What Is the N-Terminus?

Key facts

01 N-Terminus Definition

The N-terminus is the start of a peptide chain: the first amino acid residue, whose alpha-amino group is not part of a peptide bond ^[1].

Every amino acid has an amino group and a carboxyl group. Link them up and each peptide bond consumes the carboxyl group of one residue plus the amino group of the next. In a linear chain, that leaves one spare amino group at one end and one spare carboxyl group at the other. The amino end is the N-terminus, and the carboxyl end is the C-terminus.

Every separate chain gets its own amino end, so a two-chain protein such as insulin has two. That is exactly what Sanger picked up in 1945. He tagged the free amino groups, found glycine and phenylalanine at chain ends, and concluded that insulin contains two chains ^[2].

02 Why Are Sequences Written From the N-Terminus?

Partly it is convention: IUPAC-IUB says so. It also matches the direction in which cells make proteins ^[1][3].

A ribosome adds each new amino acid to the free carboxyl end of the growing chain. The N-terminal residue is made first, and the chain grows toward the C-terminus ^[3]. Writing sequences the same way keeps the page in step with the biology. In BPC-157 (GEPPPGKPADDAGLV) the N-terminal residue is glycine; in MOTS-c (MRWQEMGYIFYPRKLR) it is methionine.

In the lab it runs the other way. With solid-phase peptide synthesis the C-terminal residue is anchored to the resin and each new residue goes on toward the amino end, which makes the N-terminal residue the last one attached.

03 Charge and Chemistry at the N-Terminus

A free amino end is basic. Near neutral pH it usually carries a positive charge.

The terminal alpha-amino group in a peptide typically has a pKa of roughly 8 to 9, so at pH 7 most molecules carry it as -NH3+. That charge feeds into the peptide's net charge and solubility, and into how long it is retained in reversed-phase HPLC. Cap that amine, say with an acetyl group, and the charge disappears. Retention time and ionization behavior can shift noticeably as a result.

04 Common N-Terminal Modifications

Few positions get modified as often as this one, in natural proteins and synthetic peptides alike.

Acetylation in particular is everywhere in human cells. A proteomics study found it on 84% of the human protein N-termini analyzed, against 57% in yeast ^[4]. Among synthetic examples on Vinnix pages are PT-141, whose structure begins Ac-Nle, and Tesamorelin, whose first residue carries a trans-3-hexenoyl group that distinguishes it from natural GHRH.

05 How the N-Terminus Is Used in Analysis

Classic sequencing chemistry starts at the free amino end, and that end is one of the two anchors for reading fragment spectra.

• Edman degradation. Phenyl isothiocyanate reacts with the free N-terminal amine and clips off one residue per cycle, reading the sequence from that end inward. Automated in the 1960s as the protein sequenator ^[5], it cannot start on a blocked (acetylated or pyroglutamate) N-terminus.
• Tandem mass spectrometry. When a peptide fragments along its backbone, the pieces that keep the amino end are named a, b and c ions under the standard nomenclature ^[6]. With a complete b-ion ladder you can read the sequence from the N-terminal side. See Mass Spectrometry.
• Intact mass. An unexpected +42 Da shift often points to an acetylated N-terminus; -17 Da on an N-terminal Gln suggests pyroglutamate.

A Certificate of Analysis summarizes these checks, and our guide to reading a COA explains where to find them.

06 Finding the N-Terminus in a Written Sequence

Look at the first symbol on the left, along with any prefix attached to it. In a correctly written sequence, that is the N-terminal residue.

Watch two prefixes when you read this column. D- belongs to the residue and tells you its configuration, nothing about the terminus. Ac- belongs to the terminus and tells you the amino group is capped. For the codes themselves, see One-Letter vs. Three-Letter Amino Acid Codes.

07 N-Terminus vs Amino Group vs Side-Chain Amine

The N-terminus means one particular amino group: the one on the first residue's alpha carbon. Other amines in the chain are something else.

Lysine side chains carry amino groups too, and the reagents used to modify the terminal amine will happily react with them. Careful nomenclature handles this by writing N-alpha for the terminal amine and N-epsilon for the lysine side chain ^[1]. In CJC-1295 with DAC, for instance, the linker is attached to a lysine side chain rather than the N-terminus; see CJC-1295. The wider conventions are in Understanding Peptide Sequences.

REFScientific references

1. IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN). Nomenclature and symbolism for amino acids and peptides. Recommendations 1983. Eur J Biochem. 1984;138(1):9-37. PubMed 6692818
   nomenclature standard
2. Sanger F. The free amino groups of insulin. Biochem J. 1945;39(5):507-515. PubMed 16747948
   in vitro chemical study
3. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. Molecular Biology of the Cell. 4th ed. New York: Garland Science; 2002. From RNA to Protein. NCBI Bookshelf NBK26829. Source
   textbook (NCBI Bookshelf)
4. Arnesen T, Van Damme P, Polevoda B, et al. Proteomics analyses reveal the evolutionary conservation and divergence of N-terminal acetyltransferases from yeast and humans. Proc Natl Acad Sci U S A. 2009;106(20):8157-8162. PubMed 19420222
   proteomics study (human and yeast cells, in vitro)
5. Edman P, Begg G. A protein sequenator. Eur J Biochem. 1967;1(1):80-91. PubMed 6059350
   analytical methods paper
6. Roepstorff P, Fohlman J. Proposal for a common nomenclature for sequence ions in mass spectra of peptides. Biomed Mass Spectrom. 1984;11(11):601. PubMed 6525415
   nomenclature proposal (mass spectrometry)