Protein Identification

Proteins are among the most crucial biomolecules in living organisms, playing a pivotal role in various physiological processes. Identifying and analyzing protein properties is essential for understanding biological functions and disease mechanisms. There are numerous methods for protein identification and analysis, ranging from traditional methods like burning and chemical reactions to modern mass spectrometry techniques. This article reviews several major methods for protein identification and analysis, with a particular focus on the application of mass spectrometry in protein identification.

 

Protein Identification Process

The protein identification process typically includes three main steps: protein extraction, purification, and identification. This process is akin to "dissecting" proteins, layer by layer, to analyze their various properties.

 

1. Protein Extraction

Extract proteins from samples, ensuring that the proteins remain intact and undegraded.

 

2. Protein Purification

Purify the target proteins using various separation techniques such as gel electrophoresis and chromatography.

 

3. Protein Identification

Identify the purified proteins using high-precision instruments like mass spectrometers.

 

Mass spectrometry is currently the most commonly used platform technology for protein identification. Commonly used mass spectrometers include the Thermo Fisher Q Exactive, LTQ Orbitrap Velos, and AB SCIEX 6500 Q TRAP.

 

Traditional Protein Identification Methods

Traditional protein identification methods, such as immunoblotting, chemical sequencing of peptides, and comigration analysis, are typically time-consuming and labor-intensive, making them unsuitable for high-throughput screening. The following are some common traditional protein identification methods:

 

1. Burning Method

Observing the reaction of samples when burned to preliminarily determine the presence of proteins.

 

2. Chemical Reaction Method

Identifying proteins by using chemical reagents that react specifically with them.

 

Although these methods are simple, they lack precision and are not suitable for identifying complex protein mixtures.

 

Modern Protein Identification Methods

1. Image Analysis

Image analysis technology uses computer software to analyze 2-D electrophoresis images. Modern image analysis software like Phoretix-2D and Quest can accurately detect and compare protein spots, improving identification accuracy.

 

2. Microsequencing

Microsequencing involves sequencing the N-terminus of proteins using the Edman degradation method. While amino acid composition analysis and peptide mass fingerprinting (PMF) can also be used for protein identification, Edman degradation remains the most commonly used method.

 

3. Mass Spectrometry Techniques

Mass spectrometry has become an important tool for protein identification, particularly matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and electrospray ionization mass spectrometry (ESI-MS). These techniques can accurately determine the molecular weight of proteins and peptides and are widely used in large-scale automated protein identification.

 

4. Amino Acid Composition Analysis

Amino acid composition analysis is a "footprint" technique that uses the heterogeneity of proteins. By determining the amino acid composition of proteins labeled with radioactive amino acids, it is possible to identify proteins independently of their sequences.

 

In conclusion, the advancement of protein identification technologies, especially mass spectrometry, has greatly enhanced our ability to analyze and understand protein properties. These methods provide detailed insights into protein structure and function, aiding in the exploration of biological mechanisms and the development of therapeutic strategies.