Principle of Protein Primary Structure Characterization

Proteins are one of the most crucial functional molecules in living organisms, and their functional diversity primarily stems from their structural complexity. The structure of proteins is divided into four levels: primary, secondary, tertiary, and quaternary structures. The primary structure of a protein refers to its amino acid sequence, which is a linear chain formed by amino acids connected by peptide bonds in a specific order.

 

Composition and Characteristics of Protein Primary Structure

Proteins are linear polymers formed by 20 different amino acids linked by peptide bonds. Each amino acid consists of a central carbon atom (α-carbon) connected to an amino group (-NH2), a carboxyl group (-COOH), a hydrogen atom, and a side chain group (R group). These amino acids are arranged in a specific order and linked by dehydration condensation reactions to form peptide bonds, creating the primary structure of proteins.

 

Methods for Characterizing Protein Primary Structure

1. Edman Degradation

Edman degradation is a classic method for determining the primary structure of proteins. This method sequentially removes one amino acid from the N-terminus of the peptide chain under mild conditions and identifies it to determine the amino acid sequence of the protein. The main steps of Edman degradation include:

 

(1) Processing and purification of the protein sample.

(2) Reaction with phenylisothiocyanate (PITC) to form PTH-amino acid derivatives.

(3) Release and identification of PTH-amino acids under acidic conditions.

 

Although Edman degradation is highly accurate, it is suitable for shorter peptide sequences due to its stepwise degradation nature.

 

2. Mass Spectrometry

Mass spectrometry (MS) is currently the most widely used technique for characterizing protein primary structure. This method relies on ionizing the protein sample and separating and detecting it based on the mass-to-charge ratio (m/z) using a mass spectrometer. The main steps of mass spectrometry include:

 

(1) Sample ionization: Common ionization techniques include electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI).

(1) Mass analysis: Separation of ions using mass analyzers such as time-of-flight (TOF) or ion traps.

(1) Data processing and sequence derivation: Determining the amino acid sequence by analyzing the mass spectra.

 

Mass spectrometry is characterized by high sensitivity and high throughput, making it suitable for analyzing complex protein samples.

 

3. Nuclear Magnetic Resonance Spectroscopy (NMR)

Nuclear magnetic resonance spectroscopy (NMR) is a non-destructive technique that provides structural information about proteins by detecting the resonance frequencies of atomic nuclei in a magnetic field. NMR can be used not only for studying the tertiary structure and dynamics of proteins but also for determining primary structure. The main steps of NMR include:

 

(1) Sample preparation: High-concentration purified protein solution.

(1) Spectrum acquisition: Recording one-dimensional and multi-dimensional NMR spectra in a high-field magnet.

(1) Data analysis: Deriving the amino acid sequence by interpreting the NMR spectra.

 

NMR is suitable for studying proteins in solution but requires high concentration and purity of the samples.

 

Applications of Protein Primary Structure Characterization

Characterizing the primary structure of proteins has important applications in biological and medical research. For example, determining the primary structure of pathogen proteins aids in vaccine design and development; studying the primary structure of protein mutants helps understand the molecular mechanisms of genetic diseases; comparing protein sequences across different species reveals evolutionary relationships and functional conservation.

 

The characterization of the primary structure of proteins is fundamental to understanding protein function and biological processes. With technological advancements, methods for characterizing protein primary structure will become more precise and efficient, providing stronger support for life science research. MtoZ Biolabs provides integrate protein primary structure characterization service.