An Introduction of Intact Mass Analysis - Protein Characterization by Mass Spect

Posted by Dora West on March 24th, 2022

As the field of biotechnology continues its inexorable march towards unfathomable frontiers, the characterization of proteins emerges as a critical linchpin in this progressive process. In this context, intact mass analysis by mass spectrometry serves as an indispensable tool of the trade, empowering researchers to achieve a thorough understanding of these enigmatic molecules.

Intact mass analysis is a complex yet powerful technique that forms the cornerstone of protein characterization. Its versatility is evident in its applicability towards a diverse range of proteins, from the minuscule to the monumental. In this article, we aim to provide readers with a comprehensive introduction to this technique, its multifarious applications, and highlight examples of the intact protein. The potential of this technique is not limited to mere protein analysis, and it extends to the highly specialized realm of antibody research. In this context, we will delve into the intricacies of intact mass analysis of these remarkable biological entities, and provide readers with an illustrative protocol for conducting intact protein mass spectrometry.

What Are Intact Proteins?

Intact mass, as the name suggests, is a measurement of the aggregate mass of a molecule, encompassing all of the constituent atoms and functional groups. Moreover, this measurement assumes a pivotal role in the sphere of protein characterization, facilitating an unambiguous identification and quantification of intact proteins.

Intact proteins, the building blocks of life, represent a remarkable feat of nature, possessing towering complexity and multifarious functionality. These intricate molecules serve as the bedrock for a plethora of biological processes, inclusive of enzymatic reactions, cellular signaling, and immune response. Unsurprisingly, the study of intact proteins is of tremendous significance and represents an inexhaustible font of potential for researchers across various domains.

Examples of Intact Proteins

Intact mass analysis is an immensely potent technique that has emerged as a critical method for the characterization of proteins. Here are some examples of intact proteins that researchers can analyze, utilizing intact mass analysis to unlock subtle nuances that remain hidden in the intricate fabric of these molecules:

Monoclonal Antibodies: These remarkable biological entities have become ubiquitous in the treatment of an array of diseases, including cancer and autoimmune disorders. An analysis of monoclonal antibodies via intact mass spectrometry empowers researchers to detect and quantify their molecular weight, glycosylation patterns, and the existence of any post-translational modifications.

Insulin: This hormone is responsible for regulating blood sugar levels, a critical physiological process. Intact mass analysis of insulin offers insights into its molecular weight, as well as discerning potential modifications, such as disulfide bonds.

Hemoglobin: The protein responsible for transporting oxygen from the lungs to the tissues is a marvel of nature. Intact mass analysis of hemoglobin helps researchers understand its molecular weight, the presence of post-translational modifications, and even the detection of variant forms.

Enzymes: Enzymes are the workhorses of the biochemical world, responsible for catalyzing a host of biological processes. Intact mass analysis of these proteins unlocks a treasure trove of information regarding their molecular weight, quaternary structure, and the existence of post-translational modifications.

Immunoglobulins: These proteins play an essential role in bolstering the immune system, serving as a first line of defense. Intact mass analysis of immunoglobulins provides researchers with insights into their molecular weight, glycosylation patterns, and, crucially, their structural integrity.

Therapeutic Monoclonal Antibodies: These laboratory-generated proteins are a burgeoning domain within the pharmaceutical industry. Intact mass analysis of therapeutic monoclonal antibodies is an important step in their development and production. It involves the determination of the accurate mass of the protein, which can provide valuable information about its purity, homogeneity, and structural integrity.

One of the most common techniques used for intact mass analysis is mass spectrometry. This method involves ionizing the protein and measuring the mass-to-charge ratio of the resulting ions. The data obtained can then be used to calculate the accurate mass of the protein.

Antibody proteins may undergo post-translational modifications, such as glycosylation and oxidation, that affect their function and efficacy. By accurately determining the mass of a protein, researchers can identify and quantify any modifications that may be present and ensure high quality and consistency of the final product, so intact mass analysis is especially important for therapeutic mAbs.

Overall, intact mass analysis is a critical step in the development and production of therapeutic monoclonal antibodies, and its importance is only expected to grow as more of these drugs are developed and brought to market.

Intact Mass Analysis of Antibodies

Subunit composition analysis of antibodies:

Subunit composition analysis of antibodies is a critical step in understanding their structure and function. Antibodies have a Y-shaped structure consisting of two heavy chains and two light chains. By using intact mass analysis, we can determine the subunit composition of the antibody and identify any heterogeneity in the sample, which are valuable for ensuring a consistent manufacturing process and understanding antibody efficacy and safety.

Antibodies Post-translational modification (PTMs) analysis:

PTMs refer to covalent modifications that occur after protein synthesis, including glycosylation, phosphorylation, and oxidation, which play an essential role in the biological function of antibodies. Intact mass analysis is used for identifying and quantifying PTMs on an antibody, providing valuable insights into the glycosylation pattern and variation in glycosylation machinery between the heavy and light chains of the antibody， which is crucial in developing biosimilars and ensuring the safety and efficacy of the antibody.

The advancements in high-resolution mass spectrometry have vastly broadened the horizons of intact mass analysis. This breakthrough has enabled a more profound analysis of intricate samples and improved the accuracy and precision of PTM identification.