An Overview of Common Techniques Suitable for Protein Characterization

 

• April 4, 2023

Protein Characterization Services

Protein characterization involves performing various analyses and tests on proteins to determine their structure and function. By specifying different properties of proteins, such as their molecular weight, purity, stability, and three-dimensional structure, they can be used to identify proteins, understand their functions, and design Drugs or other treatments that target them. Not only that, but protein characterization can also help researchers understand how proteins interact with drugs, ligands, or other proteins.

Protein characterization is crucial in advancing our understanding of the molecular basis of life and developing new treatments for diseases. It is critical to research fields such as drug discovery, structural biology, and biotechnology. Common methods for characterizing proteins include mass spectrometry, nuclear magnetic resonance spectroscopy, circular dichroism analysis, proteomics analysis, etc. Today we will take stock of these protein characterization techniques.

What are Proteins and Their Importance?

Protein is the material basis of life, an organic macromolecule, the basic organic matter that constitutes a cell, and the main bearer of life activities. Without protein, there would be no life. Amino acid is the basic building block of protein.

The Basic Unit of Protein Structure - Amino Acid

There are more than 300 kinds of amino acids in nature, and protein sequences are composed of twenty different amino acid residues. Proteins are multimers formed by linking different amino acids, and through correct folding into a specific configuration, they exert the biological function of protein drugs.

Protein structure refers to the spatial structure of protein molecules. Proteins are mainly composed of chemical elements such as carbon, hydrogen, oxygen, and nitrogen. All proteins are polymers formed by connecting 20 different amino acids. After forming proteins, these amino acids are also called residues.

The characteristics and functions of a protein sequence have always depended on the nature, type, and quantity of the amino acid residues that make it up. Specific positions in the amino acid sequence can be covalently bonded to chemical groups, resulting in protein post-translational modifications, which will lead to changes in the structure of the protein, thereby affecting the biological activity of protein drugs.

Proteins have the characteristics of relatively large molecular weight, complex structure, and high heterogeneity. Therefore, their characterization is extremely challenging, and it is very important to use appropriate methods to characterize them.

What is the 3D Structure of Proteins?

 Protein molecules are covalent polypeptide chains formed by the condensation of amino acids end-to-end. Every natural protein has its own unique spatial structure or three-dimensional structure. This three-dimensional structure is usually called the conformation of the protein, that is, the structure of the protein. The molecular structure of proteins can be artificially divided into primary, secondary, tertiary, and quaternary structures.

• Primary structure: the linear sequence of amino acids that make up the polypeptide chain of a protein.
• Secondary structure: a stable structure formed by hydrogen bonds between C=O and N-H groups between different amino acids, mainly α-helix and β-sheet.
• Tertiary structure: the three-dimensional structure of a protein molecule formed by the arrangement of multiple secondary structure elements in three-dimensional space.
• Quaternary structure: used to describe functional protein complex molecules formed by the interaction between different polypeptide chains (subunits).

What is Protein Characterization?

Protein characterization analysis is to characterize the biological functions and various properties and parameters of proteins, including protein types, protein structures, content, protein purity, amino acid composition, molecular mass, amino acid composition, etc.

The most common way to perform protein characterization is mass spectrometry. Mass spectrometry (mass spectrometry, MS) analysis technology won the Nobel Prize in 2002. (In 2002, Tanaka Koichi and John B Fenn at the University of Virginia were awarded the Nobel Prize in Chemistry for their contribution to the soft adsorption ionization method.)

In 2009, Medicilon obtained the certificate of “Protein Crystallography-Based Drug Discovery and Screening Technology Service Platform” issued by Shanghai R&D Public Service Platform, which proved Medicilon’s ability in protein crystal technology.

Protein Characterization Techniques:

Mass Spectrometry:

Mass spectrometry has good sensitivity and accuracy and can accurately determine proteins. At present, mass spectrometry mainly determines the primary structure of proteins, including molecular weight, amino acid sequence of peptide chains, and the number and position of polypeptides or disulfide bonds, which occupies an important role in the study of protein structure analysis. The mass spectrometer consists of a sample injector, an ion source, a mass analyzer, an ion detector, a control computer, and a data analysis system. Traditional mass spectrometry is only used for the analysis of small molecule volatile substances, but with the emergence of new ionization techniques, such as matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionization mass spectrometry (ESI-MS). The emergence of various mass spectrometry technologies provides a new, accurate, and fast way for protein analysis. Mass spectrometer technologies, such as MALDI, TOF, CFR, and hybrid quadrupole mass analyzers, have been vigorously advanced to meet the high selectivity and high sensitivity required for the identification and characterization of new proteins and other biomolecules in drug discovery. [1]

The Principle of Mass Spectrometry Protein Analysis

The basic principle of mass spectrometry protein analysis is to convert protein molecules into ions through an ionization source and then use the electric field and magnetic field of the mass spectrometer to separate protein ions with a specific mass-to-charge ratio (M/Z), and pass through the ion detector to collect the separated ions, determine the M/Z value of the ions, and analyze and identify unknown proteins. Usually combined with corresponding processing and other techniques, proteins can be identified more accurately and quickly.

Our Structural Biology Laboratory is equipped with molecular cloning room and a drug discovery and selection solution based on protein crystallography, supporting drug development based on structures and from new targets determination to final structural determination. Learn more.

Circular Dichroism Spectral Characterization

Circular dichroism is a special absorption spectrum. It obtains the secondary structure of biological macromolecules by measuring the circular dichroism spectrum of proteins and other biological macromolecules. It is simple and fast. It is widely used in protein folding, protein conformation research, and enzyme field of dynamics.

Far-ultraviolet circular dichroism can be used to determine the secondary structure of proteins, and near-ultraviolet circular dichroism can be used to detect the tertiary structure of protein side chains.

Fourier Transform Infrared Spectroscopy Characterization

Fourier Transform Infrared Spectrometer, abbreviated as FTIR. The Fourier transform infrared spectrometer (FTIR) was born in the 1970s. It is composed of a light source (carbon rod, high-pressure mercury lamp), a Michelson interferometer, a sample chamber, a detector, a computer system, and a recording and display device. It consists of several parts. The Fourier transform infrared spectrometer not only has high resolution and scanning speed but is not limited to the use of the mid-infrared (MIR) section, and the application of the beam splitter can cover its spectral range from ultraviolet to Far-infrared segment.[2]

X-ray Crystallography Characterization

The principle of X-ray determination of protein structure is to use X-rays with a wavelength of about 1 Å in analyzing the three-dimensional structure of proteins. Since this wavelength is the same order of magnitude as the distance between atoms in protein crystals, and the molecules in the crystal structure are arranged regularly when X-rays are incident on the crystal, each atom in the crystal emits secondary X-rays that interfere with each other and superimpose and produce strong X-ray diffraction. For X-rays, although there is no material that can directly gather scattered light as an objective lens to form an image, the diffraction phenomenon of the crystal has a certain relationship with the internal structure of the crystal; that is, the diffraction direction is related to the shape and size of the unit cell in the crystal. , and the intensity of diffraction is related to the arrangement and period of heavy metal atoms in the unit cell.[3]

NMR Spectroscopy Characterization

As the main analysis method in structural biology, nuclear magnetic resonance(NMR) spectroscopy is one of the few techniques that can characterize the fine three-dimensional structure of proteins at the atomic scale. With the development of high-field NMR technology, combined with protein isotope labeling technology, it has been possible to characterize the structure of super large proteins and protein complexes with molecular weights up to hundreds of thousands.

Compared with X-ray crystallography, NMR technology has the advantage of being able to study the three-dimensional structure of proteins in a state closer to the physiological environment (pH, salt concentration, temperature, etc.). The study of protein function and the “dynamic structure” of protein molecules is very important. NMR technology can study the dynamic properties of proteins through the nuclear relaxation process at the atomic level and has an incomparable effect on other technologies.

Cryo-electron Microscopy Characterization

Cryo-electron microscopy is referred to as cryo-electron microscopy or cryo-electron microscopy. This is a technology that uses a transmission electron microscope to observe samples under low-temperature conditions. This technology, together with X-ray crystallography characterization and NMR technology, has laid the foundation for high-resolution structural biology research. 

Compared with traditional methods of studying the three-dimensional structure of protein molecules, such as X-ray diffraction technology and nuclear magnetic resonance technology, cryo-electron microscopy has the following advantages: maintaining the activity and functional state of biological samples; The determination of the three-dimensional structure of protein macromolecules is more suitable; the analysis of the three-dimensional structure of protein macromolecules has the characteristics of high throughput, fast and efficient.

X-ray-based Crystal Services

The measurement of complete three-dimensional structure of protein and its complex assembly is the scientific basis for studying the relationship between molecular structure and function in life activities and revealing the physical and chemical nature of life phenomena. The X-ray diffraction of protein and its complex crystals is one of the main methods to study the three-dimensional fine structure of biological macromolecules.

Penetrating with other disciplines and especially driven by popular disciplines like structural genomics, protein crystallography extends from analyzing the simple three-dimensional structure of protein to studying the structure of various biological macromolecules and complexes, and focus more on the relationship between structure and function. In pharmaceutical R&D, protein crystallographic structure is widely and significantly used in structure-based new drug design.

Services

• High-throughput-based selection of protein crystals
• Diffraction data collection in protein synchrotron radiation
• Protein-compound co-crystallization
• Analysis of protein crystal structure

Reference:

[1]. ChrisSutton. PROCESS. 2004(000)003: 74-75.

[2]. Jianchuan He, et al. Chinese Journal Of Clinical Medicine. 2011(018)002: 147-149.

[3]. Qiang Xiong, et al. Carcinogenesis, Teratogenesis & Mutagenesis. 2019(031)001: 82-85.