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Absolute Quantification (AQUA)

Absolute Quantification (AQUA)

Absolute Quantification is a targeted quantitative proteomics technique that exhibits robust efficacy and is being increasingly utilized for a wide variety of quantitative proteomics studies. AQUA strategy is for the absolute quantification (AQUA) of proteins and their modification states. Peptides are synthesized with incorporated stable isotopes as ideal internal standards to mimic native peptides formed by proteolysis. These synthetic peptides can also be prepared with covalent modifications (e. g. , phosphorylation, methylation, acetylation, etc.) that are chemically identical to naturally occurring posttranslational modifications. Such AQUA internal standard peptides are then used to precisely and quantitatively measure the absolute levels of proteins and post-translationally modified proteins after proteolysis by using a selected reaction monitoring analysis in a tandem mass spectrometer.

Advances in biological mass spectrometry have resulted in the development of numerous strategies for the large-scale quantification of protein expression levels within cells. Besides the measurements of protein expression accomplished through differential incorporation of stable isotopes into cellular proteins, the absolute quantification is a useful method in proteomics analysis.

The absolute quantification strategy: a general procedure for the quantification of proteins and post-translational modification. AQUA provides absolute quantification by employing synthetic peptides containing stable isotopes.

The absolute quantification method is based on the discovery of an unexpected relationship between MS signal response and protein concentration: the average MS signal response for the three most intense tryptic peptides per mole of protein is constant within a coefficient of variation of less than 10%. Given an internal standard, this relationship is used to calculate a universal signal response factor. The universal signal response factor (counts/mol) was shown to be the same for all proteins tested.

While isotope methods establish only relative quantification of expressed proteins, the absolute quantification (AQUA) strategy can provide information for the precise determination of protein expression and post-translational modification levels. The AQUA method relies on the use of a synthetic internal standard peptide that is introduced at a known concentration to cell lysates during digestion. Analysis of the proteolyzed sample by a selected reaction monitoring (SRM) experiment in a tandem mass spectrometer results in the direct detection and quantification of both the native peptide and isotope labeled AQUA internal standard peptide.

The simplicity and sensitivity of the method, coupled with the widespread availability of tandem mass spectrometers, make the AQUA strategy a highly useful procedure for measuring the levels of proteins and post-translational modifications directly from cell lysates.

View the online catalog of Isotope Labeled AQUA Internal Standard Peptide.
If the product of interest is not available in our catalog, we can synthesize for you with our quality controlled
Customized Synthesized Peptide/Proteins Service!

Creative Proteomics also provide the following bioinformatics services in Absolute Quantification:
Functional annotation and enrichment analysis
Clustering analysis
Network analysis
Statistical analysis

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