Protein Qualitative and Quantitative Analysis

Protein Qualitative Analysis refers to the determination of the types, structure, and function of proteins. Protein Quantitative Analysis refers to the determination of the protein content in the sample. 

 

Commonly Used Methods of Protein Qualitative Analysis

1. Electrophoresis

Proteins are separated and identified by their differential migration rates in an electric field. This includes sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis (2-DE).

 

Features: High sensitivity and resolution, good separation effect, it's mainly used to determine the presence of specific proteins in the sample or to determine the types of proteins in the sample; but the operation is relatively complex and time-consuming.

 

2. Immunoprecipitation (IP)

The target protein is precipitated from the mixed sample through a specific antibody, and the protein is identified by methods such as staining and mass spectrometry.

 

Features: High specificity, it not only can detect individual proteins, but also study the interaction between proteins; but the detection of low-abundance proteins is limited, with the risk of non-specific binding.

 

3. Mass Spectrometry

The protein is digested into peptides, and the mass-to-charge ratio (m/z) of the peptides is analyzed by a mass spectrometer. The peptide mass fingerprint is obtained by analyzing these data, and the protein type is determined by comparing the spectrum with the mass information of known protein peptides in the database. The commonly used mass spectrometry methods include liquid chromatography-mass spectrometry (LC-MS) and capillary electrophoresis-mass spectrometry (CE-MS).

 

Features: High throughput, high sensitivity and accuracy, it can not only identify protein types, but also obtain protein sequence information and post-translational modification conditions; but the sample preparation is complicated, the cost is high, and the operator's technical requirements are high.

 

Commonly Used Methods of Protein Quantitative Analysis

1. Kjeldahl Method

The nitrogen element in the sample is converted into ammonia nitrogen, and then the content of ammonia nitrogen is measured by titration to calculate the protein content.

 

Features: Classic and accurately quantitative, the operation is relatively simple, the cost is low, suitable for the determination of protein in various foods; but it is time-consuming, the accuracy is low, and the reagent is corrosive.

 

2. Ultraviolet Absorption Spectroscopy (Ultraviolet Spectrophotometry)

The aromatic amino acids in proteins (such as tyrosine, phenylalanine, and tryptophan) have absorption characteristics in the ultraviolet light region (such as 280nm). The protein content is calculated by measuring the absorbance of the sample at this wavelength.

 

Features: Simple and fast operation; but the accuracy is low, it's usually only used for preliminary estimation of protein content or as a rapid screening method.

 

3. Colorimetric method

The protein undergoes a specific color reaction with certain dyes or reagents, the light absorbance of these colors is proportional to the protein concentration, and the protein content is calculated by measuring its absorbance at a specific wavelength. The currently used colorimetric methods for determining protein content are the BCA method and the Coomassie Brilliant Blue method (Bradford).

 

Features: Easy to operate, fast, high sensitivity; but it's easily interfered by other substances.

 

4. Mass Spectrometry

The protein content is accurately measured by measuring the mass peak intensity of the protein digested into peptides or the mass difference caused by isotope labeling. The mass spectrometry analysis techniques commonly used to determine protein content include SILAC, iTRAQ/TMT, and Label-free.

 

Features: It can be used for both protein qualitative analysis and protein quantitative analysis, with high sensitivity and good resolution, especially suitable for studying the composition and changes of proteins in complex samples; but the operation is relatively complex and requires professional equipment and technical personnel.