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.