Introduction:
Absolute Quantification (AQUA) is an innovative and powerful targeted quantitative proteomics technique that has gained widespread popularity across various proteomics studies. This cutting-edge approach allows for precise and accurate measurement of protein levels and their post-translational modification states. By synthesizing peptides with stable isotopes as internal standards, AQUA effectively mimics native peptides generated through proteolysis. Moreover, these synthetic peptides can be custom-designed to include chemically identical covalent modifications, such as phosphorylation, methylation, or acetylation, replicating naturally occurring post-translational modifications. By utilizing AQUA internal standard peptides, researchers can quantitatively analyze the absolute levels of proteins and post-translationally modified proteins using selected reaction monitoring analysis in tandem mass spectrometry.
Advancements in biological mass spectrometry have spurred the development of numerous strategies for large-scale protein expression quantification within cells. While differential incorporation of stable isotopes has been instrumental in measuring relative protein expression, absolute quantification has emerged as an indispensable method in proteomics analysis, providing crucial insights into protein expression and post-translational modification levels.
The AQUA Strategy: A Comprehensive Quantification Approach:
The AQUA strategy represents a universal procedure for the precise quantification of proteins and their post-translational modifications. This strategy revolves around the utilization of synthetic peptides containing stable isotopes, enabling accurate and reliable absolute quantification.
The foundation of the absolute quantification method lies in the serendipitous discovery of an unexpected relationship between the mass spectrometry (MS) signal response and protein concentration. Remarkably, it has been observed that the average MS signal response for the three most intense tryptic peptides per mole of protein remains remarkably consistent, with a coefficient of variation below 10%. Capitalizing on this internal standard, researchers can calculate a universal signal response factor, which has been found to be consistent across a diverse range of proteins.
While traditional isotope methods only offer relative quantification of expressed proteins, the AQUA strategy provides an exceptional advantage by enabling precise determination of protein expression levels and post-translational modification states. This groundbreaking method relies on the use of synthetic internal standard peptides, which are introduced at a known concentration into cell lysates during the digestion process. The subsequent analysis of the proteolyzed sample using a selected reaction monitoring (SRM) experiment in a tandem mass spectrometer facilitates the direct detection and quantification of both the native peptide and the isotope-labeled AQUA internal standard peptide.
The Simplicity, Sensitivity, and Versatility of the AQUA Strategy:
The AQUA strategy is renowned for its simplicity, sensitivity, and versatility. Coupled with the widespread availability of tandem mass spectrometers, this approach has become an invaluable tool for directly measuring protein levels and post-translational modifications from cell lysates.
In addition to the revolutionary Absolute Quantification (AQUA) technique, Creative Proteomics offers a comprehensive range of bioinformatics services to enhance your research:
Functional annotation and enrichment analysis: Uncover the functional significance of your proteomic data through comprehensive annotation and enrichment analysis.
Clustering analysis: Identify patterns and relationships within your proteomics data through advanced clustering algorithms.
Network analysis: Explore protein-protein interaction networks to gain insights into complex biological processes and pathways.
Statistical analysis: Apply rigorous statistical methods to validate and interpret your proteomics data accurately.
We encourage you to reach out to us and discuss your projects with our team of experts. We are confident that we can fulfill your research needs effectively, providing you with exceptional support and insights throughout your proteomics journey.
