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

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.


Furthermore, the application of intact mass analysis is not limited to understanding the structure and function of antibodies. It can also be used to assess the stability of these proteins under varying conditions such as shifts in temperature, pH, and storage time. Stability studies are integral in ensuring the reliability and safety of antibody-based therapeutics. Intact mass analysis is, therefore, a fundamental tool in this critical process.

In summary, intact mass analysis by mass spectrometry is a crucial technique in the scrutiny of antibodies. Its unique ability to offer a comprehensive picture of the structure, function, and stability of antibodies renders it an indispensable tool in the evaluation of existing therapies and the development of new ones. The escalating application of monoclonal antibodies in therapeutics amplifies the importance of intact mass analysis. It is expected that this technique will play an even more substantial role in ensuring the safety and effectiveness of these intricate proteins.

Intact Protein Mass Spectrometry Protocol

Intact protein mass spectrometry is an analytical technique of great potency which paves the way for protein characterization without prior digestion. To achieve high-quality data, this technique mandates precise sample preparation and analysis protocols. A standard intact protein mass spectrometry protocol entails the following steps:

Sample Preparation

The initial step of intact protein mass spectrometry protocol, protein extraction, takes advantage of carefully designed extraction buffers and protocols to ensure maximal efficacy. The proteins are carefully extracted from the sample, and apt protocols have been crafted to optimize protein yields for subsequent analysis.

Protein purification follows the protein extraction step and is a crucial step in intact protein mass spectrometry. In this step, diverse chromatography techniques, including size-exclusion, ion-exchange, or reversed-phase chromatography, are employed to ensure optimal efficacy in the purification of the extracted proteins. This step ensures that the protein samples are devoid of impurities and contaminants that may affect subsequent steps.

Protein Mass Spectrometry Analysis

Protein Denaturation: Next up, the concentrated protein samples are subjected to denaturation using suitable denaturants, such as urea, or guanidine hydrochloride.

Protein Reduction: Using reducing agents like dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP), the denatured proteins are reduced.

Protein Alkylation: After reduction, the proteins are alkylated via the use of iodoacetamide or other alkylating agents deemed suitable by the specific protocols.

Protein Desalting: The alkylated protein samples are then desalted, with C18 or other desalting columns being utilized to achieve the desired effect.

Protein Ionization: The desalted protein samples are ionized using electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI) techniques, which are important to ensure optimal spectroscopic performance.

Protein Mass Analysis: Finally, high-resolution mass spectrometry, such as Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) or Orbitrap mass spectrometry, is employed to analyze the ionized protein samples.

Data Analysis

The data obtained during the above steps is then analyzed using requisite software tools which facilitate multiple functions including the visualization of the intact protein mass spectra, deconvolution of the mass spectra, and identification of protein modifications. The software tools used in data analysis are designed to process large amounts of data which result from intact protein mass spectrometry.

What can we offer

Creative Proteomics is equipped with both of these mass spectrometry systems. Whether you need to confirm the protein spectrum or peptide fingerprint analysis and protein identification of a variety of protein molecular weight analysis, Creative Proteomics can meet your needs to provide customized solutions, including Intact Mass Analysis Service, ESI-Q TOF-MS Intact Mass Analysis, MALDI-TOF-MS Intact Mass Analysis, Protein Molecular Weight Determination, Protein characterization et al,.


  1. Kenneth R. Durbin et al,. Intact mass detection, interpretation, and visualization to automate Top-Down proteomics on a large scale Proteomics. 2010
  2. Donnelly, D.P., Rawlins, C.M., DeHart, C.J. et al. Best practices and benchmarks for intact protein analysis for top-down mass spectrometry. Nat Methods 16, 587–594 (2019)
  3. Luca FornelliTimothy K. Toby Characterization of large intact protein ions by mass spectrometry: What. BBA - Proteins and Proteomics 2022
  4. Shay Vimer et al,. Mass Spectrometry Analysis of Intact Proteins from Crude Samples Anal. Chem. 2020, 92, 19, 12741–12749 Publication Date:September 8, 2020
  5. Daniel P. Donnelly et al,. Best practices and benchmarks for intact protein analysis for top-down mass spectrometry Nature Methods
  6. Mei Han et al,. Intact mass analysis of monoclonal antibodies by capillary electrophoresis—Mass spectrometry. Journal of Chromatography B 2016

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