Workflow of AQUA in Quantitative Proteomics

AQUA (Absolute Quantification) is an isotope-labeled mass spectrometry-based technique used for the precise absolute quantification of specific peptides from target proteins. By incorporating known amounts of synthetic peptides as internal standards, AQUA provides absolute quantification of target peptides in a sample. The workflow of AQUA involves multiple critical steps, ranging from sample preparation to data processing and analysis, which ensures accuracy and reproducibility.

 

Sample Preparation

The first step in the AQUA workflow is sample preparation. This involves extracting proteins from the sample, followed by denaturation, reduction, and alkylation to ensure that disulfide bonds are reduced and do not reform. The proteins are then digested into peptides, typically using trypsin, for mass spectrometry analysis. To increase quantification accuracy, isotopically labeled peptides with known concentrations are added as internal standards during this step.

 

Selecting the appropriate isotope-labeled peptides is crucial. These peptides must closely resemble the target peptides in their mass spectrometry behavior, including retention time and ionization efficiency. Internal standard peptides are chemically synthesized to match the sequence of the target peptides exactly, but with stable isotope-labeled amino acids (e.g., ^15N or ^13C), which causes a slight difference in the mass-to-charge ratio (m/z) compared to the endogenous peptides, allowing them to be distinguished in the mass spectrometer.

 

Mass Spectrometry Analysis

Mass spectrometry analysis is the core of the AQUA workflow. Prepared samples are analyzed using liquid chromatography (LC) coupled with mass spectrometry (MS). The LC system separates the complex peptide mixtures, while the MS system measures the mass-to-charge ratio and abundance of the peptides.

 

During analysis, the mass spectrometer detects the signals of both the internal standard peptides and the target peptides based on their specific mass-to-charge ratios. Since the concentration of the internal standards is known, the absolute amount of the target peptides can be calculated by comparing the peak areas of the internal standards and the target peptides. This method is highly sensitive and can quantify proteins present at very low concentrations with high precision.

 

Data Processing and Analysis

After collecting mass spectrometry data, the next step is data processing and analysis. Specialized software identifies the signals of the internal standard and target peptides and performs quantification. Using the known concentration of the internal standards as a reference, the software calculates the actual concentration of the target peptides using linear regression or other statistical models.

 

During data processing, background subtraction and peak shape corrections are applied to ensure accurate quantification. Additionally, to improve reproducibility, multiple replicates are typically performed, followed by statistical analysis of the data.

 

Data Validation and Reporting

Data validation is a crucial step in the AQUA workflow. Researchers often use various methods to validate the reliability of the results, such as verifying the linear range of quantification using standard curves and performing parallel experiments to assess the stability of the results.

 

The final step involves reporting the results. The report typically includes the absolute concentrations of the target proteins or peptides, statistical data from the analysis, and the quantification error margins. These results are not only useful in fundamental research, such as protein function studies but are also applicable in clinical fields for tasks like drug target quantification and biomarker detection.

 

AQUA is a highly reliable and sensitive technique in quantitative proteomics, offering unparalleled precision in absolute protein quantification through the use of isotope-labeled internal standards in mass spectrometry. Every step in the workflow, from sample preparation to data analysis, demands accuracy and rigor. Although the workflow is complex, AQUA offers significant advantages in absolute quantification.