N-Terminus vs C-Terminus: How to Read the Two Ends of a Peptide

Short answer: Every linear peptide has two different ends. The N-terminus is the residue whose amino group is free (not acylated by another residue); the C-terminus is the residue whose carboxyl group is free (it does not acylate another residue) [1]. By convention, sequences and formulas are written from the N-terminus on the left to the C-terminus on the right [1][4], so Ala-Ser and Ser-Ala are two different dipeptides [4].

This article is educational chemistry for laboratory readers. It does not describe biological effects, and it is not guidance for use in people or animals.

N-terminus and C-terminus: definitions

The IUPAC-IUB Joint Commission on Biochemical Nomenclature defines the ends of a peptide by what their terminal groups are doing [1]:

N-terminal residue: the residue with an amino group that is free, or at least not acylated by another amino-acid residue. It may still carry another group, for example an acetyl or formyl group, and it is still called N-terminal [1].
C-terminal residue: the residue with a free carboxyl group, or at least one that does not acylate another amino-acid residue. It may, for example, be converted to an amide (–CO–NH₂) and still be called C-terminal [1].

OpenStax puts the same idea in practical terms: the N-terminal amino acid is the one with the free –NH₃⁺ group, and the C-terminal amino acid is the one with the free –CO₂⁻ group [4]. Between the two ends runs the repeating –N–CH–CO– chain called the backbone [4]. For why each link in that backbone is a rigid, planar amide, see our guide to the peptide bond.

N-terminus vs C-terminus at a glance

Terminal group: N-terminus = free α-amino group; C-terminus = free α-carboxyl group [1][4].
Where it is written: N-terminus on the left; C-terminus on the right [1][4].
In a peptide name: the N-terminal residue starts the name as an acyl ("-yl") name; the C-terminal residue ends the name and is the only residue that keeps its full amino-acid name [1].
Typical charge in water near pH 7: the free amino end is protonated (–NH₃⁺) and the free carboxyl end is deprotonated (–CO₂⁻), the same pattern as free amino acids [4][5].
Common modifications: N-terminal acetylation (Ac-) and formylation; C-terminal amides (–NH₂) and esters (–OMe, –OEt) [1][3].
Direction of assembly: ribosomes build polypeptides from the N-terminus to the C-terminus [8]; chemical synthesis builds from the C-terminus toward the N-terminus [9].

How sequences and names follow the N-to-C rule

IUPAC's naming rule makes the direction explicit. When glycine acylates alanine, the dipeptide is glycylalanine; when they condense the other way, the product is alanylglycine [1]. The name of any peptide begins with the acyl name of the N-terminal residue, continues through the internal residues, and ends with the C-terminal residue, the only one named as a full amino acid [1].

The same direction applies to symbols. OpenStax's example: alanylserine is written Ala-Ser or A-S, and serylalanine is Ser-Ala or S-A [4]. Bachem states the rule from the manufacturing side: whatever notation is used, a peptide sequence is written starting at the N-terminus [9]. Residue numbering follows suit: IUPAC numbers residues from the N-terminus when atoms in a peptide need locants [1].

The hyphens in three-letter notation carry meaning too [2]:

Gly- means the glycine has given up the OH of its carboxyl group to a peptide bond (it is acylating the next residue) [2].
-Gly means the glycine has given up an H from its amino group (it is the acylated partner) [2].
-Gly- is an internal residue, bonded on both sides [2].

So Gly-Glu, with no hyphens at its ends, is a complete dipeptide with a free N-terminus and a free C-terminus, while -Gly-Glu- is a stretch from inside a longer chain [2]. For the two-residue case in more depth, see what is a dipeptide.

Charges at the two ends

At pH 7.3, a carboxyl group exists mainly as the carboxylate anion and an amino group mainly as the ammonium cation, which is why free amino acids exist in water primarily as zwitterions [5]. A peptide's free termini follow the same pattern: the N-terminal residue carries –NH₃⁺ and the C-terminal residue carries –CO₂⁻ [4].

For reference, OpenStax's Table 26.1 lists, for free amino acids, an α-CO₂H pKa of 2.34 and an α-NH₃⁺ pKa of 9.69 for alanine, and 2.34 and 9.60 for glycine [5]. These are values for the free amino acids, not for a residue inside a peptide. The Henderson–Hasselbalch equation links pH and pKa: when pH equals pKa, the acid and its conjugate base are present in equal amounts [6]. Just as single amino acids have isoelectric points, proteins have an overall isoelectric point set by the cumulative effect of the acidic and basic amino acids they contain [6]. Changing a terminus removes one of those ionizable groups from the count: an amide nitrogen is nonbasic [4], so an acetylated N-terminus no longer carries –NH₃⁺, and a C-terminal amide no longer has a –CO₂⁻ group.

Common N-terminal and C-terminal modifications

IUPAC symbols show terminal substitutions directly on the end of the sequence [3]:

N-acetyl (Ac-): N-acetylglycine is written Ac-Gly [3].
Ester at the carboxyl end: glycine ethyl ester is Gly-OEt; serine methyl ester is Ser-OMe [3].
C-terminal amide: -Ala-NH₂ shows the amide of a C-terminal alanine [3].
Unsubstituted ends, shown explicitly: H-Ala can be contrasted with Ac-Ala, and Ala-OH with Ala-OMe, to emphasize that a terminus carries no substituent [3].
Cyclized N-terminus: IUPAC provides symbols for 5-oxoproline (also known as pyroglutamic acid), a cyclic amino-acid derivative [2]; Unimod lists its formation from glutamine at a peptide N-terminus [12].

So a label that reads "H-…-OH" describes a peptide with a free amine and a free acid, while "Ac-…-NH₂" describes one capped at both ends [3].

Why the termini change the molecular weight

Terminal groups are part of the formula, so modifications show up as fixed mass shifts. The Unimod modification database lists them [10][11][12]:

Acetylation: adds C₂H₂O — +42.010565 Da monoisotopic, +42.0367 average. Unimod lists peptide N-termini among its sites [10].
Amidation (C-terminal amide): composition change H N O(−1) — −0.984016 Da monoisotopic, −0.9848 average, specified at the C-terminus [11].
Pyroglutamate from an N-terminal glutamine (Gln→pyro-Glu): composition change H(−3) N(−1), i.e., loss of NH₃ — −17.026549 Da monoisotopic, −17.0305 average [12].

Worked example: if a peptide's free-acid form (H-…-OH) has an average mass of 1,000.00 Da, the C-terminal amide (H-…-NH₂) has an average mass of 1,000.00 − 0.9848 = 999.02 Da, and the N-acetylated amide (Ac-…-NH₂) is 1,000.00 − 0.9848 + 42.0367 = 1,041.05 Da [10][11]. When a certificate of analysis gives an observed mass, comparing it with the theoretical mass for the stated terminal groups is a quick consistency check. Our peptide calculator guide covers mass math, and mass spectrometry peptide testing explains how observed masses are measured.

Direction of synthesis: N-to-C in cells, C-to-N in the lab

The two ends are also where chains are built from — in opposite directions depending on the method:

Ribosomes: all ribosomes translating an mRNA synthesize the polypeptide from the N-terminus to the C-terminus. When translation ends, a release factor catalyzes hydrolysis between the last amino acid and its tRNA, which releases the free carboxyl terminus [8].
Chemical synthesis: Bachem explains that in chemical synthesis the peptide is constructed from the C-terminus toward the N-terminus, so the terminal carboxyl group must stay protected throughout the synthesis [9]. Bachem also still uses solution-phase synthesis for some peptides modified at the C-terminus [9].

The sequence is still written N-to-C in both cases [9]. See our peptide synthesis guide for how solid-phase assembly and protecting groups work.

Reading from the N-terminus: Edman degradation

The N-terminus is also the classic handle for chemical sequencing. In Edman degradation, phenyl isothiocyanate (PITC) attaches to the –NH₂ group of the N-terminal amino acid, and treatment with trifluoroacetic acid then splits that residue off as an anilinothiazolinone (ATZ) derivative, leaving a peptide one residue shorter [7]. The ATZ derivative is converted into a phenylthiohydantoin (PTH), which is identified by its elution time, and the cycle repeats [7]. Automated sequencers can run as many as 50 cycles before by-products interfere [7].

Because the first step attaches PITC to the N-terminal –NH₂ group [7], a peptide whose N-terminus is acylated — for example acetylated, which IUPAC notes still counts as N-terminal [1] — does not have that free –NH₂ for PITC to attach to. Much peptide sequencing today is done by mass spectrometry using ESI or MALDI [7].

Why this matters for lab readers

Read sequences left to right as N→C. Ala-Ser and Ser-Ala are different compounds [4].
Check the end groups on the label. "H-" and "-OH" mean free ends; "Ac-" and "-NH₂" mean capped ends [3].
Expect a mass shift for terminal modifications. Amidation is about −0.98 Da and acetylation about +42.04 Da (average) [10][11].
Know which end a method needs. Edman chemistry starts at a free N-terminal amine [7].

See how to read a peptide COA for where sequence, mass and identity data appear on a certificate of analysis.

Frequently Asked Questions

What is the difference between the N-terminus and the C-terminus?
The N-terminus is the end of a peptide with a free (or at least non-acylated-by-another-residue) amino group; the C-terminus is the end with a free carboxyl group, or one that does not acylate another residue [1].

Which end of a peptide is written first?
The N-terminus. Formulas and sequences are written with the N-terminal residue on the left and the C-terminal residue on the right [1][4].

Is the N-terminus positive and the C-terminus negative?
In water near pH 7, typically yes: the free amino end is protonated (–NH₃⁺) and the free carboxyl end is deprotonated (–CO₂⁻) [4][5]. Capping an end removes that charge: an acetylated N-terminus is an amide, and an amide nitrogen is nonbasic [4]; a C-terminal amide has no carboxylate group [3].

What does "-NH₂" at the end of a peptide sequence mean?
It shows a C-terminal amide, as in -Ala-NH₂ for the amide of a C-terminal alanine [3]. Amidation lowers the mass by 0.984016 Da (monoisotopic) compared with the free acid [11].

What does "Ac-" at the start of a sequence mean?
An acetyl group on the N-terminal amino group, as in Ac-Gly for N-acetylglycine [3]. It adds 42.010565 Da (monoisotopic) [10].

Are peptides made from the N-terminus or the C-terminus?
Ribosomes build polypeptides from the N-terminus to the C-terminus [8]; chemical synthesis builds from the C-terminus toward the N-terminus [9].

Bottom line

The N-terminus is the end with the free amino group and the C-terminus is the end with the free carboxyl group [1]. Every name, sequence and residue number runs from N to C [1][4][9], terminal groups carry their own charges [4][5], and capping or modifying an end changes the formula and mass by known amounts [10][11][12]. For lab readers, checking the end groups is part of reading any sequence, label or COA correctly.

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