Peptide Modifications: N-Terminal, Internal, and C-Terminal

1.0 N-Terminal Modifications

1.1 Acetylation

This modification removes the positive charge on the N-terminal of peptides, thus mimicking natural proteins. In some cases, it increases peptide stability by preventing N-terminal degradation ^1-2

1.2 Biotin

Biotin has a very strong affinity for streptavidin and avidin. Biotin-labeled peptides are commonly used in immunoassays¹, histocytochemistry², and fluorescence based flow cytometry³

1.3 Dansyl

Dansyl labeled peptides are used in fluorescence based assays.

1.4 2, 4-Dinitrophenyl

2, 4-DNP is used as a quencher for (7-methoxy coumarin-4-yl) acetyl (MCA) and sometimes tryptophan. This modification can be attached at the N-terminal of a peptide or as an internal modification through lysine side chain.

1.5 Fluorescein

Fluorescein-labeled peptides have many fluorescein based biomolecular applications including protein-protein interaction, flow cytometry and localization studies^1-3.

1.6 7-methoxycoumarin acetic acid (Mca)

7-methoxy coumarin-labeled peptides have applications in protein-protein interaction and localization studies^1-3.

1.7 Palmitic Acid

Palmitic acid is 16-carbon fatty acid that is conjugated to peptides to increase their cell permeability and help binding of the peptides to cell membrane ^1-2.

2.0 Internal Modifications

2.1 Cyclization (Disulfide Bonds)

This process involves formation of disulfide bond between two cysteine residues. Peptides can be cyclized to stabilize the peptide conformation, increase bioactivity, and enzyme stability^1-2.

2.2 Cysteine Carbamidomethylation (CAM)

Carbamidomethylation (CAM) is a deliberate post-translational modification introduced to cysteine residues by reacting with iodoacetamide. Peptides with this modification are mainly used in Peptide Mass Fingerprinting for identification and characterization of proteins¹. In other assays, this process is used to block Cysteine from oxidation².

2.3 Isotope labeled Amino Acids

AQUA peptides are synthetic peptides with amino acids enriched in ¹⁸O, ¹³C, and/or ¹⁴N. They are similar to their native peptides in terms of chemical, physical properties and also their biological activities¹. Main applications for these peptides are to study protein interactions, proteins, post translation modifications such as ubiquitination and phosphorylation^2-5.

2.4 Phosphorylation

Phosphorylation can be performed on Tyr, Ser and Thr residues as a posttranslational modification (PTM) on peptides. Phosphorylated peptides have application in many cellular processes such as gene expression, protein-protein interaction and signal transduction in plants and animals^1,2.

2.5 Spacers

Spacers are used to create a distance between the peptide and the cargo to reduce steric hindrance at the binding sites of the peptide. In this case cargo can be a drug, dye, tag.

2.5.1 PEGylation

Attachment of poly (ethylene glycol) to a peptide is called PEGylation. Short bifunctional PEG (Poly (ethylene glycol)) can be used as a spacer in bioconjugation of peptides with other molecules. PEG bioconjugation has also been used to improve proteolytic stability, biodistribution and solubility of peptides^1-2.

2.5.2 Amino hexanoic acid

Amino hexanoic acid is a hydrophobic spacer to which a molecule- either a fluorophore, tag or any biological molecule - can be attached to a peptide ^1-2.

3.0 C-Terminal Modifications

3.1 Amide (Amidation)

The C-terminal of the peptide is synthesized as an amide to neutralize negative charge created by the C-terminal COOH. This modification is added to prevent enzyme degradation, to mimic native proteins, and in some cases to remove hydrogen bonding at the C-terminal of the peptides which may interfere with the assays.¹