Chemically synthesized peptides of optimized sequence have been used for more than 30 years as in vitro
phosphorylation substrates using both purified kinases and cell lysates30–32
. Our single-reaction KAYAK strategy simultaneously measures 90 peptide-based phosphorylation rates using high-resolution MS. We selected substrate peptides from optimized targets or from uncharacterized sites on interesting proteins9
to encompass diverse signaling pathways and enhanced measurement accuracy by using stable isotope–labeled internal standards. The purified peptides used in single-reaction KAYAK profiling ensure absolute quantification of activities that are linear over several logs of lysate amounts. The approach also partially addresses the kinase-specificity problem inevitably associated with peptide-based measurements (Supplementary Note
). Finally, based on the many cellular settings investigated, the assay appears to faithfully and simultaneously report the core activation states for many pathways, including the PI3K and MAPK pathways, which are most frequently altered in cancer.
Compared to other strategies, the KAYAK strategy9
has several advantages. First, KAYAK directly monitors kinase enzymatic activities, thereby averting the need for an activity-indicating antibody. Although commonly used, phosphorylation-activity relationships are known to be far from ideal. Moreover, activation-state phospho-antibodies are not available for many kinases. Second, single-reaction KAYAK measures the intrinsic activity of multiple kinases reflecting the complex cellular context. Although high-throughput kinase assays using large kinase panels are becoming feasible33
, those approaches often use truncated or recombinant purified enzymes, which may not reflect the actual conformational or kinase activity state as they appear in cells. Third, KAYAK has high sensitivity owing to the signal-amplifying nature of enzymatic reactions. Using our single-reaction KAYAK strategy with 20-fold lower peptide concentrations than used previously9
, two KAYAK peptides showed detectable phosphorylation from as little as 1 ng of cell lysate, which corresponds near single-cell levels ( and Supplementary Fig. 2
). The sensitivity of the single-reaction KAYAK approach permits very low sample consumption. Practically, 10–20 μg of cell lysate produce reliable signals for about 50 simultaneous peptide reactions (). Fourth, the approach measures site-specific phosphorylation rates. Commonly, additional phosphorylatable residues are available34
. As the internal standard peptides were synthesized with phosphorylation at known positions, the co-elution of lysate-phosphorylated peptides and the standard phosphopeptides in conjunction with fragmentation sequencing ensures that site-specific phosphorylation is measured. This is not accomplished by any alternative method. Fifth, single-reaction KAYAK is highly quantitative, with exceptional reproducibility (Supplementary Figs. 3 and 4
). Internal standards of heavy peptides, which are added upon quenching the kinase reaction, cancel any downstream sample manipulation and measurement variations and provide the basis for absolute activity measurements (that is, fmol phosphorylation/μg lysate/min). Western blot analysis does not offer a similar level of quantitative quality. Sixth, the assay and protocol can be applied across a wide range of cellular settings, including recombinant purified enzymes (Supplementary Fig. 1
), cell line lysates (–), mouse primary tissue cultures (Supplementary Fig. 9
) and clinical tissue samples (Supplementary Fig. 10
). Seventh, KAYAK is radio-isotope free. Finally, single- reaction KAYAK is flexible in terms of substrate concentration and peptide number. We believe that the single-reaction KAYAK protocol could exploit >90 peptides by using shallower gradients during LC-MS analyses. However, our current focus is to remove redundant or less useful peptides while adding peptides that are monospecific for underreported kinases and pathways.
Single-reaction KAYAK is not without disadvantages. As it is based on an in vitro kinase assay, the current implementation does not measure kinase activities within an intact cell. Moreover, owing to its peptide-based nature, the activity of a kinase that requires a conformational substrate may not be suitably monitored. Finally, the specificities of many kinases toward a peptide substrate may not be unique even at the 5 μM level. Therefore, some peptides may still be phosphorylated by more than one kinase, which complicates pathway identification.
Although in vitro
phosphorylation using purified kinases (Supplementary Fig. 1
) cataloged likely kinase candidates for many phosphorylation events, identification of the responsible kinase directly from cell lysates confirms the physiological relevance of the kinase-substrate pairs. Whereas identifying a phosphorylation event using a specific kinase is relatively straightforward35
, developing a general methodology to identify a kinase responsible for a specific phosphorylation event is far more challenging36–39
. A series of chemical reagents that can cross-link a kinase and its substrate may meet this need, but the reagents have not been shown to work in complex situations, such as those involving cell lysates40,41
. Although traditionally, identification of a responsible enzyme for a specific activity has been accomplished by comparing enzymatic activity and a protein band after SDS-PAGE gel separation, this correlation requires multiple purification steps. Owing to the advancement of protein quantification by MS42
, correlation profiles have been used to determine protein localization by MS43–45
. This has renewed interest in applying modern quantitative proteomics using the classic concept of comparing enzyme activities and protein profiles. Our strategy is a general methodology to decipher kinase-substrate relationships starting with a phosphorylated peptide substrate and a minimally fractionated lysate.
Phosphoproteomics projects have delivered atlases of experimentally mapped phosphorylation sites10,11,46–50
. Yet, many phosphorylation sites and/or motifs have not been associated with a kinase and may be referred to as “orphan”41
. Indeed, during this proof-of-concept study, we found that one unpredicted peptide was phosphorylated by Cdc2/Cyclin B1 complex in a specific cellular context. Although a fraction of these sites may be phosphorylated in the context of the appropriate three-dimensional protein fold, most would be expected to be phosphorylated with a high degree of specificity owing to primary sequence determinants. We predict that the combination of activity profiles and protein correlation profiling will bridge the gap between large-scale phosphoproteomics efforts to characterize phosphorylation events and better insight into their biological context and function.
Although considerable challenges remain, the single-reaction KAYAK strategy has the potential to impact kinase-based therapies on many levels. KAYAK profiles might influence a lead compound decision at an early phase by revealing off-target effects or potency differences in highly related molecules or suggest a tractable biomarker of drug efficacy needed in clinical trials. Finally, single-reaction KAYAK profiles could enable physicians to tailor drug choice or dosing to address the aberrant signaling events underlying a patient’s particular pathology.