AQUA Proteomics
AQUA proteomics (absolute quantification) is a mass spectrometry-based technique designed to achieve precise absolute quantification of target proteins in complex samples using stable isotope-labeled peptides as internal standards. The method involves synthesizing isotope-labeled peptides that are chemically identical to the target peptides in the sample but distinguishable in mass spectrometry due to isotope labeling. By comparing the peak intensities of endogenous peptides with their isotope-labeled counterparts, AQUA proteomics enables accurate determination of target protein concentrations. This approach has widespread applications in clinical biomarker discovery, disease mechanism studies, drug target validation, and the analysis of protein post-translational modifications. Compared to traditional relative quantification methods, AQUA proteomics offers superior accuracy, reproducibility, and cross-laboratory data consistency, making it an essential tool in quantitative proteomics.
In disease biomarker discovery, AQUA proteomics supports early diagnosis by precisely quantifying disease-associated proteins and identifying specific biomarkers. This capability provides critical data for developing diagnostic tools and monitoring disease progression. In drug development, the technique aids in validating the efficacy of drug targets, optimizing research strategies, and improving therapeutic outcomes. For post-translational modification analysis, AQUA proteomics excels in quantifying modifications such as phosphorylation and acetylation, shedding light on their regulatory roles in biological processes. Furthermore, in personalized medicine, this method facilitates the development of tailored diagnostic and therapeutic strategies by quantifying specific protein expression levels, driving advancements in precision medicine.
Common Methods in AQUA Proteomics (Absolute Quantification)
1. Synthesis of Stable Isotope-Labeled Peptides
A critical step in AQUA proteomics involves synthesizing stable isotope-labeled peptides. These peptides incorporate isotopes such as ¹³C or ¹⁵N into specific amino acid residues, allowing clear differentiation from endogenous peptides during mass spectrometry analysis.
2. Sample Preparation
Effective sample preparation, including extraction, enzymatic digestion, and purification, forms the foundation of AQUA proteomics. The enzymatic digestion process must selectively degrade target proteins into specific peptides while minimizing non-specific cleavage to ensure reliable results.
3. Mass Spectrometry Detection
High-resolution mass spectrometry, such as LC-MS/MS, is employed to detect both target peptides and isotope-labeled peptides. This step generates precise data on their mass-to-charge ratios (m/z) and peak intensities, enabling accurate comparison.
4. Quantitative Data Analysis
Quantification is achieved by comparing the peak area ratios of endogenous peptides to isotope-labeled peptides. Using the known concentration of labeled peptides as a reference, the absolute concentration of target proteins in the sample is calculated.
Technical Workflow of AQUA Proteomics (Absolute Quantification)
1. Target Peptide Selection
Peptides that are stable, highly specific, and readily detectable by mass spectrometry are selected from the target protein to serve as quantitative signature peptides.
2. Synthesis of Labeled Peptides
Stable isotope-labeled peptides are chemically synthesized to ensure high purity and reliable quantification characteristics.
3. Sample Digestion
The sample is digested using specific proteases, such as trypsin, to produce a peptide mixture suitable for mass spectrometry analysis.
4. Addition of Internal Standards
Isotope-labeled peptides of known concentrations are added to the digested sample, serving as internal standards for accurate quantification.
5. Mass Spectrometry Detection
High-resolution mass spectrometry platforms, such as LC-MS/MS, are employed to detect and measure the signals of both target peptides and their isotope-labeled counterparts.
6. Data Analysis
Mass spectrometry signals are processed and analyzed using specialized software, enabling the absolute quantification of proteins based on the comparison of peptide signal intensities.
Technical Advantages of AQUA Proteomics (Absolute Quantification)
1. High-Precision Quantification
AQUA proteomics delivers accurate protein concentration measurements, reducing biases typically encountered with relative quantification methods.
2. Excellent Reproducibility Across Laboratories
The use of isotope-labeled peptides as internal standards minimizes variability between experimental batches and laboratories, ensuring consistent and reproducible results.
3. High Specificity
By targeting specific peptides, this technique effectively distinguishes between closely related or homologous proteins, enhancing quantification accuracy.
4. Broad Quantitative Range
AQUA proteomics supports protein quantification across diverse concentration ranges and is particularly effective for precise detection of low-abundance proteins.
5. Simultaneous Quantification of Multiple Targets
The technique enables the concurrent quantification of multiple proteins within a single analysis, significantly increasing experimental throughput and efficiency.
MtoZ Biolabs specializes in AQUA proteomics services, offering a comprehensive workflow that includes target peptide selection, stable isotope labeling, sample preparation, mass spectrometry analysis, and data interpretation. We ensure high-precision, reproducible quantification tailored to the specific needs of our clients. Whether for disease biomarker discovery, drug target validation, or post-translational modification analysis, MtoZ Biolabs provides customized solutions designed to advance research outcomes.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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