Absolute Quantification Mass Spectrometry
Absolute quantification mass spectrometry is a method used for the precise measurement of the absolute levels of specific proteins in biological samples, leveraging mass spectrometry technology. Unlike traditional relative quantification techniques such as iTRAQ, TMT, or label-free quantification, this approach provides precise quantification of target proteins by employing standards or isotopically labeled internal standards. This method circumvents potential errors introduced by sample variability, sample preparation processes, or fluctuations in mass spectrometry instrumentation. It holds significant application potential in fields like biomedical research, disease biomarker discovery, drug development, and biotechnology, making it a valuable tool in quantitative proteomics. The fundamental principle of absolute quantification mass spectrometry involves introducing internal standards of known concentration, which are analyzed under the same experimental conditions as the target proteins or peptides in mass spectrometry. Common strategies for internal standards include stable isotope-labeled standards and isotope dilution mass spectrometry. In the stable isotope-labeled standard method, isotopically labeled standards for the target peptide-incorporating stable isotopes such as ^13C, ^15N, or ^2H-are synthesized and added to the sample. Although the labeled peptide and the target peptide share nearly identical chemical properties, they exhibit distinctive mass differences in mass spectrometry, allowing for accurate calculation of the target protein’s absolute content by comparing their peak area ratios. In isotope dilution mass spectrometry, the target protein or peptide is mixed with an isotopically labeled internal standard of known concentration, and quantification is achieved by comparing the two through mass spectrometry.
Key Steps in Absolute Quantification Mass Spectrometry
1. Sample Preparation
Sample preparation forms the basis of absolute quantification mass spectrometry and includes steps such as protein extraction, peptide degradation, and desalting. The quality of these samples directly impacts the accuracy of subsequent quantification results.
2. Introduction of Internal Standards
Isotopically labeled standard peptides of known concentration are added to the samples to ensure simultaneous detection of the internal standard and the target peptide during mass spectrometry analysis.
3. Liquid Chromatography-Mass Spectrometry (LC-MS/MS) Analysis
Peptides are separated using liquid chromatography, and characteristic ion fragments are detected through tandem mass spectrometry (MS/MS).
4. Data Analysis
The peak areas of the target peptide and internal standard peptide are compared, and the absolute concentration of the target protein is determined using a standard curve.
Advantages of Absolute Quantification Mass Spectrometry
1. High Accuracy and Sensitivity
This method allows for highly specific and sensitive quantification of proteins in complex biological samples, making it particularly suitable for detecting low-abundance proteins.
2. Cross-Sample Comparison
Absolute quantification mass spectrometry facilitates direct comparison across different samples due to the use of internal standards, minimizing errors from sample processing differences.
3. Broad Application Spectrum
This technique provides reliable quantitative data suitable for biomedical research, drug development, and disease biomarker screening.
Applications of Absolute Quantification Mass Spectrometry
1. Disease Biomarker Discovery
By quantifying specific proteins in plasma, tissue, or cell samples, this method aids in discovering disease-related biomarkers, supporting early diagnosis.
2. Drug Target Validation
In the context of drug development, the method precisely quantifies interactions between drugs and target proteins, helping to assess drug efficacy and safety.
3. Quality Control in Biopharmaceuticals
In biopharmaceuticals, it is used to quantify active protein content in drugs, ensuring product quality and stability.
4. Metabolic Pathway Research
The method provides insights into cellular metabolic mechanisms by quantifying enzymes and proteins involved in specific metabolic pathways.
MtoZ Biolabs, leveraging extensive technical expertise and a professional team, offers high-precision mass spectrometry analysis services. We provide a comprehensive solution encompassing sample preparation, internal standard introduction, mass spectrometry detection, and data analysis to ensure the accuracy and reproducibility of quantitative results.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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