Proteins, those fundamental elements that construct the essence of life, are the prime actors in many biological processes. Nonetheless, in order to scrutinize the functions of these proteins, it becomes crucial to separate them from their intricate biological milieu. Among various methodologies utilized to isolate specific proteins from complex biological mixtures, affinity purification is considered one of the most influential and ubiquitous techniques. In this write-up, we will explore the rudimentary aspects of affinity purification, including its variant, namely affinity purification-mass spectrometry (AP-MS), and the perks it offers in the domain of protein analysis.
What is Affinity Chromatography?
Affinity chromatography is a well-established and respected separation technique that is widely used to isolate and purify target proteins or biomolecules from complex mixtures. This technique is based on the principle of selective binding between a protein of interest and a specific ligand or affinity matrix, resulting in the separation and purification of the target protein with high specificity and selectivity.
To achieve this, the ligand is immobilized onto a solid support, typically a column or a resin, and the mixture containing the target protein is then passed through the column. As the mixture flows through the column, the target protein selectively binds to the immobilized ligand, while the unwanted components flow through the column unimpeded. This results in a highly enriched sample of the target protein, free from unwanted contaminants or impurities.
The (a) sample application/washing steps and (b) examples of elution methods that are used in the on/off mode of affinity chromatography (Rodriguez et al., 2020)
How Does Affinity Purification Work?
Affinity purification is a specific type of affinity chromatography used to isolate and purify proteins with high specificity and yield. It involves the use of a protein-specific affinity tag, which is fused to the target protein of interest. The tag allows for selective binding of the protein to a specific ligand or matrix, enabling efficient purification of the protein from a complex biological sample.
The most commonly used affinity tag is the polyhistidine (His-tag), which consists of a short stretch of histidine residues that bind to immobilized metal ions, such as nickel or cobalt ions. Other commonly used affinity tags include the glutathione S-transferase (GST) tag, maltose-binding protein (MBP) tag, and streptavidin-binding peptide (SBP) tag.
What Are the Advantages of Affinity Purification?
Affinity purification offers several advantages over other purification techniques, including high specificity, yield, and purity. It allows for the isolation of a specific protein of interest from a complex mixture, without the need for extensive and time-consuming fractionation steps. Furthermore, affinity purification is a gentle technique, which preserves the biological activity and conformation of the purified protein.
What is Affinity Purification Mass Spectrometry (AP-MS)?
Affinity purification-mass spectrometry (AP-MS), a formidable technique employed to scrutinize protein-protein interactions in a high-throughput fashion, presents an amalgamation of the specificity and selectivity of affinity purification with the sensitivity and accuracy of mass spectrometry. In this approach, the protein of interest undergoes purification by means of an affinity tag, as explicated above. Subsequently, the purified protein is subject to enzymatic digestion, and the resulting peptides undergo mass spectrometry analysis.
By identifying the proteins that co-purify with the protein of interest, AP-MS provides a comprehensive and unbiased identification of protein-protein interactions. Notably, AP-MS can be utilized to pinpoint post-translational modifications, such as phosphorylation or acetylation, on the target protein or its interacting partners. This demonstrates the versatility of AP-MS as a valuable technique for investigating the intricacies of protein interactions.
Identification of interactors using AP-MS (Chen et al., 2007)
What Are the Advantages of Affinity Purification Mass Spectrometry?
It is noteworthy that AP-MS boasts various advantages over conventional approaches employed for exploring protein-protein interactions, such as yeast two-hybrid or co-immunoprecipitation. Specifically, AP-MS enables the identification of interacting partners in a comprehensive and unbiased manner, without the prerequisite of prior knowledge or validation of the interacting partner. Moreover, AP-MS can unravel novel interacting partners or post-translational modifications, which might elude detection by other techniques. This illustrates the potency of AP-MS as a valuable tool for unraveling the complex and diverse aspects of protein interactions.
What is an Affinity Tag and Why is it Necessary in Protein Purification?
An affinity tag is a short peptide or protein sequence that is genetically fused to the protein of interest. The affinity tag serves as a handle for purification and enables the specific binding of the protein to an immobilized ligand or matrix. Affinity tags are necessary for protein purification as they enable the selective and efficient isolation of the protein of interest from complex biological samples.
The choice of affinity tag depends on several factors, including the size, stability, and solubility of the protein of interest, as well as the downstream application. For example, the His-tag is widely used in protein purification due to its small size and high affinity for metal ions. However, it may not be suitable for large or unstable proteins, which may require a more robust affinity tag, such as the GST tag or MBP-tag.
Creative Proteomics offers a comprehensive range of affinity purification-mass spectrometry services, including protein identification, interaction analysis, and post-translational modification analysis. Our team of experienced scientists utilizes state-of-the-art equipment and protocols to deliver high-quality and reliable results. Contact us today to learn more about our AP-MS services and how we can assist with your research needs.
Read More: Co-immunoprecipitation
References
- Chen, Ginny I., and Anne-Claude Gingras. “Affinity-purification mass spectrometry (AP-MS) of serine/threonine phosphatases.” Methods 42.3 (2007): 298-305.
- Rodriguez, Elliott L., et al. “Affinity chromatography: A review of trends and developments over the past 50 years.” Journal of Chromatography B 1157 (2020): 122332.
