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

 

Peptide Modification Applications
Acetylation Increase peptide stability by preventing N-terminal degradation
Biotin Commonly used in immunoassays, histocytochemistry, and fluorescence based flow cytometry
Dansyl and 2, 4-Dinitrophenyl Fluorescence based assays
Fluorescein and 7-methoxycoumarin acetic acid Protein-protein interaction and localization studies
Palmitic Acid Increase their cell permeability and help binding of the peptides to cell membrane
Cyclization (Disulfide bridge) Stabilize the peptide conformation, increase bioactivity, and enzyme stability
Cysteine Carbamidomethylation (CAM) Peptide Mass Fingerprinting for identification and characterization of peptides. To block cysteine residues from oxidation in protein assays
Phosphorylation Gene expression, protein-protein interaction and signal transduction in plants and animals
Isotope labeled Amino Acids Study protein interactions, proteins, post translation modifications such as ubiquitination and phosphorylation
Spacers Reduce steric hindrance at the binding sites of the peptide and its load

 

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



Molecular Weight 43 g/mol
Availability: PEPscreen®

          
References:

  1. Thomas, A., Towards a Functional Understanding of Protein N-Terminal Acetylation. PLOS Biol. 2011, 9(5).
  2. Wallace, R. J., Acetylation of peptides inhibits their degradation by rumen micro-organisms. British Journal of Nutrition. 1992, 68, 365-372.

 

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



Molecular Weight 340 g/mol
Availability: PEPscreen®

References:

  1. Selo, et. al., Preferential labeling of alpha-amino N-terminal groups in peptide by biotin: application to the detection of specific anti-peptide antibodies by enzyme immune assays. J. Immunol. Methods, 1996, 199, 127-138.
  2. Howl, et. al., Fluorescent and biotinylated linear peptides as selective bifunctional ligands for V1a vasopressin receptor. Eur. J. Biochem.1993, 213, 711-719.
  3. Buranda, et. al. Peptide, antibodies and FRET on beads in flow cytometry: a model system using fluoresceinated and biotinylated β-endorphin. Cytometry 1991, 37, 21-31.

 

1.3  Dansyl

Dansyl labeled peptides are used in fluorescence based assays.



Molecular Weight 249 g/mol
Excitation Wavelength 372 nm
Availability: PEPscreen®

References:

  1. Glukhov, et. al., Basis for selectivity of Cationic Antimicrobial Peptides for Bacteria Versus Mammalian Membranes. Journal of Biological Chemistry 2005, 280 (40), 33960-33967.
  2. Matsuzaki, et. al., Mechanism of Synergism between Antimicrobial Peptides Magainin 2 and PGLa. Biochem. 1998, 37, 15144-15153.
  3. Pecht, et. al., Specific Excitation Energy Transfer for Antibodies to Dansyl labeled. Antigen. Eur. J. Biochem. 1971, 19, 368-371.

 

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.



Molecular Weight 167 g/mol
Excitation Wavelength 354-400 nm
Availability: PEPscreen®

References:

  1. Vickers, et. al., Hydrolysis of Biological Peptides by Human Angiotensin-converting Enzyme –related carboxypeptidase. J. Biol. Chemistry 2002, 277 (17), 14838-14843.
  2. Knight, et. al., A novel coumarin labelled peptide for sensitive continuous assays of the matrix metalloproteinases. FEBS letters 1992, 296 (3), 263-266.

 

1.5  Fluorescein

Fluorescein-labeled peptides have many fluorescein based biomolecular applications including protein-protein interaction, flow cytometry and localization studies1-3.



Molecular Weight 359 g/mol
Excitation/Emission Wavelength 494/518 nm
Availability: PEPscreen®

References:

  1. Richard, et. al., Cell-penetrating peptides, J. Biol. Chem. 2003, 278 (3), 585-590.
  2. Farley. et. al., The amino acid sequence of a Fluorescein labeled peptide for active site of (Na,K)-ATPase. J. Biol. Chem. 1984, 259 (15), 9532-9535.
  3. Futaki, et. al., Arginine rich peptides. J. Biol. Chem. 2001, 276 (8) 5836-5840.
  4. Foerg, et. al., Metabolic cleavage and translocation efficiency of selected cell penetrating peptides: a comparative study with epithelial cell cultures. AAPS Journal 2008, 10 (2) 349-359.

 

1.6  7-methoxycoumarin acetic acid (Mca)

7-methoxy coumarin-labeled peptides have applications in protein-protein interaction and localization studies1-3.



Molecular Weight 217 g/mol
Excitation/Emission Wavelength 323/382 nm
Availability: PEPscreen®

References:

  1. Vidal, et. al., Solid-Phase Synthesis and Cellular Localization of a C-and/or N-terminal Labeled Peptide. Journal of Peptide Science, 1996, 2, 125-133.
  2. Yandek, et. al., Mechanism of the Cell-Penetrating Peptide Transportan 10 Permeation of Lipid Bilayers. Biophysical Journal 2007, 92 (7) 2434-2444.

 

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.



Molecular Weight 239 g/mol
Availability: PEPscreen®

References:

  1. Avrahami, D., Shai, Y., A new group of antifungal and antibacterial lipopeptides derived from non-membrane active peptides conjugated to palmitic acid. J. Biol. Chem. 2004, 279(13), 12277-12285.
  2. Buss, J.E, Sefton, B.M., Direct identification of Palmitic Acid at the lipid attached to p21ras. Mol.and Cellular Biol. 1986, 116-122.

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 stability1-2.

Molecular Weight N/A
Availability: PEPscreen®, AQUA™ Peptides

References:

  1. Albericio, et. al., Multifaceted Roles of Disulfide Bonds. Peptides as Therapeutics. Chem Rev. 2014, 114 (2) 901-926.
  2. Schmelz, et. al., An Amino Acids Substitution Inhibits Specialist Herbivore Production of an Antagonist Effector and Recovers Insect-Induced Plant defenses. Plant Physiology, 2012, 160 (3) 1468-1478.

 

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.

 

Molecular Weight 160 g/mol
Availability: PEPscreen®, AQUA™ Peptides

References:

  1. Marc R. Wilkins, Ron D Appel, Keith L.Williams, Denis F. Hochstrasser, Proteome Research Concepts, Technology and Application. ISBN978-3-540-71241-1.
  2. Koehler P. et al., Improved identification of wheat gluten proteins through alkylation of cysteine residues and peptide-based mass spectrometry. Scientific Reports 3, 2013, 2279.

 

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 phosphorylation2-5.

References:

  1. Gerbe, et. al., Absolute quantitation of protein and post-translation modification with stable isotope-labeled synthetic peptides. Nat. Protoc. 2011, 6(2) 175-186.
  2. Hioe, et. al., Proximal Glycan Outside of the Epitopes Regulate the Presentation of HIV-1 Envelope gp120 Helper Epitopes. The Journal of Immunol. 2009, 182, 6369-6378.
  3. Le Bihan, T., et al., Quantitative analysis of low-abundance peptides in HeLa cell cytoplasm by targeted liquid chromatography/mass spectrometry and stable isotope dilution: emphasizing the distinction between peptide detection and peptide identification. Rapid Commun. Mass Spectrom., 2010, 24(7), 1093-1104.
  4. Santhoskumar, P., et al., αA-Crystallin Peptide 66SDRDKFVIFLDVKHF80 Accumulating in Aging Lens Impairs the Function of α-Crystallin and Induces Lens Protein Aggregation. PLoS One 2011, 6(4), e19291.
  5. Sato, Y., et al., Simultaneous Absolute Protein Quantification of Carboxylesterases 1 and 2 in Human Liver Tissue Fractions using Liquid Chromatography-Tandem Mass Spectrometry.  Drug Metab Dispos., 2012, 40(7), 1389-1396.
  6. Brun, V., et al., Isotope dilution strategies for absolute quantitative proteomics. Journal of proteomics 2009, 72, 740-749.

 

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 animals1,2.



Molecular Weight 82 g/mol
Availability: PEPscreen®, AQUA™ Peptides

References:

  1. Hunter, T., Signaling – 2000 and Beyond. Cell 2000, 100, 113-127.
  2. Roberts, et. al., Phosphorylation of Soybean Nodulin26 on Serine 262 Enhances Water Permeability and Is Regulated Developmentally and by Osmotic Signals. The Plant Cell 2003, 15, 981-991.

 

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 peptides1-2.




Molecular Weight 146 g/mol
Availability: PEPscreen®

References:

  1. Huck W. T.S. et. al., Forced Peptide Synthesis under elastomeric stamps. Angew. Chem. 2004, 116, 4286-4289.
  2. F.M. Veronese, Peptide and protein PEGylation: a review of problems and solutions. Biomaterials 2001, 22, 405-417

 

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.



Molecular Weight 113 g/mol
Availability: PEPscreen®

References:

  1. Rothbard et. al., Polyarginine enters cells more efficiently than other poly cationic homopolymers. J. Peptide Res. 2000, 56, 318-325.
  2. Hoheisel et. al., Hybridisation based DNA screening on peptide nucleic acid (PNA) oligomer arrays. Nucleic acid Research 1997, 25 (14)2792-2799.


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

References:

  1. Kim K, -H. Seong, B. L. Peptide Amidation: Production of Peptide Hormones in vivo and in vitro. Biotechnol. Bioprocess Eng. 2001, 6 (4), 244-251.
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