bond. Residue masses are what is added up to estimate a peptide’s mass. Guide: Mass Spectrometry for Peptide Identity See also: Amino acid sequence See also: Molecular weight
Resin#: The insoluble polymer beads on which a peptide chain is assembled in solid-phase synthesis. Guide: Solid-Phase Peptide Synthesis: An Overview of the Chemistry See also: Solid-phase peptide synthesis (SPPS)See also: Cleavage
Retention time#: The time from the start of a chromatographic run to the top of a peak. It depends on the column, gradient, temperature and mobile phase, and is not proof of identity. Guide: How to Read an HPLC Chromatogram and a Purity Value See also: Chromatogram See also: Gradient
RP-HPLC#: Reversed-phase HPLC: HPLC with a hydrophobic column and a polar mobile phase, in which molecules leave the column in order of increasing hydrophobicity. Guide: How to Read an HPLC Chromatogram and a Purity Value See also: HPLC See also: Mobile phase

S

Serial dilution#: A series of dilutions in which each solution is made from the previous one by the same dilution factor, so the concentration falls geometrically. See also: Dilution See also: Stock solution
Solid-phase peptide synthesis (SPPS)#: A method of making peptides in which the chain is assembled one residue at a time on an insoluble resin, with excess reagents washed away between steps. Guide: Solid-Phase Peptide Synthesis: An Overview of the Chemistry See also: Fmoc See also: Resin
Solubility#: The amount of a substance that dissolves in a given amount of solvent under stated conditions, such as temperature and pH. Guide: Peptide Solubility and Aggregation: Laboratory Basics See also: Isoelectric point (pI)See also: Cosolvent
Solvent#: The liquid in which a substance is dissolved. Water, buffers and mixtures with organic solvents are common in peptide work. Guide: Peptide Solubility and Aggregation: Laboratory Basics See also: Cosolvent See also: Diluent
Stock solution#: A concentrated solution prepared in bulk and diluted to the working concentration when needed. Guide: Peptide Solubility and Aggregation: Laboratory Basics See also: Dilution See also: Aliquot
Stopper#: The rubber closure that seals a vial and keeps the contents dry and clean. Once it has been pierced or removed, the sealed environment is gone. Guide: Lyophilization: Why Research Peptides Are Freeze-Dried See also: Vial See also: Desiccant

T

Tandem mass spectrometry (MS/MS)#: A mass spectrometry experiment in which an ion is selected, fragmented and its fragments measured. For peptides the fragments are mostly b and y ions that reveal the sequence. Guide: Mass Spectrometry for Peptide Identity See also: Mass spectrometry See also: Residue
TFA salt#: A peptide salt in which trifluoroacetate is the counter-ion. It is the usual result of cleavage with TFA and purification by HPLC with a TFA-containing mobile phase. Guide: Counter-Ions in Synthetic Peptides: TFA Salts and Acetate Salts See also: Counter-ion See also: Acetate salt
TFA (trifluoroacetic acid)#: A strong organic acid used to cleave peptides from the resin in synthesis and as an ion-pairing additive in reversed-phase HPLC. Its molar mass is about 114.02 g/mol. Guide: Counter-Ions in Synthetic Peptides: TFA Salts and Acetate Salts See also: TFA salt See also: Ion-pairing reagent

V

Vial#: A small glass or plastic container with a closure, used to hold a sample. A sealed vial protects the contents from moisture and contamination. Guide: Lyophilization: Why Research Peptides Are Freeze-Dried See also: Stopper

For laboratory research use only. Not for human or veterinary use. Definitions describe chemistry and laboratory practice in general and make no statement about any product or its use. See also the laboratory guides, the laboratory tools and the free printable lab sheets.