How to Know C and N Terminus in a Primary Sequence
The primary sequence of a protein is a linear chain of amino acids covalently linked by peptide bonds in a defined order. At the two ends of this polypeptide chain lie the N-terminus (amino terminus, N-terminal) and the C-terminus (carboxyl terminus, C-terminal), respectively. Accurate identification of the N-terminus and C-terminus is essential for protein characterization, functional analysis, studies of post-translational modifications (PTMs), and quality control in biopharmaceutical development. In both experimental and bioinformatic workflows, the termini of protein sequences are typically determined using strategies based on chemical characteristics, sequencing techniques, and database searching.
Identification of N-Terminus and C-Terminus Based on Chemical Properties
In all proteins, the N-terminus and C-terminus exhibit distinct chemical characteristics, which enable their clear distinction within the primary sequence:
1. N-Terminal
The N-terminus marks the beginning of the protein sequence, where the first amino acid typically possesses a free α-amino group (-NH₂), unless chemically modified (e.g., acetylation).
2. C-Terminal
The C-terminus denotes the end of the protein sequence, where the carboxyl group (-COOH) of the terminal amino acid is generally unbound, although it may be modified (e.g., amidation).
Identification of N-Terminus and C-Terminus Using Experimental Approaches
A variety of experimental techniques can be employed to determine and confirm the N- and C-terminal regions of proteins.
1. N-Terminus Identification
(1) Edman Degradation: This classical method utilizes phenyl isothiocyanate (PITC) to selectively label the N-terminal amino acid, followed by stepwise chemical cleavage and identification of successive residues. Edman degradation allows direct determination of the N-terminal sequence, but is ineffective when the N-terminus is blocked by modifications such as acetylation or formylation.
(2) Mass Spectrometry (LC-MS/MS): High-resolution mass spectrometry, combined with digestion using specific proteases (e.g., Lys-N), can detect N-terminal peptides, providing sequence information and revealing potential N-terminal modifications.
(3) Chemical Labeling: Reagents such as dansyl chloride or dabsyl chloride can specifically label N-terminal amino groups, which are then analyzed using spectroscopic or chromatographic methods.
2. C-Terminal Identification
(1) Carboxypeptidase Digestion: Sequential hydrolysis of amino acids from the C-terminus using specific carboxypeptidases enables identification of the terminal residues. The released amino acids are analyzed using chromatographic or mass spectrometric techniques to deduce the C-terminal sequence.
(2) Mass Spectrometry Analysis (LC-MS/MS): C-terminal-specific proteases (such as Glu-C or Carboxypeptidase Y) are employed for targeted enzymatic cleavage. The resulting C-terminal peptides are then analyzed by LC-MS/MS, allowing for accurate determination and validation of the C-terminal sequence.
(3) Chemical Labeling: Reagents that selectively react with the free carboxyl group at the C-terminus—such as those used in Click Chemistry—can be applied in combination with mass spectrometry or fluorescence detection to enable precise characterization of the C-terminal end.
Bioinformatics-based Identification of N- and C-Termini
In proteomics research, bioinformatics tools and sequence databases provide valuable support for determining protein N- and C-termini:
1. Database Search
Primary amino acid sequences of known proteins are archived in public databases (e.g., UniProt, PDB, NCBI), where alignment or sequence matching enables prediction of the N- and C-terminal sequences.
2. Prediction of Translation Initiation Site
mRNA sequences can be translated in silico using tools such as ExPASy Translate, allowing for prediction of the translation start site, typically initiating with methionine (Met).
3. Prediction of C-Terminal Signals
Certain proteins contain characteristic C-terminal signal motifs, such as ubiquitination sites or transmembrane anchoring sequences. These functional and post-translational features can be predicted using specialized bioinformatics tools (e.g., SignalP, NetPhos).
The N- and C-termini of proteins can also be directly characterized based on their distinct chemical properties—the N-terminus possesses a free amino group (-NH₂), while the C-terminus contains a free carboxyl group (-COOH). Experimentally, Edman degradation and mass spectrometry remain the primary techniques for N-terminal sequencing, while C-terminal sequencing often relies on carboxypeptidase digestion and mass spectrometry. Additionally, chemical labeling strategies and computational approaches serve as valuable complementary methods. In modern proteomics, high-resolution mass spectrometry integrated with database searching has become the most widely used technique for both N-terminal and C-terminal sequencing. Looking forward, innovations in chemical derivatization methods and artificial intelligence-assisted data analysis will further enhance the precision and efficiency of terminus identification, offering robust tools for protein function analysis, disease research, and biopharmaceutical development. MtoZ Biolabs offers professional N-terminal and C-terminal sequencing services. Our comprehensive “one-stop” platform is designed to streamline workflows and support efficient advancement of your research projects.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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