AQUA is a method for absolute quantification of proteins and phosphoproteins from cell lysates using tandem mass spectrometry. The method involves the synthesis of synthetic peptides with stable isotopes as internal standards to mimic native peptides. These peptides are used to measure the absolute levels of proteins and their modifications after proteolysis by selected reaction monitoring (SRM) in a tandem mass spectrometer. The AQUA strategy was used to quantify low abundance yeast proteins involved in gene silencing, determine the cell cycle-dependent phosphorylation of Ser-1126 of human separase, and identify kinases capable of phosphorylating Ser-1501 of separase in an in vitro kinase assay. The methods described represent focused, alternative approaches for studying the dynamically changing proteome. Proteomics is the systematic identification and characterization of proteins for their structure, function, activity, quantity, and molecular interactions. Quantitative proteomics seeks to provide information about both protein and modified protein expression levels. Two-dimensional gel electrophoresis (2DE) has been used to separate and quantify many protein products simultaneously in a single gel. However, 2DE is highly selective for abundant proteins and does not allow for the detection of regulatory proteins. Alternative strategies for proteome analysis generally involve increasing levels of protein and peptide separation coupled with amino acid sequence analysis by tandem MS. These techniques have been combined with stable isotope labeling to conduct quantitative studies. However, comparative analysis of posttranslationally modified proteins has proven to be a more difficult challenge. In contrast to relative quantification strategies, synthetic peptides with incorporated stable isotopes can provide absolute protein quantification. The AQUA strategy was extended to measure the precise amounts of posttranslationally modified proteins and to perform analyses directly from whole-cell lysates separated by SDS/PAGE. The method was validated by analyzing horse heart myoglobin in a yeast background and by quantifying yeast silent information regulatory (Sir)2 and Sir4 proteins. The AQUA strategy was also used to quantify the cell cycle-dependent phosphorylation of Ser-1126 of human separase and to determine the in vitro kinase activity of separase at Ser-1501. The results demonstrated the capacity of the AQUA strategy for measuring dynamic changes in phosphorylation state directly from cell lysates. The method has the potential to assist the study of systems biology in diverse ways, including the evaluation of protein expression changes without the need for producing antibodies.AQUA is a method for absolute quantification of proteins and phosphoproteins from cell lysates using tandem mass spectrometry. The method involves the synthesis of synthetic peptides with stable isotopes as internal standards to mimic native peptides. These peptides are used to measure the absolute levels of proteins and their modifications after proteolysis by selected reaction monitoring (SRM) in a tandem mass spectrometer. The AQUA strategy was used to quantify low abundance yeast proteins involved in gene silencing, determine the cell cycle-dependent phosphorylation of Ser-1126 of human separase, and identify kinases capable of phosphorylating Ser-1501 of separase in an in vitro kinase assay. The methods described represent focused, alternative approaches for studying the dynamically changing proteome. Proteomics is the systematic identification and characterization of proteins for their structure, function, activity, quantity, and molecular interactions. Quantitative proteomics seeks to provide information about both protein and modified protein expression levels. Two-dimensional gel electrophoresis (2DE) has been used to separate and quantify many protein products simultaneously in a single gel. However, 2DE is highly selective for abundant proteins and does not allow for the detection of regulatory proteins. Alternative strategies for proteome analysis generally involve increasing levels of protein and peptide separation coupled with amino acid sequence analysis by tandem MS. These techniques have been combined with stable isotope labeling to conduct quantitative studies. However, comparative analysis of posttranslationally modified proteins has proven to be a more difficult challenge. In contrast to relative quantification strategies, synthetic peptides with incorporated stable isotopes can provide absolute protein quantification. The AQUA strategy was extended to measure the precise amounts of posttranslationally modified proteins and to perform analyses directly from whole-cell lysates separated by SDS/PAGE. The method was validated by analyzing horse heart myoglobin in a yeast background and by quantifying yeast silent information regulatory (Sir)2 and Sir4 proteins. The AQUA strategy was also used to quantify the cell cycle-dependent phosphorylation of Ser-1126 of human separase and to determine the in vitro kinase activity of separase at Ser-1501. The results demonstrated the capacity of the AQUA strategy for measuring dynamic changes in phosphorylation state directly from cell lysates. The method has the potential to assist the study of systems biology in diverse ways, including the evaluation of protein expression changes without the need for producing antibodies.