Protein Characterization & Identification: What To Know

Protein characterization and identification play a critical role in drug discovery, development, and manufacturing, especially when it comes to determining the safety and efficacy of potential biological products. Although in recent years there have been many innovations and advancements in the analytical techniques used by biopharma companies to characterize and identify proteins, the process remains challenging, owing mostly to the breadth and complexity of protein expression. 

Let’s take a closer look at what you need to know about protein characterization and identification.

What is protein characterization and identification?

Though proteins are built with just 20 amino acids, their functionality and structural characteristics can vary quite a bit from one complex molecule to another, helping to form thousands of distinct proteins within the human body. These proteins are built for a range of different jobs and functions – to respond to stimuli, to replicate DNA, to form antibodies, to provide structure within cells and tissues, and so on. Biochemists use protein characterization techniques as a way of profiling these long, polypeptide chains of amino acids in order to better understand their individual use and function.

Protein characterization is the process of analyzing an individual protein through separation and detection. The unique protein is then identified by the defining characteristics of its structure and function (i.e. molecular weight, composition, purity, activity, and so on). To do this, chemists must first isolate or extract the protein from surrounding cells using a range of appropriate purification techniques.

What is the character of a protein?

To begin analysis of the character of a protein, scientists first look at the primary, secondary, tertiary and quaternary structures of a protein. 

• Primary Structure - The first step of the protein characterization process is looking at the primary structure, or the unique linear sequence in which amino acids appear. The primary structure determines the intramolecular bonds that form along the polypeptide amino acid chain, in addition to the protein’s folding pattern.
  
  
• Secondary Structure - Next, scientists will observe the secondary structure of the protein, which is the type of folding patterns that have formed along the polypeptide chain (i.e. alpha helices or beta sheets).
  

• Tertiary Structure - Next, scientists observe the combination of folds and formations that appear along the amino acid chain to inform its final three-dimensional shape, also known as its tertiary structure.
  

• Quaternary Structure - Some proteins have a quaternary structure and contain more than one polypeptide chain. Scientists will look at the arrangement of any additional chains within the fully-formed protein, which is known as its quaternary structure.
  
  
• Post-translational modifications (PTMs) - Finally, any changes that take place after protein biosynthesis are known as post-translational modifications (PTMs). Identifying PTMs can be critically important in understanding cell biology and disease pathogenesis.

How do you analyze proteins?

Today, the scope of methods used for protein characterization and identification is fairly wide-ranging – from one-step procedures to large-scale productions. 

For example, biochemists may select a sample to be fractionated, lyse the cells (break down or destroy the cell membrane) and then extract the sample through differential centrifugation. In this method, the purified supernatant is separated from the rest of the sample debris. Even after several passes in a centrifuge, the supernatant may still contain thousands of distinct proteins.

This process, known as the purification of the protein of interest, takes place only after the sample protein has undergone an additional separation technique. Chromatography, for example, is one of the most common technologies used in protein purification and has become central to the process. 

After a protein has been highly purified and finalized, biochemists can begin their characterization, which involves identification of many variables that impact protein physicochemical property and structure including:

• Aggregation state
• Extinction coefficient 
• Homogeneity/heterogeneity
• Molecular weight
• Protein Modifications 
• Purity/impurity
• Spectroscopic characteristics

What are some protein characterization techniques?

There are a range of different protein characterization techniques that can be used to detect, isolate and map the unique amino acids that make up a protein chain. These methods allow for separation based on a broad assortment of characteristic properties. 

• Amino Acid Analysis  - This technique is often complementary to protein characterization, giving researchers a method to discover unique characteristics of a sample using chromatographic techniques that isolate, analyze and quantify amino acids.
  
  
• Centrifugation - This mechanical process separates proteins from a solution. Variables like shape, size, density, viscosity and other factors inform the ultimate sedimentation rate. In this way, centrifugal force is used to isolate proteins, which can then undergo individual analysis.
  
  
• Dynamic Light Scattering (DLS) - This speedy and user friendly technique delivers results in just minutes without the need for high sample volumes.
  
  
• Gel Electrophoresis - This technique uses movement within an electric field to separate proteins according to their molecular size and charge. Proteins are transferred onto a membrane for analysis using techniques such as Western blotting or immunoblotting. These methods allow researchers to isolate and identify individual proteins.
  
  
• Mass Spectrometry (MS) - This technique allows detailed characterization of PTMs using sophisticated instruments capable of identifying thousands of proteins and peptides and measuring millions of spectra.
  
  
• Quadrupole Time-of-Flight (QTOF) Mass Spectrometry - This high-resolution, advanced hybrid technique can be used to characterize proteins and profile other complex mixtures. Because of its speed and the capacity for quantitative analysis, QTOF is regularly used in drug discovery laboratories, clinical research, environmental screening, toxicology and other applications.
  
  
• Trapped Ion Mobility Spectrometry (TIMS) - This gas-phase technique captures a wide molecular weight range of signals, allowing researchers to separate ions according to their mobility for protein analysis.
  
  
• Ultra High Performance Liquid Chromatography (UHPLC)  - This high-resolution technique uses pressure and speed for protein analysis and separation.
  

Why is protein characterization and identification important?

Advances in biotechnology and bioprocessing have created new pathways for researchers to study proteins. Biologics manufacturers must deeply understand their therapeutic proteins – from their structure, to their physicochemical properties, and their biological activity – in order to connect these learnings to clinical performance. This makes protein characterization critical to the biomedical research sector. Similarly, protein characterization methods and information have been applied to many aspects of drug development, formulation and treatments, and in creating diagnostic reagents used to detect and screen for diseases. Overall, identifying the different aspects of a protein and connecting that understanding to its clinical performance is central to successful product development.