How to Accurately Determine the Molecular Weight of Proteins Using Mass Spectrometry?

Proteins are the core functional molecules of cellular life activities. Their molecular weight directly reflects the amino acid sequence and structural integrity, which is a key parameter in protein identification, function prediction, and quality control studies. In the field of biomedicine, slight changes in protein molecular weight often indicate the presence of post-translational modifications (such as glycosylation, oxidation) or degradation products, which are of great significance for drug development and quality release. Therefore, establishing a high-precision, high-sensitivity molecular weight measurement technology applicable to different types of proteins is of irreplaceable value for researchers and biopharmaceutical companies. Mass Spectrometry (MS), with its excellent resolution and molecular recognition capabilities, is becoming the preferred technology for determining protein molecular weight.

1. Basic Principles of Mass Spectrometry for Protein Molecular Weight Measurement

The core logic of mass spectrometry in determining protein molecular weight is to ionize the protein into charged ions. After entering the mass analyzer, the real molecular mass is calculated through its mass-to-charge ratio (m/z). Modern protein mass spectrometry analysis mainly uses two soft ionization techniques:

• Electrospray Ionization (ESI): Suitable for liquid phase samples, can introduce proteins into multiple charge states, aiding in high-quality resolution.
• Matrix-Assisted Laser Desorption/Ionization (MALDI): Suitable for dry samples, forming single-charge ion peaks, facilitating rapid identification.

After ionization, the ions enter the mass analyzer (such as Orbitrap, TOF, or Q-TOF) and are analyzed for their m/z based on principles like flight time, electric field deflection, or Fourier transform. For the multi-charge distribution ESI spectrum, deconvolution algorithms are needed to restore multiple signals to the actual neutral molecular weight.

2. Overview of Common Platforms and Applicable Ionization Strategies

In protein molecular weight measurement, the choice of mass spectrometry platform has a significant impact on the accuracy and applicability of the results. Below is a brief comparison based on different technical characteristics:

1. Electrospray Ionization (ESI) Platform

Often paired with Orbitrap, Q-TOF, and triple quadrupole mass spectrometry systems. Its greatest advantage lies in producing multi-charge ion peaks, bringing the m/z values of high molecular weight proteins within the detection range of mass spectrometers. ESI is very suitable for analyzing recombinant proteins, fusion proteins, and monoclonal antibodies in prokaryotic or eukaryotic expression systems, especially when retaining the native conformation or performing non-denaturing mass spectrometry.

2. MALDI Platform

Usually combined with TOF/TOF systems, suitable for high-throughput, single-charge detection scenarios. This technology is widely applied in protein fingerprinting and rapid quality control detection, but its resolving power is relatively limited for complex modifications or high molecular weight proteins.

3. From m/z to Molecular Weight: Interpreting the Calculation Logic and Technical Challenges

1. Brief Overview of Calculation Formula

After protein analysis by mass spectrometry, multiple m/z signals are obtained rather than direct molecular mass. The commonly used reverse calculation formula is as follows:

Molecular Weight = (m/z × z) - z × Mass Unit (usually the mass of a proton)

Where z is the charge number, which can be deduced by observing the differences between different m/z values. For proteins with multiple charges, professional software is needed to perform deconvolution to restore the m/z peak groups to the true neutral molecular weight.

2. Detailed Explanation of Practical Challenges

(1) Mass shift caused by post-translational modifications: For example, N-terminal acetylation (+42 Da), glycosylation (+162–2000+ Da), or oxidation (+16 Da) will cause deviations in molecular weight from the theoretical value, requiring comprehensive analysis combined with databases and modification prediction tools.

(2) Interference from buffer components: Some samples contain high concentrations of salts or detergents (such as SDS, Tris), which can seriously inhibit ionization efficiency, leading to decreased signal intensity or even complete undetectability. Therefore, strict desalting and purification are necessary before analysis.

(3) Low ionization efficiency for high molecular weight proteins: Proteins with molecular weights exceeding 100 kDa show significantly reduced ionization efficiency in ESI mode, requiring optimization of electrospray parameters, increased organic solvent ratio, or even combination with pre-treatment strategies to enhance signal intensity.

4. Standard Operating Procedure for Protein Molecular Weight Measurement

To ensure the accuracy and reproducibility of mass spectrometry results in protein molecular weight measurement, it is recommended to strictly follow the following experimental procedures:

1. Sample Preparation

(1) Concentration recommendation: 0.5–2 μg/μL;

(2) Buffer system requirements: Avoid ionization inhibitors such as SDS, EDTA; if salts or glycerol are present, perform C18 or Zeba desalting treatment;

(3) Purity requirements: The higher the protein purity, the clearer the mass spectrometry signal. It is recommended to use SDS-PAGE or SEC for preliminary sample purity checks.

2. Ionization Method Selection

(1) For small molecular proteins (such as peptides, enzymes): ESI is recommended, which can be directly analyzed with LC-MS;

(2) For high molecular weight proteins or structurally rigid protein complexes: MALDI can be used, provided sufficient desalting and spot optimization are performed.

3. Data Collection and Analysis

(1) It is recommended to use a high-resolution mass spectrometry platform with a resolution of ≥60,000 (such as the Orbitrap Exploris series) to improve mass accuracy;

(2) For complex samples or protein mixtures, a combined MS1 (molecular weight determination) and MS2 (peptide information) strategy can be used;

(3) Data deconvolution can be performed using software tools such as Protein Deconvolution, MaxEnt1, or BioPharma Finder to improve the accuracy of molecular weight restoration.

Mass spectrometry for protein molecular weight measurement is a key link between protein expression, structural analysis, and quality control. With the continuous advancement in instrument sensitivity and data algorithms, mass spectrometry will play a greater role in future life sciences research and drug development. BGI Tech will continue to optimize mass spectrometry platforms and data interpretation capabilities to support more research projects and industry applications in achieving leaps from qualitative to quantitative analyses.

BGI Tech - Characterization of Bioproducts, High-Quality Service Provider of Multi-Omics Mass Spectrometry Detection

Related Services:

High-resolution Mass Spectrometry Molecular Weight Identification