How to Determine the Molecular Weight of Protein?

What is Protein Molecular Weight

Protein molecular weight is the primary parameter for protein identification , which defines the mass of a protein in daltons (Da) or kilodaltons (kDa). This measurement is determined by summing the individual masses of all the atoms within the protein molecule, taking into account its amino acid sequence and the chemical structure of any post-translational modifications . The molecular weight of a protein is not a fixed value in all cases, as proteins often exhibit heterogeneity in their composition.

Proteins are polymers made up of amino acids linked by peptide bonds, and their molecular weight is directly influenced by the number and types of amino acids present. For instance, a protein made up of only small amino acids like glycine or alanine will have a lower molecular weight compared to one with larger and more complex amino acids like tryptophan or tyrosine. The molecular weight can also provide insights into the protein's potential function, folding patterns, and interaction capabilities with other biomolecules.

SDS-PAGE for Protein Weight Determination

SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) enables the separation of proteins based solely on their molecular weight. In this method, proteins are first treated with SDS, an anionic detergent that binds uniformly along the polypeptide chain. This binding effectively masks the protein's native charge and confers a nearly constant negative charge-to-mass ratio, ensuring that separation during electrophoresis is determined only by size. Additionally, reducing agents such as 2-mercaptoethanol or dithiothreitol are employed to break disulfide bonds, leading to complete denaturation and linearization of the protein structure. Once denatured, the proteins are loaded into wells of a polyacrylamide gel, which functions as a molecular sieve. Smaller proteins traverse the gel matrix more rapidly than larger ones, which face greater steric hindrance. After the run, proteins are visualized using staining techniques and their migration distances are compared against known molecular weight standards, allowing accurate estimation of protein size and providing essential data for further structural and functional analyses.

Major steps:

Sample Preparation : Proteins are extracted and mixed with SDS and reducing agents to ensure complete denaturation and disruption of disulfide bonds.

Denaturation : The protein samples are heated, unfolding the proteins into linear chains and conferring a uniform negative charge.

Gel Casting : A polyacrylamide gel with an appropriate pore size is prepared, serving as a molecular sieve for protein separation.

Loading : Protein samples and molecular weight markers are loaded into wells within the gel.

Electrophoresis : An electric field is applied, causing the negatively charged proteins to migrate through the gel; smaller proteins move faster than larger ones.

Visualization : After separation, proteins are stained and visualized; their migration distances are compared with standards to estimate molecular weights accurately.

Figure 1.  Schematic diagram of SDS-PAGE. (Gülay, et al, 2018)

Mass Spectrometry Techniques in Protein Weight Determination

MALDI-TOF Mass Spectrometry

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is a high-precision analytical method used to determine protein molecular weights with exceptional accuracy and speed. In this technique, proteins are first co-crystallized with a matrix compound—commonly an organic acid such as sinapinic acid or α-cyano-4-hydroxycinnamic acid—that efficiently absorbs laser energy. When a short, intense laser pulse strikes the sample, the matrix undergoes rapid energy transfer, causing simultaneous desorption and ionization of the embedded protein molecules with minimal fragmentation. The resulting ions are then accelerated into a time-of-flight analyzer, where they travel through a field-free drift region. Ions are separated based on their mass-to-charge (m/z) ratios; lighter ions reach the detector faster than heavier ones, and the precise flight time is recorded to calculate the protein's molecular weight.

Major steps:

Sample Preparation : Mix the protein sample with a suitable matrix compound to facilitate ionization and co-crystallize them on a target plate.

Laser Irradiation : Use a laser to irradiate the co-crystallized sample, causing rapid energy absorption by the matrix.

Desorption and Ionization : The matrix assists in the desorption and ionization of protein molecules, generating singly charged ions.

Time-of-Flight Analysis : Accelerate the ions in an electric field; they travel through a flight tube where separation occurs based on mass-to-charge (m/z) ratios.

Detection and Data Analysis : Detect ions as they reach the detector; measure their time-of-flight to determine the m/z values and thus the protein's molecular weight.

ESI Mass Spectrometrys

Electrospray ionization (ESI) mass spectrometry stands as a cutting-edge technique for determining protein mass with exceptional accuracy and sensitivity. In this process, a high voltage is applied to a liquid sample containing the protein, resulting in the formation of a fine aerosol composed of charged droplets. As these droplets travel through a heated desolvation region, the solvent gradually evaporates, leaving behind gas-phase ions. These ions frequently acquire multiple charges, which effectively reduces their mass-to-charge (m/z) ratios into the optimal detection range of the mass spectrometer. The mass analyzer then precisely measures these m/z ratios, generating a spectrum that offers comprehensive insights into the protein's molecular weight. This spectrum can reveal detailed information about post-translational modifications, isoforms, and oligomeric states.

Major steps:

Sample Preparation : Proteins are dissolved in an appropriate solvent, often with additives (e.g., acids) to facilitate ionization.

Generation of Charged Droplets : The prepared solution is introduced into a capillary under high voltage, producing a fine aerosol of charged droplets.

Desolvation and Ion Formation : As the solvent evaporates from the droplets, the droplets shrink, and Coulombic repulsion causes them to disperse, ultimately releasing multiply charged ions into the gas phase.

Ion Transfer and Focusing : Ion optics guide and focus the generated ions, efficiently transferring them from the ionization source into the mass analyzer.

Mass Analysis : Ions are separated based on their mass-to-charge (m/z) ratios using analyzers such as quadrupole, time-of-flight, or ion trap systems.

Detection and Data Analysis : The separated ions are detected, and the resulting spectra are processed to determine the molecular weight and structural information of the proteins.

Figure 2.  MALDI-MS and ESI-MS procedures. (Lin Y, et al., 2009)

Size Exclusion Chromatography(SEC) for Protein Weight Determination

SEC is a powerful, non-denaturing technique used to estimate the molecular weight of proteins by measuring their hydrodynamic volume. In SEC, a protein sample is injected onto a chromatography column packed with porous beads that serve as a molecular sieve. As the sample passes through the column, molecules separate based on their ability to penetrate the pores of the beads. Smaller proteins can access these pores and therefore travel a longer, more convoluted path, resulting in a delayed elution. In contrast, larger proteins are largely excluded from the pores and elute more rapidly. This differential elution creates a distinct profile, where the elution volume is directly correlated with molecular size. By calibrating the column with standard proteins of known molecular weights, the elution volumes of unknown proteins can be accurately compared to these benchmarks.

Major steps:

Sample Preparation : Purify and pre-treat the protein sample to remove aggregates and impurities.

Column Loading:  Inject the prepared protein sample into an SEC column packed with porous beads.

Separation Mechanism : Proteins are separated based on their hydrodynamic volume; larger proteins elute earlier as they are excluded from the bead pores, while smaller proteins enter the pores and elute later.

Elution Profile Monitoring:  Collect the elution fractions and monitor protein concentration to generate an elution profile.

Calibration and Molecular Weight Estimation : Compare the elution times of the protein sample with those of standard proteins of known molecular weights to construct a calibration curve. Use the calibration curve to estimate the molecular weight of the protein based on its retention time.

Data Analysis : Analyze the elution data to confirm the protein's molecular weight and assess its purity and aggregation state.

Advanced Analytical Techniques in Protein Weight Determination

Analytical Ultracentrifugation (AUC)

AUC measures protein sedimentation behavior under high centrifugal forces. In an AUC experiment, proteins are spun at extremely high speeds in an ultracentrifuge, generating centrifugal fields that cause the proteins to sediment based on their mass, shape, and density. Sedimentation velocity experiments track how fast proteins move through the solution, providing insights into their hydrodynamic properties and potential aggregation states. Conversely, sedimentation equilibrium experiments allow the system to reach a balance between sedimentation and diffusion, enabling the direct determination of molecular weight without the need for external calibration standards. Advanced mathematical models, such as the Lamm equation, are applied to the sedimentation data to extract parameters including the sedimentation coefficient, frictional ratio, and partial specific volume.

Major steps:

Sample Preparation : Prepare the protein solution under optimal buffer conditions and concentration to ensure accurate sedimentation behavior.

Loading into AUC Cells:  Introduce the sample into specialized analytical ultracentrifugation cells, ensuring proper sealing to prevent leakage.

Centrifugation : Subject the sample to high centrifugal forces in an ultracentrifuge, causing proteins to sediment based on their mass, shape, and density.

Data Acquisition:  Monitor the sedimentation process using optical detection systems (e.g., absorbance or interference) to record sedimentation profiles over time.

Data Analysis : Analyze the sedimentation velocity and/or equilibrium profiles to determine sedimentation coefficients, which are directly related to molecular weight.

Molecular Weight Calculation:  Apply mathematical models that account for factors such as shape and frictional properties to accurately calculate the protein's molecular weight.

Dynamic Light Scattering (DLS)

DLS is a powerful, non-invasive technique that quantifies the fluctuations in scattered light caused by the Brownian motion of particles in solution. As proteins and other macromolecules undergo random thermal motion, their movement induces temporal changes in the intensity of scattered light. These fluctuations are captured and analyzed using an autocorrelation function, which allows for the precise determination of the translational diffusion coefficient. Utilizing the Stokes-Einstein equation, this diffusion coefficient is then directly related to the hydrodynamic radius of the particles. With the hydrodynamic radius established, and assuming a known shape and density, researchers can estimate the molecular weight of proteins.

Major steps:

Sample Preparation : Purify the protein solution and remove particulate contaminants to ensure accurate scattering measurements. Adjust the protein concentration.

Laser Illumination : Direct a laser beam at the protein solution, which serves as the light source for scattering.

Light Scattering Detection : Measure the intensity fluctuations of light scattered by proteins undergoing Brownian motion in the solution.

Autocorrelation Analysis : Process the detected light fluctuations using an autocorrelation function to determine the diffusion behavior of the protein particles.

Calculation of Diffusion Coefficient:  Derive the translational diffusion coefficient from the autocorrelation data, which reflects how fast the proteins move in solution.

Hydrodynamic Radius Determination : Apply the Stokes-Einstein equation to convert the diffusion coefficient into the hydrodynamic radius of the protein particles.

Molecular Weight Estimation : Infer the protein molecular weight based on the hydrodynamic radius, incorporating considerations of the protein's shape and the solution's viscosity.

Advantages and Limitations of Protein Molecular Weight Methods

Importance of Accurate Protein Characterization

Biological Significance : Precise molecular weight determination ensures correct protein identification, avoiding misclassification that could misinterpret function, structural properties, or interactions. Protein size influences enzymatic activity, binding specificity, and cellular localization, making accurate characterization essential for functional studies.

Role in Drug Development : The development of monoclonal antibodies, fusion proteins, and therapeutic enzymes relies on precise molecular weight assessment to confirm structural integrity.

Quality Control in Bioprocessing : In recombinant protein production, deviations in molecular weight can indicate post-translational modifications, degradation, or improper folding.

Stability and Aggregation Assessment : Protein aggregation can impact drug efficacy and immunogenicity, making molecular weight determination essential for formulation studies.

Applications of Protein Weight Determination

Protein molecular weight determination has broad applications across various fields of research and industry:

• Proteomics : Protein molecular weight is a key parameter in proteomic studies, where it helps in protein identification , quantification , and characterization of post-translational modifications .
• Biopharmaceutical Development : Accurate molecular weight determination is essential for ensuring the correct identity and purity of therapeutic proteins and monoclonal antibodies.
• Structural Biology : Protein molecular weight data, when combined with other techniques like X-ray crystallography or NMR spectroscopy, can provide valuable insights into protein structure and folding.

References

• Matsumoto H, Haniu H, Komori N. Determination of protein molecular weights on SDS-PAGE. Electrophoretic Separation of Proteins: Methods and Protocols , 2019: 101-105. DOI: 10.1007/978-1-4939-8793-1_10
• Lin Y, et al. The current state of proteomics in GI oncology. Digestive diseases and sciences , 2009, 54: 431-457. DOI: 10.1007/s10620-008-0656-5
• Prabhu G R D, et al. Mass spectrometry using electrospray ionization[J]. Nature Reviews Methods Primers , 2023, 3(1): 23. DOI: s43586-023-00203-4