A need exists for technologies that permit the direct quantification of differences in protein and posttranslationally modified protein expression levels. Here we present a strategy for the absolute quantification (termed 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 posttranslationally modified proteins after proteolysis by using a selected reaction monitoring analysis in a tandem mass spectrometer. In the present work, the AQUA strategy was used to (i) quantify low abundance yeast proteins involved in gene silencing, (ii) quantitatively determine the cell cycle-dependent phosphorylation of Ser-1126 of human separase protein, and (iii) identify kinases capable of phosphorylating Ser-1501 of separase in an in vitro kinase assay. The methods described here represent focused, alternative approaches for studying the dynamically changing proteome.

Sister-chromatid separation is triggered by a speci¢c proteolytic cleavage of chromosomal cohesins catalyzed by the endopeptidase separase. Prior to anaphase, separase is inhibited independently by a⁄nity binding to securin and by speci¢c inhibitory phosphorylation. Here we show that separase itself is also subjected to proteolytic cleavages at three adjacent sites. The cleavages are auto-catalyzed and occur speci¢cally at anaphase coincident with separase activation. The cleaved fragments remain associated with each other and are catalytically active. Mapping of the cleavage sites reveals that all three sites are conserved in vertebrates underlining a signi¢cant function for this regulation. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.