Principle of Absolute Quantitative Analysis

In proteomics research, quantitative analysis is essential for understanding the changes in protein expression levels under different biological conditions. Absolute Quantification (AQUA) is a precise and reliable method widely used in proteomics for accurate quantification. The AQUA technique relies on mass spectrometry (MS) and isotopically labeled standards to measure the amount of target proteins or peptides in an absolute manner.

 

Principle of AQUA

The core principle of the AQUA absolute quantification method involves using isotope-labeled synthetic peptides (AQUA peptides) as internal standards to accurately quantify the concentration of target peptides or proteins. The key steps include the following:

 

1. Synthesis of Isotope-Labeled Peptides

First, synthetic peptides with the same sequence as the target peptide are chemically synthesized, with stable isotopes (e.g., carbon-13 or nitrogen-15) incorporated at one or more amino acid positions. These labeled peptides are known as AQUA peptides, which share similar physical properties with the target peptides but can be distinguished by mass spectrometry due to their isotopic labeling.

 

2. Addition of Internal Standards

Before sample analysis, known concentrations of AQUA peptides are added to the sample. Since AQUA peptides have the same chemical properties as the target peptides, they behave similarly during protein digestion, separation, and detection.

 

3. Mass Spectrometry Detection

The target peptides and AQUA peptides are detected simultaneously using liquid chromatography-mass spectrometry (LC-MS/MS). The mass spectrometer differentiates between the internal standard peptides and the endogenous peptides in the sample based on their mass-to-charge ratios (m/z).

 

4. Data Analysis

The absolute quantity of the target peptide or protein is calculated using the known concentration of the internal standard peptide. By comparing the mass spectrometric signal intensities of the target peptide and the AQUA peptide, the absolute quantification of the target peptide is obtained.

 

Advantages of AQUA

1. High Precision and Accuracy

Since the AQUA technique uses isotope-labeled internal standard peptides, the chemical properties of the target peptide and the internal standard peptide are almost identical, ensuring the precision and accuracy of the quantification results.

 

2. Absolute Quantification

Unlike relative quantification methods, AQUA provides absolute quantification information, which allows researchers to determine the exact amount of the target protein or peptide, not just relative changes.

 

3. High Sensitivity

AQUA can accurately quantify low-abundance proteins in complex samples, making it highly useful in biomarker discovery and validation.

 

Disadvantages of AQUA

1. High Cost

Synthesizing stable isotope-labeled peptides can be expensive, especially in experiments that involve multiple target proteins.

 

2. Time-Consuming

AQUA requires synthesizing specific peptides for each target protein, and the simultaneous internal standard addition and mass spectrometric detection increase the complexity and time required for the experiment.

 

3. Peptide Selection

The success of AQUA depends on selecting appropriate peptides to synthesize as internal standards. Some peptides from the target protein may not perform well in mass spectrometric detection, so careful selection is necessary.

 

Applications of AQUA

AQUA is widely used in various biological research fields, particularly in proteomics, disease biomarker studies, and drug target validation. For example, in cancer research, AQUA is employed to quantify tumor biomarkers, helping to reveal differences in protein expression across different cancer subtypes. Additionally, AQUA can be used to quantify drug targets, assisting in the precise measurement of target proteins during drug development.

 

The AQUA absolute quantification technique, through the use of isotope-labeled internal peptides, provides high-precision protein quantification. Although it is more expensive and complex, its advantages in precision, absolute quantification, and high sensitivity make it a powerful tool in proteomics research.