Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS

TLDR

There is a need for technologies that directly quantify differences in protein and post‑translationally modified protein expression, and the methods described here offer focused, alternative approaches to study the dynamically changing proteome. The study presents AQUA, a strategy for absolute quantification of proteins and their modification states. AQUA uses synthetic peptides labeled with stable isotopes, including covalently modified forms that are chemically identical to natural post‑translational modifications, as internal standards to precisely quantify absolute protein and modified protein levels by selected reaction monitoring in a tandem mass spectrometer after proteolysis. Using AQUA, the authors quantified low‑abundance yeast gene‑silencing proteins, measured cell‑cycle‑dependent phosphorylation of Ser‑1126 in human separase, and identified kinases that phosphorylate Ser‑1501 of separase in vitro.

Abstract

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.