Absolute quantification of proteins and phosphoproteins using the AQUA strategy. The strategy has two stages. Stage 1 involves the selection and standard synthesis of a peptide (or phosphopeptide denoted by pS) from the protein of interest. During synthesis, stable isotopes are incorporated (e.g., ¹³C, ¹⁵N, etc.) at a single amino acid residue such as the leucine shown here denoted by *. These peptide internal standards are analyzed by MS/MS to examine peptide fragmentation patterns. The mass spectrometer is next set up to perform a SRM analysis in which a specific precursor-to-product ion transition is measured. Stage 2 is the implementation of the new peptide internal standard for precise quantification. Protein is harvested from a biological sample and proteolyzed with trypsin in the presence of the AQUA internal standard peptide/phosphopeptide. An LC–SRM experiment then measures the abundance of a specific fragment ion from both the native peptide and the synthesized peptide as a function of reverse-phase chromatographic retention time. The absolute quantification is determined by comparing the abundance of the known AQUA internal standard peptide with the native peptide.

AQUA method development process for validation protein horse heart myoglobin. (A) MS/MS mass spectra of two synthetic, isotopically labeled peptides corresponding to native peptides from myoglobin formed by trypsin proteolysis. L* indicates a leucine with six ¹³C and one ¹⁵N atoms. The fragmentation pattern of each synthetic peptide revealed prominent y-type fragment ions suitable for monitoring. (B) LC–SRM traces for the specific parent-to-product ion transitions of 200 fmol of the two AQUA internal standard peptides for myoglobin using a 100-μm ID microcapillary LC column.

Validation of the AQUA method for horse heart myoglobin. (A) SDS/PAGE gel separation of 50 μg of yeast lysate spiked with standardized myoglobin protein at different amounts (300 amol to 30 pmol). Regions corresponding to migrated myoglobin were generously excised from the gel and digested with trypsin in the presence of 500 fmol of each AQUA internal standard peptide. (B) Analysis of 300 amol of myoglobin from a yeast background using the two AQUA peptides for reference and quantification by LC-SRM. The top trace of each pair represents the response for the “native” peptide formed by trypsinization, and the bottom trace corresponds to the same peptide synthesized with stable isotopes to have a mass difference of 7 Da. The peak area signal-to-noise is indicated for each determination. (C) Observed vs. expected response curve for the myoglobin quantification. (Inset) The low end of the curve (300 amol–300 fmol). (D) Effect of trypsin amount (50 pg to 1 μg) on the levels of myoglobin detected from the SDS/PAGE gel during a 6-h digestion. The same experiment as in A was performed, except the amount of spiked myoglobin (300 fmol) and AQUA peptides (500 fmol) was held constant while the amount of trypsin added for in-gel digestion varied. The curve plateaus when >250 ng trypsin was added, suggesting complete trypsinization.

AQUA analysis of endogenous yeast Sir proteins 2 and 4. (A) MS/MS mass spectra of the synthetic internal standard peptides IYSPL*HSFIK (Sir2) and QFDSIF*NSNK (Sir4) for product ion selection. The leucine and phenylalanine marked by * contained stable isotopes. (B) SDS/PAGE separation of 50 μg of yeast lysate per lane (1.8 × 10⁷ cells) visualized by Coomassie staining. Molecular mass regions corresponding to Sir2 (55–66 kDa) and Sir4 (120–180 kDa) were excised from both lanes and digested in-gel in the presence of 150 fmol of each internal standard peptide. (C)LC–SRM analysis for Sir2 and Sir4 expression levels. (Upper) The response for the native peptide formed by trypsinization. (Lower) The response for the respective AQUA internal standard peptide (150 fmol). Assuming no losses, the analysis resulted in a calculated expression level of 1,750 and 1,150 copies per cell for Sir2 and Sir4 protein, respectively, for yeast in an exponential growth phase.

Quantitative analysis of the phosphorylation state of Ser-1126 phosphorylation from human separase protein as a function of cell cycle. (A) Percent of phosphorylated Ser-1126 measured before, during, and after exit from mitosis in HeLa cells (▪). Percent of cells in G₂/M phase of the cell cycle was also determined by fluorescence-activated cell sorting analysis (⋄). Separase is quantitatively (>98%) phosphorylated at the anaphase-metaphase transition (nocodozole arrest). The percent of phosphorylated Ser-1126 decreases after mitosis. (B) LC–SRM analysis of phosphorylated and nonphosphorylated separase Ser-1126. These data were acquired from the equivalent of only 16 μg of starting material and 10 fmol of each AQUA peptide on a high-resolution triple quadrupole mass spectrometer. The calculated percent of phosphorylation was 34%. Some of the data in this figure have been published previously. [Reproduced with permission from ref. 15 (Copyright 2001, Elsevier Science).]

AQUA methodology for determining site-specific, kinase-dependent protein phosphorylation in vitro. Application to separase Ser-1501 affinity-purified separase, ATP, and a normalized quantity of each kinase were incubated for a defined interval. Reactions were purified by gel electrophoresis and quantified by in-gel trypsin digestion in the presence of 200 fmol of each synthetic internal standard peptides LTDNWRKMSFEIL*R and LTDNWRKM(pS)FEIL*R. (A) LC–SRM trace for phosphorylated and nonphosphorylated separase Ser-1501 after treatment with a mutant (inactive) PKA. The basal level of phosphorylation was determined to be 16%. (B) LC–SRM trace for phosphorylated and nonphosphorylated separase Ser-1501 after treatment with active PKA. The percent of phosphorylation was determined to be 98%. (C) Percent of phosphorylation for separase Ser-1501 after treatment with a bank of kinases. Data shown are the result of triplicate analyses with ranges in standard deviation from 0.5% to 9.1% with a median value of 3.6%. Both calmodulin-dependent kinase II (CaMKII) and PKA successfully phosphorylated Ser-1501 in vitro.