title: Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS.
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.
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Gerber
authors.forename: John
authors.initials: J
authors.lastname: Rush
authors.forename: Olaf
authors.initials: O
authors.lastname: Stemman
authors.forename: Marc W
authors.initials: MW
authors.lastname: Kirschner
authors.forename: Steven P
authors.initials: SP
authors.lastname: Gygi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D010450:Endopeptidases; D006367:HeLa Cells; D006655:Histone Deacetylases / Q000032:analysis; D006736:Horses; D006801:Humans; D066298:In Vitro Techniques; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000032:analysis; D010750:Phosphoproteins / Q000032:analysis*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011506:Proteins / Q000032:analysis*; D029701:Saccharomyces cerevisiae Proteins; D064247:Separase; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae / Q000032:analysis; D056565:Sirtuin 2; D037761:Sirtuins / Q000032:analysis
chemical_list: D018797:Cell Cycle Proteins; D009211:Myoglobin; D010750:Phosphoproteins; D011506:Proteins; C061573:SIR4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; D011494:Protein Kinases; D010450:Endopeptidases; C577986:ESP1 protein, S cerevisiae; C577987:ESPL1 protein, human; D064247:Separase; C087778:SIR2 protein, S cerevisiae; D056565:Sirtuin 2; D037761:Sirtuins; D006655:Histone Deacetylases