How to Identify the Structure of Peptides?

Peptides are a class of compounds composed of 11 to 50 amino acids linked by peptide bonds, which are distinguished from proteins only by the length of the peptide chain, and there is no strict distinction between the two. Like proteins, its structural levels are also divided into four levels: primary structure, secondary structure, tertiary structure, and quaternary structure. The primary structure refers to the arrangement of amino acids from the N-terminus to the C-terminus in the peptide molecule; the secondary structure refers to the way the peptide chain itself folds and winds; the tertiary structure is the three-dimensional spatial structure formed by the entire peptide chain further folding, curling, and winding on the basis of the secondary structure; the quaternary structure is the three-dimensional structure formed by the aggregation of multiple peptides in multi-subunit proteins in an appropriate manner. Apart from the primary structure, the other three layers of structure are usually collectively referred to as the advanced structure or spatial structure of the peptide.

 

The structure of peptides is closely related to their function. By identifying the structure of peptides, we can gain a deeper understanding of their biological activity, interaction with other molecules, and the biological processes they participate in, and decipher the specific role of peptides in the body. This can help us better utilize them in research fields such as medicine and biotechnology. Here, the identification of peptide structure is divided into primary structure identification and spatial structure identification, and the corresponding methods mainly include the following.

 

Identification of Primary Structure of Peptides

The identification of the primary structure of peptides, also known as peptide sequence analysis, commonly used sequence analysis methods include:

 

1. Edman Degradation

This is the most traditional method, which can continuously and sequentially determine the N-terminal amino acid sequence of the peptide. In each round, the N-terminal amino acid of the peptide is selectively labeled, then cleaved and identified. This process can be repeated multiple times to gradually determine the amino acid sequence.

 

2. Mass Spectrometry (MS)

In recent years, mass spectrometry has become the preferred method for peptide sequencing. It can determine the molecular weight of the peptide by measuring the mass-to-charge ratio (m/z), and further infer the sequence of the peptide through fragment ion analysis.

 

3. DNA Sequencing

Since it is easier to determine the nucleotide sequence of DNA than the amino acid sequence of a peptide, for longer peptide chains, the gene encoding the peptide chain can be isolated, its nucleotide sequence determined, and the amino acid sequence inferred using the genetic code.

 

Identification of Spatial Structure of Peptides

1. Infrared Spectroscopy

Infrared spectroscopy is an important method for identifying the structure of organic compounds, with features such as small sample size and no need for high-purity crystals. This method uses the interaction between infrared radiation and molecular substances to study the structure of peptides. Infrared spectroscopy can provide information about the amide bonds, carboxyl groups, amino groups, and other groups in the peptide, thereby inferring the conformation and secondary structure of the peptide.

 

2. Nuclear Magnetic Resonance (NMR)

NMR uses the nuclear magnetic resonance phenomenon to study the structure of peptides. By measuring the nuclear magnetic resonance signals of atomic nuclei (such as 1H, 13C, 15N, etc.) in peptides, the stereostructure and dynamic information of peptides can be obtained.

 

3. Circular Dichroism (CD)

This method mainly uses circular dichroism to study the stereostructure of peptides. Circular dichroism is sensitive to the main chain conformation of peptides and can reflect the electronic transition of the peptide backbone, thereby providing stereostructure information of the peptide.

 

4. X-Ray Crystallography

The structure of the peptide crystal is determined by measuring the diffraction pattern of X-rays in the crystal. The structural information of the peptide crystal can be analyzed from the diffraction pattern. This method is also the most effective method to study the structure of peptides to date, and the accuracy it can achieve is unparalleled by any other method.

 

In actual applications, it is usually necessary to choose the appropriate identification method according to the specific experimental purpose and the properties of the peptide. MtoZ Biolabs uses Thermo Fisher's Q ExactiveHF mass spectrometry platform combined with Nano-LC nanoscale chromatography to provide efficient and accurate protein/peptide structural identification service packages. It can analyze the primary and secondary structures of various protein/peptide samples, including the type, number, and order of amino acids in the peptide chain, as well as the location and number of disulfide bonds within or between the peptide chains. Free consultation is welcome.