Application of Multiple Reaction Monitoring (MRM) in Protein Quantitative Analysis
Quantitative analysis of proteins in life sciences and biomedical research is crucial for understanding biological processes, discovering disease biomarkers, and developing new therapies. Multiple Reaction Monitoring (MRM), a highly specific and sensitive quantification method based on mass spectrometry, has become an essential tool for targeted protein quantification analysis.
I. Overview of Multiple Reaction Monitoring (MRM) Technology
1. What is Multiple Reaction Monitoring (MRM)?
MRM is a mass spectrometry scanning mode that uses a triple quadrupole mass spectrometer (Triple Quadrupole MS) to monitor specific ion pairs (precursor ion → product ion). By setting specific ion transition pathways, MRM can selectively detect target peptides in complex biological samples, achieving high sensitivity and specificity for quantitative analysis.
2. Brief Description of the Working Principle of MRM
The MRM mode typically involves a three-step selection process: the first quadrupole (Q1) selects the target precursor ion, the collision cell (q2) induces fragmentation of the precursor ion, and the second quadrupole (Q3) selects specific product ions. Only ions that meet preset mass transitions are recorded, significantly reducing background noise and improving detection sensitivity and quantitative accuracy.
II. Advantages of Multiple Reaction Monitoring (MRM) in Protein Quantification Analysis
1. High Specificity and Sensitivity
By selectively monitoring specific ion transition pathways, MRM can effectively distinguish target peptides from other background molecules, achieving detection limits at the attomole level. This feature makes it particularly suitable for analyzing low-abundance proteins and complex biological matrix samples.
2. Wide Dynamic Range
MRM can achieve a dynamic detection range of 4-5 orders of magnitude, covering the quantification needs from high to low abundance proteins, especially suitable for multi-target analysis in complex samples such as plasma and tissue extracts.
3. High Reproducibility and Quantitative Accuracy
Thanks to a stable ion monitoring mode and the introduction of endogenous/exogenous standards, MRM demonstrates good reproducibility across multiple batches of experiments and has the potential for absolute quantification, providing reliable data support for biomarker validation and clinical research.
III. Practical Applications of Multiple Reaction Monitoring (MRM) in Different Protein Quantification Scenarios
1. Targeted Proteomics Research
In exploring disease mechanisms, drug responses, and signal pathway regulation, researchers often need to validate candidate proteins identified. Due to its high throughput and quantitative precision, MRM has become an indispensable tool in the validation phase. For example, in tumor biomarker research, MRM can simultaneously quantify dozens to hundreds of candidate proteins, greatly accelerating the verification of candidate molecules.
2. Clinical Biomarker Validation
Compared to Discovery Proteomics, MRM provides more clinically translatable quantitative data. In the development of biomarkers for cardiovascular diseases, tumors, and autoimmune diseases, MRM is widely used in the validation phase of large cohort samples, such as accurately quantifying low-abundance proteins (such as cytokines, inflammatory factors) in plasma.
3. Drug Mechanism of Action Studies
Understanding the interaction between drugs and target proteins is crucial in drug development. MRM can be used to monitor changes in target protein levels before and after drug treatment, helping to elucidate mechanisms of drug efficacy and resistance development. For example, MRM can quantify changes in the expression of specific pathway proteins in cell lines after drug exposure, providing a direct reflection of the drug's effects.
4. Simultaneous Detection of Multiple Targets
MRM can monitor multiple target proteins simultaneously without relying on antibody resources, enhancing experimental efficiency and data throughput. In recent years, MRM-based multi-target protein quantification methods have been widely applied in the comprehensive diagnosis of infectious diseases, multiple diseases, and the development of biomarker panels.
IV. How to Optimize MRM Protein Quantification Experimental Design?
1. Rational Selection of Surrogate Peptides
Selecting representative, stable, unmodified, and easily ionizable peptides is a prerequisite for successful MRM quantification. Typically, 2-3 high-quality surrogate peptides are screened for each target protein to ensure data reliability.
2. Optimization of Ion Pair Selection and Parameter Settings
For each peptide, at least 2-3 high-response, low-background product ions should be selected for monitoring (i.e., transition), while optimizing mass spectrometry parameters such as collision energy to obtain the best signal-to-noise ratio.
3. Use of Stable Isotope-Labeled Internal Standards
Introducing stable isotope-labeled peptides (SIS peptides) as internal standards can effectively correct errors introduced by sample processing, ionization efficiency, and instrument fluctuations, thereby achieving more accurate absolute quantification.
4. Standardization of Sample Preprocessing Workflow
The standardization of sample preparation directly affects the accuracy and reproducibility of MRM quantification. Unified steps for protein extraction, digestion, purification, and concentration, combined with the introduction of quality control samples (QC samples), are key to ensuring experimental reliability.
As protein quantification analysis becomes increasingly refined and high-throughput, Multiple Reaction Monitoring (MRM), with its excellent specificity, sensitivity, and stability, has become an indispensable technical tool for researchers and biopharmaceutical companies. Biotech company Biotye Parker continuously optimizes mass spectrometry platforms and quantitative methodology development to provide global researchers with high-quality, highly reliable quantitative proteomics analysis services, supporting every step of scientific discovery and clinical application.
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