Mechanism of AQUA in Protein Detection

Absolute Quantification of proteins (AQUA) is a key technology in proteomics, utilizing stable isotope-labeled peptides for precise protein quantification. Combined with mass spectrometry (MS), AQUA enables the absolute quantification of target proteins, providing crucial insights into biomarker discovery, drug target validation, and functional analysis of proteins.

 

AQUA operates by using mass spectrometry and synthetic stable isotope-labeled peptides. Proteins of interest are digested into peptides by proteases (typically trypsin), and specific peptides matching target proteins are synthesized with stable isotopes (such as 13C or 15N). These isotopically labeled peptides differ in mass from natural peptides, allowing the comparison of labeled and endogenous peptides in mass spectrometry, leading to absolute protein quantification.

 

Mechanism of Quantification in AQUA

1. Design and Synthesis of Stable Isotope-Labeled Peptides

Peptides matching the specific sequences of the target protein are synthesized, incorporating stable isotope labels through chemical synthesis. The stable isotope does not alter the peptide's chemical properties, allowing the labeled peptide to behave similarly to the natural peptide in the sample. Commonly used isotope labels include 13C or 15N-labeled lysine or arginine.

 

2. Sample Preparation and Protein Digestion

The target protein in the sample is digested by a protease (usually trypsin) into peptides. At the same time, a known amount of the synthesized stable isotope-labeled peptide is added as an internal standard. This labeled peptide mirrors the natural peptide in every respect except for mass, allowing it to be distinguished during mass spectrometry.

 

3. Mass Spectrometry Analysis and Signal Comparison

Mass spectrometry is employed to detect both the digested peptides from the sample and the internal standard peptide. Since the labeled and natural peptides are chemically identical, they have nearly identical ionization efficiencies and behavior during mass spectrometry. However, the slight difference in mass results in distinct signal peaks. By comparing the peak areas of the natural peptide and the labeled peptide, the absolute quantity of the target protein in the sample can be calculated.

 

4. Calibration and Data Analysis

During mass spectrometry, the known concentration of the labeled peptide is used to calibrate the signal from the natural peptide. By integrating and comparing signal peaks, the absolute amount of the target protein in the sample can be precisely calculated. The calibration step in AQUA ensures high accuracy in quantification across different samples and experimental conditions.

 

Accuracy and Sensitivity of AQUA Quantification

AQUA achieves high accuracy in quantification primarily due to the use of stable isotope-labeled peptides. These peptides share almost identical chemical properties with the target peptides, eliminating variability during sample handling and mass spectrometry analysis. Additionally, mass spectrometry's high resolution and sensitivity allow AQUA to detect even low-abundance proteins in complex biological samples, with detection limits reaching the femtomolar range.

 

Application of AQUA in Complex Biological Samples

AQUA is widely applied in the analysis of complex biological samples, such as blood, tissues, or cell lysates. These samples often contain numerous non-target proteins, but AQUA can specifically quantify target proteins without interference from other molecules. By directly comparing the stable isotope-labeled peptide with the target peptide, AQUA minimizes matrix effects, ensuring more accurate quantification.

 

Despite its advantages in protein quantification, AQUA has some limitations. The synthesis of stable isotope-labeled peptides can be expensive, and the technique is limited to pre-selected protein targets. Additionally, AQUA relies on highly specialized mass spectrometry equipment and technical expertise, requiring significant initial investment.