BACKGROUND
adaptation to changes in the environment is crucial for the survival of any organism and adjustment of protein levels and activities is pivotal for this process.
long-term adaptations via transcriptional regulation or irreversible protein modification typically affect protein abundance.
fast adaptations are mediated by allosteric regulators and a chemically diverse group of covalent post-translational modifications .
in eukaryotes, phosphorylation stands out among ptms for the broad scope of regulation and the complexity of its network with over  <dig> known kinases and phosphatases in saccharomyces cerevisiae  <cit> .
recent advances in proteomics allow for deep interrogation of phosphoproteomes, leading to large catalogues of phosphorylation sites  in different species , suggesting that as many as 45% of all proteins in eukaryotes may be phosphorylated  <cit> .
while the number of mapped phosphosites is ever increasing, we still rarely know whether phosphorylation events have a functional role.
a substantial fraction of all phosphosites was suggested to be non-functional, resulting from stochastic off-target kinase activity  <cit> .
typically, only a handful of all observed phosphosites in any organism has been functionally validated and characterized  <cit>  because the success in phosphoproteomics in detecting phosphosites has largely outpaced the performance and throughput of the available approaches for analysis of phosphosites functionality.
present workflows for functional analysis of phosphosites typically start with phosphoproteomics to generate catalogues of modified amino acids.
computational approaches then distinguish between relevant and non-functional phosphosites on the basis of different criteria including kinase recognition motives  <cit> , conservation patterns of phosphosites across species  <cit> , correlations of changes in phosphorylation state with metabolic flux  <cit> , or temporal patterns of phosphosite appearances upon disturbance  <cit> .
after prioritization of interesting phosphosites, their functionality remains to be determined in laborious follow-up experiments.
typically, these experiments take advantage of site specific phosphosite mutants and include a combination of phosphorylation assays, growth and viability assays, localization studies, expression studies and enzymatic in vitro assays .
targeted measurements of intracellular metabolites have been used in some cases to investigate phosphoregulation of metabolism  <cit> .
all these methods have been powerful in elucidating the importance of single phosphosites.
however, they are not suitable for high throughput analysis as the readout of most approaches is specific to the investigated protein and would imply tedious laboratory work to make them applicable in larger scale.
although growth and viability assays allow high throughput, their sensitivity for assessing functionality is unclear.
given that phosphorylation often only fine-tunes the activity of a protein, interfering with phosphorylation might not necessarily penetrate to the phenotypic level.
the recent developments in genome editing render the generation of large numbers of mutants with modified phosphosites for functional analysis feasible .
hence, the limitation shifts to a suitable high throughput approach for subsequent mutant analysis, in particular when phenotypes are more subtle, as can be expected for most point mutations.
in principle the various omics technologies would be applicable, but in practice they are typically too laborious or expensive.
a notable exception is mass spectrometry-based metabolomics.
in particular, it offers the potential to identify small differences in metabolite profiles well before a change of the phenotype is observed  <cit> .
since many of the phosphoproteins listed in phosphosite databases are part of the metabolic network, metabolomics is a promising approach for probing their functionality.
here we investigate the potential of untargeted metabolomics for higher throughput identification and characterization of phosphosite functionality.
we introduced pointmutations into  <dig> phosphosites from  <dig> proteins in s. cerevisiae, several of which were known to be functionally regulated by phosphorylation.
the proteins included metabolic enzymes and regulatory proteins implicated in the tor or hog signaling, two pathways closely related to metabolism .
the phosphoresidues were mutated to amino acids that mimic a constitutively dephosphorylated and, for a selected subset, phosphorylated state, resulting in a total of  <dig> phosphomutants.
applying untargeted metabolomics, we retrieved most previously characterized phosphoproteins as functionally relevant and suggest functionality for additional phosphosites.
we also showed that untargeted metabolomics aids in unveiling the biological roles of functional phosphosites in regulating metabolic pathways.
our results demonstrate that untargeted metabolomics is a highly sensitive method for high throughput detection of functionalities of phosphosites.
RESULTS
phenotypic analysis reveals functionalities of strong-impact phosphosites
for the purpose of defining a set of phosphomutants for phenotypic and metabolic analysis, we selected single phosphosites or multiple phosphosites in close proximity  on metabolic enzymes or proteins involved in tor or hog signaling.
the  <dig> chosen phosphosites were located on  <dig> proteins that represent different functional classes, such as metabolic enzymes, kinases or transcription factors .
the two signaling pathways were selected for their metabolic function, rendering them amenable for a metabolomics approach.
tor signaling controls the cellular response to nitrogen availability, having a substantial impact on the state of metabolism when metabolizing nitrogen sources of different quality  <cit> .
the main role of the hog signaling pathway is sensing of extracellular hyperosmolarity and eliciting a stress response, which in s. cerevisiae primarily consists of a metabolic adaptation to increase the concentration of the osmolyte glycerol together with cell cycle and transcriptional modulation  <cit> .
the total set of the  <dig> phosphosites contained  <dig> functionally characterized and  <dig> uncharacterized phosphosites.
we classified phosphosites as characterized when a phosphosite mutant has been reported to have an effect in any type of functional analysis.
most uncharacterized phosphosites were selected based on differential phosphorylation data from phosphoproteomics studies in either different growth conditions or upon kinase deletion  <cit>  .
the chosen phosphosites were mutated to alanine whenever serine or threonine was the phosphoresidue, or phenylalanine if tyrosine was the phosphoresidue, thereby abolishing phosphorylation of the target amino acid .
for six out of the  <dig> phosphosites the residues were additionally mutated to glutamic acid to mimic a constitutively phosphorylated state of the protein, resulting in a total of  <dig> mutants.
throughout this study, mutants with abolished phosphorylation are referred to as “out” mutants, while phosphomimic mutants are referred to as “in” mutants.
up to three independent mutant replicates per phosphosite were generated .
phosphomutant replicates with broad metabolic effects exhibited highly similar metabolic profiles , suggesting that genetic manipulations were precise.table  <dig> phosphoprotein mutant set
tf transcription factor, ch previously characterized phosphosite, dif phosphosite shown in previous phosphoproteomics studies to change in degree of phosphorylation in different conditions, htp phosphosite detected in high throughput phosphoproteomics studies, no data on differential phosphorylation
athe previously shown functionality of phosphorylation of pfk <dig> s <dig> is potentially a result of increased expression from the plasmid used in that study
to identify condition-specific regulation, the  <dig> phosphomutants were grown on four combinations of carbon and nitrogen sources that require different activities of the mutated proteins, such as glycolytic or gluconeogenic fluxes, nitrogen sources of different quality, and low osmotic stress on  <dig>  m sodium pyruvate .
we quantified growth rates and maximum cell density and qualitatively assessed lag times and other abnormalities in the growth curves of microtiter plate cultures .
six mutants in four different phosphosites exhibited an aberrant phenotype under at least one condition .
three of these phosphosites were already previously characterized, but functionality of the tip <dig> phosphosite was hitherto unknown.
the phosphomutants of pda <dig> and pbs <dig> showed varying effects under most conditions, whereby the in mutant was more strongly affected in both proteins.
the in mutant of the kinase pbs <dig> showed a very similar behavior as its target hog <dig> out, supporting the hypothesis that phosphorylation of pbs <dig> s <dig> inactivates the protein, while in hog <dig>  phosphorylation of s <dig> and t <dig> in the activation loop activates this kinase  <cit> .
overall, we retrieved three out of the  <dig> known phosphoregulated proteins under the tested conditions.
thus, growth phenotypes provide strong evidence for phosphosite functionality but would require testing of many conditions.table  <dig> carbon and nitrogen sources of media for growth and metabolomics experiments
glucose/nh4
+
+
pyruvate/nh4
+
+
fig.
<dig> growth phenotype of s. cerevisiae phosphomutants.
a example curves for analyzed growth traits of wild-type  and mutant .
b growth phenotype in different growth conditions.
results that differ significantly from the wild-type are highlighted.
red and green boxes indicate higher or lower results for the respective growth trait of the mutant compared to the wild-type.
black boxes indicate a general difference in shape.
mutants with a growth phenotype are highlighted in blue.
abbreviations: μ: growth rate.
cd: maximum cell density in one growth phase.
l: lag time.
s: abnormalities in shape of growth curve
untargeted metabolomics reveals changes in phenotypically silent phosphomutants
next, we tested whether metabolic functionality of phosphosites whose genetic perturbation does not propagate to physiological phenotypes could be identified by untargeted metabolomics.
intracellular metabolites were extracted with hot ethanol from exponentially growing cultures in the four before used conditions using 96-well cultivation.
the extracts were injected into a time of flight mass spectrometer  and metabolic profiles in the m/z range of  <dig> to  <dig> dalton were recorded  <cit> .
across all conditions, 122– <dig> ions were annotated to 183– <dig> metabolites using a genome-wide reconstruction model of s. cerevisiae  <cit>  .
the data was processed to remove intensity drifts during measurements and an od-specific fold change was calculated for every ion of each metabolic profile.
the fold changes of the replicates of one mutant were compared to the fold changes of all other samples using a 2-sample t-test and the median log <dig> fold change over all replicates was determined.
ions with a corrected p-value < 10− <dig> and a |log <dig> fold change| >  <dig>  were considered as changing significantly.
the selected log <dig> fold change cutoff corresponds to a 30% change and represents the  <dig> % quantile across the fold changes of all ions from the wild-type dataset .
typically, metabolite concentrations changed less than two-fold in the phosphomutants  and the majority of changes were in arginine, proline, pyrimidine, purine or lysine metabolism .
while 31% of the mutants exhibited no significant  metabolic response, most exhibited changes in some metabolites, and five of the six mutants with physiological phenotypes  showed changes in more than  <dig> metabolites in at least one condition .
in many cases these broadly changing metabolic profiles were presumably the consequence of altered growth rates .
generally, phosphomutants with previously characterized phosphosites featured more changes than the unknown subset, suggesting that the latter contains more nonfunctional sites .
metabolic profiles of phosphosites with strong metabolic impact often showed a tendency towards condition-specific effects, demonstrating the regulatory role of phosphosites under particular conditions.
hog <dig> out for example showed broad effects only when grown on pyruvate as the carbon source, presumably because of the elevated osmotic stress in this condition, and tip <dig> showed almost exclusively effects when growing on proline as sole nitrogen source .
overall, our data suggest that metabolomics can detect subtle consequences of deregulated phosphorylation that is without detectable growth phenotypes.fig.
<dig> overview on changing ions in metabolic profiles.
a number of changing ions of each mutant and condition.
only mutants with significantly changing ions are depicted .
b relation between number of changing ions and mutant growth rate.
c distribution of changing ions in different mutant subclasses with no , little , intermediate  or many  changing ions
metabolic profile analysis indicates phosphosite functionality
while we did not detect changes in direct reactants of the  <dig> phospho-mutated enzymes after applying a strict cutoff, we found metabolite changes for  <dig> mutants to be enriched in specific pathways, suggesting phosphorylation to be functionally important for these pathways .
for example, the out mutant of yap <dig>  a transcription factor implicated in stress responses, showed changes enriched in purine metabolism when growing on glucose/glutamine .
metabolic effects of the pda <dig> phosphomutants predominantly affected arginine and proline metabolism .
a particularly clear case was lys <dig>  one of two homocitrate synthase isoenzymes that catalyze the conversion of oxoglutarate to homocitrate, the first step in lysine biosynthesis.
lysine metabolism was found to be exclusively enriched  in lys <dig> out when growing on glucose/proline.
while lysine biosynthesis is known to be subject to transcriptional and allosteric control, it was recently shown that lys <dig> activity is also regulated by phosphorylation  <cit> .
metabolite responses in lys <dig> out were observed only during growth on the poor nitrogen source proline and significant  changes were restricted to the final pathway product lysine and its precursor saccharopine .
consistent with previous results  <cit> , increased lysine levels in lys <dig> out suggest phosphorylation at position s395/t <dig> to deactivate the enzyme.
since lysine biosynthesis flux is rather low during slow growth on proline, our data indicates that the pathway capacity is down-regulated by phosphorylation in this condition.fig.
<dig> metabolite level changes in lysine biosynthesis of the lys <dig> out mutant growing on glucose/proline.
coloring indicates the strength and direction of the fold changes.
significant fold changes are highlighted with a bold frame .
metabolites in grey were not detected
to identify more global impact of phosphosites on metabolism, we used two correlation approaches to analyze the metabolic profiles.
in the first, we correlated log <dig> fold changes between all  <dig> possible mutant-pairs for each condition.
the correlation coefficients of all mutant-pairs showed a normal distribution around  <dig>  with a mean of  <dig>  .
the four correlation coefficients from each mutant-pair were compared against the distribution of all derived coefficients to identify significantly correlation pairs.
five mutant-pairs correlated significantly across the four conditions , suggesting these phosphosites to exhibit similar effects on metabolism .
two of these five pairs were mutant variants of the same protein , emphasizing similar functionality of the introduced mutations.
rim <dig> out, implicated in cell cycle regulation in response to nutrient, and pda <dig> in were negatively correlated in all conditions, whereby the strongest opposing effects were largely in amino acid metabolism.
the two other cases were pfk <dig> out associating with ste <dig> out as well as yap <dig> out.
in the second correlation, we quantified the similarity of each mutant with itself across all four conditions to identify phosphosite functionalities based on subtle but consistent changes in metabolites across conditions.
the correlation coefficients of all condition-pairs showed an average above zero, emphasizing that our phosphomutant set contains mutants with condition-independent metabolic effects .
out of the eight phosphomutants that were recovered in this analysis, we already described strong metabolic changes predominantly under one condition for three mutants .
the calculated correlation coefficients were particularly weak for hog <dig> out and pbs <dig> in.
we suspect that despite the predominant effects in specific conditions, this weak similarity is driven by underlying subtle, but condition-independent, metabolic changes.
one such example in pbs <dig> in is the slight depletion of glycerol across conditions, emphasizing the role of pbs <dig> in controlling glycerol accumulation via the hog pathway even under non-stress conditions  <cit> .
additionally, the gpd <dig> out, gut <dig> out, sko <dig> out and lys <dig> out mutants associated with themselves across conditions.
these mutants are cases without strong effects under any tested condition, but with subtle condition-independent changes, which suggest a possible fine-tuning function of the phosphosites.
the respective metabolites driving condition-independent correlations were diverse, e.g.
affecting nucleotide metabolism in gut <dig> out and lysine in lys <dig> out.
as expected, we also recovered pda <dig> in that has effects throughout all conditions.
to systematically interpret the evidence for functionality of a given mutated phosphosite, we ranked the functionality evidence based on a score that reflects the number of changing ions, enriched pathways, correlation with other mutants, and correlation with itself for each phosphosite .
the number of changing ions was scored between  <dig> and  <dig>  the changes were classified as either little , affecting equal or less ions than in the wild-type, where  <dig>  ions were changing on average, intermediate , or many  changes, affecting a broad part of metabolism with changes in > 10% of all averagely detected ions.
the growth condition with the most changes was considered for scoring.
the three other aspects were scored between  <dig> and  <dig> according to p-values.
a score was attributed based on the most significant p-value obtained in any growth condition.
a p-value of at least 10− <dig> was scored with a  <dig>  while a p-value below 10− <dig> was scored with a  <dig>  supporting our approach, most of the already characterized phosphosites scored very high .
the three characterized cases with low scores can be explained by incomplete mutation of all relevant phosphosites  or lack of protein relevance under the tested condition   <cit> .
our data suggests that the reported phosphoregulation of pfk <dig> at position s <dig>  <cit>  resulted, at least in part, from a non-endogenous increase of pfk <dig> levels from the employed plasmid construct .
since we cannot exclude that the reported phenotype of this pfk <dig> variant originated from increased protein abundance, we did not consider pfk <dig> s <dig> as a known functional phosphosite and found only weak functionality evidence in the pfk <dig> in mutant.
overall, the metabolomics data identified most of the already characterized phosphosites as functional with a total score of  <dig> or higher, one novel regulatory phosphosite with high confidence , and several other novel functional phosphosites with lower confidence.
the much higher number of phosphosite functionality identification by metabolomics compared to growth phenotype analysis underlines the value of metabolomics in assessing at least metabolic functionality.
in the next sections, we exploit the metabolomics data to hypothesize specific functions for phosphosites in the glycerol dehydrogenases gpd <dig> and gpd <dig>  and the mak kinase pbs <dig> fig.
<dig> phosphomutant functionality scoring based on metabolic profiles.
the number of changing ions was scored between  <dig> and  <dig> according to the following scheme:  <dig> for no change;  <dig> for 1– <dig> changes;  <dig> for 5– <dig> changes;  <dig> for >  <dig> changes.
the remaining three criteria were scored between  <dig> and  <dig> based to on the strongest p-value:  <dig> for p-value > 10−2;  <dig> for p-value < 10−2;  <dig> for p-value < 10− <dig>  mutants exhibiting a growth phenotype in the growth analysis are highlighted in blue
phosphorylation of pbs <dig> at s <dig> negatively regulates hog <dig> activity
phosphorylation at position s <dig> and t <dig> activates the mapk kinase pbs <dig>  which in turn activates the hog <dig> kinase in response to osmotic stress  <cit>  .
besides these two well-studies phosphosites, the here investigated s <dig> phosphosite was suggested to be important for the inactivation of pbs <dig> after adaptation to osmotic stress  <cit> .
our approach scored both pbs <dig> in and its target hog <dig> out as regulated by phosphorylation .
the strongest metabolic responses of both mutants occurred during growth on pyruvate, presumably due to the osmotic stress of increased sodium concentration in this medium.
the striking similarity of the hog <dig> out and pbs <dig> in profiles and the very different pbs <dig> out profiles strongly support the hypothesis that phosphorylation at position s <dig> deactivates pbs <dig>  which causes reduced hog <dig> activity .fig.
<dig> metabolic profiles, hog signaling activation and phenotype of pbs <dig> phosphomutants.
a log <dig> fold changes of all ions corresponding to deprotonated metabolites of hog out compared to pbs <dig> in in pyruvate/nh4
+ media.
b log <dig> fold changes of all ions corresponding to deprotonated metabolites of hog out compared to pbs <dig> out in pyruvate/nh4
+ media.
c time course western blot of hog <dig> activity in wild-type and pbs <dig> phosphomutants, measured as phosphorylated hog <dig>  upon osmotic stress.
total hog <dig>  was measured as control.
d phenotype analysis upon osmotic stress and exposure to different temperatures.
spotting assay of pbs <dig> phosphomutants and hog <dig> out in synthetic complete -glucose, sc-glucose supplemented with  <dig>  m nacl, sc-glucose at 42 °c and sc-glucose at 16 °c.
the assay was done three times, the results of one exemplary experiment are shown
we further investigated the presumably inactivating role of s <dig> by exposing the pbs <dig> mutants to elevated concentrations of nacl and monitored hog pathway activity by following hog <dig> phosphorylation over time .
consistent with the above reasoning, pbs <dig> in failed to phosphorylate and activate hog <dig> upon osmotic stress.
however, no delay in inactivation of hog <dig> was observed for pbs <dig> out compared to wild-type dynamics, suggesting that deactivation of pbs <dig> was not impaired in the pbs <dig> out mutant.
since the hog signaling pathway is known to cross-talk with other signaling pathways, we wondered whether phosphorylation of s <dig> had other physiological roles besides the deactivation of pbs <dig> after adaptation to osmotic stress.
one such interconnected pathway is the cell wall integrity pathway  <cit> .
to test a link of pbs <dig> s <dig> phosphorylation to cell wall integrity, we grew the mutants at different temperatures to impose different cell wall stress levels.
we found that growth of pbs <dig> out was exclusively impaired at elevated temperatures, while pbs <dig> in and hog <dig> out showed growth defect most strongly when growing at 16 °c .
a change in cell wall stress leading to conditional phenotypes of the pbs <dig> phosphomutants suggests that phosphorylation of s <dig> is important for proper activation of hog signaling under specific cell wall stress conditions and plays a critical role in the cross-talk between the two signaling pathways.
phosphorylation of gpd <dig> and gpd <dig> influences arginine metabolism
the glycerol dehydrogenases gpd <dig> and gpd <dig> catalyze the conversion of dihydroxyacetone phosphate to glycerol-3-phosphate, the first step of glycerol synthesis.
although gpd <dig> and gpd <dig> have distinct roles, they can at least partially compensate for each other  <cit> .
in addition to transcriptional regulation, both enzymes are subject to phosphoregulation  <cit> .
in our screen, both glycerol dehydrogenases were detected as regulated phosphoproteins, with gpd <dig> out ranking slightly higher in the metabolic profile analysis than gpd <dig> out .
although we did not detect changes in the direct reactants of either of the mutated isoenzymes, both out mutants exhibited specific metabolic responses throughout most conditions with an enrichment in arginine and proline metabolism , in particular when growing on proline as nitrogen source .
unexpectedly, we detected the strongest changes in citrulline and ornithine, along with some less pronounced changes in arginine that we confirmed by targeted lc-ms/ms measurements .
the striking correlation of gpd <dig> out with itself across all conditions demonstrates that this phosphoregulation is not specific to the proline condition .fig.
<dig> metabolite responses in arginine biosynthesis of the gpd <dig> out and gpd <dig> out mutants.
a metabolite response in arginine biosynthesis under all tested conditions.
coloring indicates strength and direction of fold changes.
metabolites in grey were not detected in the respective conditions.
metabolites without color boxes were not detected.
b quantification of arginine and citrulline levels by lc-ms/ms for gpd <dig> out and wild-type in glucose/nh4
+ medium.
c possible metabolic routes for g3p
how glycerol metabolism influences arginine metabolism remains unclear.
the out mutants have more active glycerol dehydrogenases and hence generate more g3p than the wild-type  <cit> , but neither of the known routes from g3p connects to arginine metabolism .
although the observed metabolite changes suggest a possible blockage at arg <dig> or arg <dig> in the glycerol dehydrogenases phosphomutants , the influence is more likely to occur indirectly via a regulatory interaction.
previously, transcriptional and translational regulation of arginine metabolism has been suggested to be implicated in adaptation to osmotic stress  <cit> .
in our study, pbs <dig> in and hog <dig> out, which have impaired hog signaling and cannot activate gpd <dig> by dephosphorylation, showed strong changes mostly in arginine but also citrulline upon osmotic stress .
these metabolites change in opposite directions compared to the gdp <dig> out and gpd <dig> out mutants where arginine is decreased while citrulline is increased, supporting a phosphorylation-dependent influence of the glycerol dehydrogenases on arginine metabolism.
discussion
the large networks of kinases and phosphatases represent possibly the most complex post-translational regulation system and play key roles in essentially all cellular processes.
given the rapidly growing collection of reported phosphosites, a major limitation is assessing phosphosite functionality and generating hypotheses on their precise roles, at a scale and speed that matches the high-throughput of modern mapping methods  <cit> .
here we demonstrate that untargeted metabolomics of point mutants with phospho in or out modifications has the potential to address this functionality problem.
from phosphosite mutants of  <dig> proteins involved in metabolism, tor and hog signaling as a proof-of-concept, we were able to recover  <dig> of the  <dig> positive controls by metabolomics, compared to only three when relying on quantitative growth assays.
importantly, metabolomics was able to detect also more subtle metabolic effects that were phenotypically silent.
the other three known phosphoproteins were presumably not detected because we did not mutate all relevant phosphosites  or did not chose the appropriate conditions that required activity of these proteins   <cit> .
of the remaining  <dig> so far uncharacterized phosphosites, metabolomics provides evidence that those on cys <dig>  gln <dig>  gut <dig>  nbp <dig>  pfk <dig>  tco <dig> and tip <dig> are generally functional.
at least gln <dig> and tip <dig> had been suspected to be regulated by phosphorylation, although no phosphosites were identified so far .
the strongest evidence in our data was for s <dig> on tip <dig>  an essential regulatory protein in tor signaling whose role upon activation of tor is not yet clearly established  <cit> .
the exclusively strong effect of tip <dig> out during growth on a poor nitrogen source suggests that s <dig> is phosphorylated when the tor complex is inactive and becomes dephosphorylated upon activation of tor.
since our recovery of positive controls was fairly good, we believe that the newly tested phosphosites for which we could not obtain functionality evidence are indeed mostly without functional relevance.
the employed, metabolomics-based scoring system allowed classification of functionality, but does not directly identify the regulatory consequences of phosphorylation.
nevertheless, in several cases the metabolite profiles provided starting hypotheses for the specific biological function without prior knowledge.
for instance accumulation of pathway end products suggested that phosphorylation of lys <dig>  the first enzyme in lysine biosynthesis, specifically regulates lysine pathway flux.
the correlation of pbs <dig> in and hog <dig> out metabolite profiles strongly suggested that the two phosphorylation events have a similar function in mediating hog signal flow.
however, changes in metabolism were not always intuitive and possibly not direct consequences of the mutated phosphosites, as observed in the gpd <dig> and gpd <dig> mutants.
the main benefit of the metabolomics data in the process of understanding phosphosite functionality is to point out relevant cases efficiently, which can then be investigated in more depth by other methods or in combination with proteomics and transcriptomics data.
even though about half of the tested phosphosites appeared to be functional, most phosphomutants had no detectable growth phenotype and exhibited relatively modest metabolic changes, both in number and magnitude.
this apparent resilience to point mutations does not come entirely unexpected given that the much more drastic intervention of  <dig> kinase and phosphatase deletions in s. cerevisiae resulted in few detectable growth phenotypes where only 26% of the mutants exhibited a significant transcriptional response and about 40% had no detectable metabolite response  <cit> .
several reasons are likely to contribute to this robustness, including the large overlap and redundancy both in kinase targets and phosphosites on a given protein .
additionally, adaptation to a new steady state in our experiments will mask many direct regulatory effects and our population-based measurements will have missed, for example, cell cycle specific regulation events.
CONCLUSIONS
in this study, we investigated the potential of untargeted metabolomics along with growth analysis to identify functionality of phosphosites in a higher throughput.
while growth analysis was suitable to detect functionality of a small number of phosphosites, metabolomics allowed recovering most of the previously characterized sites, along with some novel functional phosphosites and aided in hypothesis generation on biological roles of functionally relevant sites.
our results demonstrate the potential of untargeted metabolomics to experimentally identify functionality and conditional relevance of post-translational modifications at high throughput to complement current computational approaches .

 adaptation to changes in the environment is crucial for the survival of any organism and adjustment of protein levels and activities is pivotal for this process. long-term adaptations via transcriptional regulation or irreversible protein modification typically affect protein abundance. fast adaptations are mediated by allosteric regulators and a chemically diverse group of covalent post-translational modifications. in eukaryotes, phosphorylation stands out among ptms for the broad scope of regulation and the complexity of its network with over known kinases and phosphatases in saccharomyces cerevisiae. recent advances in proteomics allow for deep interrogation of phosphoproteomes, leading to large catalogues of phosphorylation sites in different species, suggesting that as many as 45% of all proteins in eukaryotes may be phosphorylated. while the number of mapped phosphosites is ever increasing, we still rarely know whether phosphorylation events have a functional role. a substantial fraction of all phosphosites was suggested to be non-functional, resulting from stochastic off-target kinase activity. typically, only a handful of all observed phosphosites in any organism has been functionally validated and characterized because the success in phosphoproteomics in detecting phosphosites has largely outpaced the performance and throughput of the available approaches for analysis of phosphosites functionality. present workflows for functional analysis of phosphosites typically start with phosphoproteomics to generate catalogues of modified amino acids. computational approaches then distinguish between relevant and non-functional phosphosites on the basis of different criteria including kinase recognition motives, conservation patterns of phosphosites across species, correlations of changes in phosphorylation state with metabolic flux, or temporal patterns of phosphosite appearances upon disturbance. after prioritization of interesting phosphosites, their functionality remains to be determined in laborious follow-up experiments. typically, these experiments take advantage of site specific phosphosite mutants and include a combination of phosphorylation assays, growth and viability assays, localization studies, expression studies and enzymatic in vitro assays. targeted measurements of intracellular metabolites have been used in some cases to investigate phosphoregulation of metabolism. all these methods have been powerful in elucidating the importance of single phosphosites. however, they are not suitable for high throughput analysis as the readout of most approaches is specific to the investigated protein and would imply tedious laboratory work to make them applicable in larger scale. although growth and viability assays allow high throughput, their sensitivity for assessing functionality is unclear. given that phosphorylation often only fine-tunes the activity of a protein, interfering with phosphorylation might not necessarily penetrate to the phenotypic level. the recent developments in genome editing render the generation of large numbers of mutants with modified phosphosites for functional analysis feasible. hence, the limitation shifts to a suitable high throughput approach for subsequent mutant analysis, in particular when phenotypes are more subtle, as can be expected for most point mutations. in principle the various omics technologies would be applicable, but in practice they are typically too laborious or expensive. a notable exception is mass spectrometry-based metabolomics. in particular, it offers the potential to identify small differences in metabolite profiles well before a change of the phenotype is observed. since many of the phosphoproteins listed in phosphosite databases are part of the metabolic network, metabolomics is a promising approach for probing their functionality. here we investigate the potential of untargeted metabolomics for higher throughput identification and characterization of phosphosite functionality. we introduced pointmutations into phosphosites from proteins in s. cerevisiae, several of which were known to be functionally regulated by phosphorylation. the proteins included metabolic enzymes and regulatory proteins implicated in the tor or hog signaling, two pathways closely related to metabolism. the phosphoresidues were mutated to amino acids that mimic a constitutively dephosphorylated and, for a selected subset, phosphorylated state, resulting in a total of phosphomutants. applying untargeted metabolomics, we retrieved most previously characterized phosphoproteins as functionally relevant and suggest functionality for additional phosphosites. we also showed that untargeted metabolomics aids in unveiling the biological roles of functional phosphosites in regulating metabolic pathways. our results demonstrate that untargeted metabolomics is a highly sensitive method for high throughput detection of functionalities of phosphosites.  phenotypic analysis reveals functionalities of strong-impact phosphosites for the purpose of defining a set of phosphomutants for phenotypic and metabolic analysis, we selected single phosphosites or multiple phosphosites in close proximity on metabolic enzymes or proteins involved in tor or hog signaling. the chosen phosphosites were located on proteins that represent different functional classes, such as metabolic enzymes, kinases or transcription factors. the two signaling pathways were selected for their metabolic function, rendering them amenable for a metabolomics approach. tor signaling controls the cellular response to nitrogen availability, having a substantial impact on the state of metabolism when metabolizing nitrogen sources of different quality. the main role of the hog signaling pathway is sensing of extracellular hyperosmolarity and eliciting a stress response, which in s. cerevisiae primarily consists of a metabolic adaptation to increase the concentration of the osmolyte glycerol together with cell cycle and transcriptional modulation. the total set of the phosphosites contained functionally characterized and uncharacterized phosphosites. we classified phosphosites as characterized when a phosphosite mutant has been reported to have an effect in any type of functional analysis. most uncharacterized phosphosites were selected based on differential phosphorylation data from phosphoproteomics studies in either different growth conditions or upon kinase deletion. the chosen phosphosites were mutated to alanine whenever serine or threonine was the phosphoresidue, or phenylalanine if tyrosine was the phosphoresidue, thereby abolishing phosphorylation of the target amino acid. for six out of the phosphosites the residues were additionally mutated to glutamic acid to mimic a constitutively phosphorylated state of the protein, resulting in a total of mutants. throughout this study, mutants with abolished phosphorylation are referred to as “out” mutants, while phosphomimic mutants are referred to as “in” mutants. up to three independent mutant replicates per phosphosite were generated. phosphomutant replicates with broad metabolic effects exhibited highly similar metabolic profiles, suggesting that genetic manipulations were precise.table phosphoprotein mutant set tf transcription factor, ch previously characterized phosphosite, dif phosphosite shown in previous phosphoproteomics studies to change in degree of phosphorylation in different conditions, htp phosphosite detected in high throughput phosphoproteomics studies, no data on differential phosphorylation athe previously shown functionality of phosphorylation of pfk s is potentially a result of increased expression from the plasmid used in that study to identify condition-specific regulation, the phosphomutants were grown on four combinations of carbon and nitrogen sources that require different activities of the mutated proteins, such as glycolytic or gluconeogenic fluxes, nitrogen sources of different quality, and low osmotic stress on  m sodium pyruvate. we quantified growth rates and maximum cell density and qualitatively assessed lag times and other abnormalities in the growth curves of microtiter plate cultures. six mutants in four different phosphosites exhibited an aberrant phenotype under at least one condition. three of these phosphosites were already previously characterized, but functionality of the tip phosphosite was hitherto unknown. the phosphomutants of pda and pbs showed varying effects under most conditions, whereby the in mutant was more strongly affected in both proteins. the in mutant of the kinase pbs showed a very similar behavior as its target hog out, supporting the hypothesis that phosphorylation of pbs s inactivates the protein, while in hog phosphorylation of s and t in the activation loop activates this kinase. overall, we retrieved three out of the known phosphoregulated proteins under the tested conditions. thus, growth phenotypes provide strong evidence for phosphosite functionality but would require testing of many conditions.table carbon and nitrogen sources of media for growth and metabolomics experiments glucose/nh4 + + pyruvate/nh4 + + fig. growth phenotype of s. cerevisiae phosphomutants. a example curves for analyzed growth traits of wild-type and mutant. b growth phenotype in different growth conditions. results that differ significantly from the wild-type are highlighted. red and green boxes indicate higher or lower results for the respective growth trait of the mutant compared to the wild-type. black boxes indicate a general difference in shape. mutants with a growth phenotype are highlighted in blue. abbreviations: μ: growth rate. cd: maximum cell density in one growth phase. l: lag time. s: abnormalities in shape of growth curve untargeted metabolomics reveals changes in phenotypically silent phosphomutants next, we tested whether metabolic functionality of phosphosites whose genetic perturbation does not propagate to physiological phenotypes could be identified by untargeted metabolomics. intracellular metabolites were extracted with hot ethanol from exponentially growing cultures in the four before used conditions using 96-well cultivation. the extracts were injected into a time of flight mass spectrometer and metabolic profiles in the m/z range of to dalton were recorded. across all conditions, 122– ions were annotated to 183– metabolites using a genome-wide reconstruction model of s. cerevisiae. the data was processed to remove intensity drifts during measurements and an od-specific fold change was calculated for every ion of each metabolic profile. the fold changes of the replicates of one mutant were compared to the fold changes of all other samples using a 2-sample t-test and the median log fold change over all replicates was determined. ions with a corrected p-value < 10− and a |log fold change| >  were considered as changing significantly. the selected log fold change cutoff corresponds to a 30% change and represents the % quantile across the fold changes of all ions from the wild-type dataset. typically, metabolite concentrations changed less than two-fold in the phosphomutants and the majority of changes were in arginine, proline, pyrimidine, purine or lysine metabolism. while 31% of the mutants exhibited no significant metabolic response, most exhibited changes in some metabolites, and five of the six mutants with physiological phenotypes showed changes in more than metabolites in at least one condition. in many cases these broadly changing metabolic profiles were presumably the consequence of altered growth rates. generally, phosphomutants with previously characterized phosphosites featured more changes than the unknown subset, suggesting that the latter contains more nonfunctional sites. metabolic profiles of phosphosites with strong metabolic impact often showed a tendency towards condition-specific effects, demonstrating the regulatory role of phosphosites under particular conditions. hog out for example showed broad effects only when grown on pyruvate as the carbon source, presumably because of the elevated osmotic stress in this condition, and tip showed almost exclusively effects when growing on proline as sole nitrogen source. overall, our data suggest that metabolomics can detect subtle consequences of deregulated phosphorylation that is without detectable growth phenotypes.fig. overview on changing ions in metabolic profiles. a number of changing ions of each mutant and condition. only mutants with significantly changing ions are depicted. b relation between number of changing ions and mutant growth rate. c distribution of changing ions in different mutant subclasses with no, little, intermediate or many changing ions metabolic profile analysis indicates phosphosite functionality while we did not detect changes in direct reactants of the phospho-mutated enzymes after applying a strict cutoff, we found metabolite changes for mutants to be enriched in specific pathways, suggesting phosphorylation to be functionally important for these pathways. for example, the out mutant of yap a transcription factor implicated in stress responses, showed changes enriched in purine metabolism when growing on glucose/glutamine. metabolic effects of the pda phosphomutants predominantly affected arginine and proline metabolism. a particularly clear case was lys one of two homocitrate synthase isoenzymes that catalyze the conversion of oxoglutarate to homocitrate, the first step in lysine biosynthesis. lysine metabolism was found to be exclusively enriched in lys out when growing on glucose/proline. while lysine biosynthesis is known to be subject to transcriptional and allosteric control, it was recently shown that lys activity is also regulated by phosphorylation. metabolite responses in lys out were observed only during growth on the poor nitrogen source proline and significant changes were restricted to the final pathway product lysine and its precursor saccharopine. consistent with previous results, increased lysine levels in lys out suggest phosphorylation at position s395/t to deactivate the enzyme. since lysine biosynthesis flux is rather low during slow growth on proline, our data indicates that the pathway capacity is down-regulated by phosphorylation in this condition.fig. metabolite level changes in lysine biosynthesis of the lys out mutant growing on glucose/proline. coloring indicates the strength and direction of the fold changes. significant fold changes are highlighted with a bold frame. metabolites in grey were not detected to identify more global impact of phosphosites on metabolism, we used two correlation approaches to analyze the metabolic profiles. in the first, we correlated log fold changes between all possible mutant-pairs for each condition. the correlation coefficients of all mutant-pairs showed a normal distribution around with a mean of. the four correlation coefficients from each mutant-pair were compared against the distribution of all derived coefficients to identify significantly correlation pairs. five mutant-pairs correlated significantly across the four conditions, suggesting these phosphosites to exhibit similar effects on metabolism. two of these five pairs were mutant variants of the same protein, emphasizing similar functionality of the introduced mutations. rim out, implicated in cell cycle regulation in response to nutrient, and pda in were negatively correlated in all conditions, whereby the strongest opposing effects were largely in amino acid metabolism. the two other cases were pfk out associating with ste out as well as yap out. in the second correlation, we quantified the similarity of each mutant with itself across all four conditions to identify phosphosite functionalities based on subtle but consistent changes in metabolites across conditions. the correlation coefficients of all condition-pairs showed an average above zero, emphasizing that our phosphomutant set contains mutants with condition-independent metabolic effects. out of the eight phosphomutants that were recovered in this analysis, we already described strong metabolic changes predominantly under one condition for three mutants. the calculated correlation coefficients were particularly weak for hog out and pbs in. we suspect that despite the predominant effects in specific conditions, this weak similarity is driven by underlying subtle, but condition-independent, metabolic changes. one such example in pbs in is the slight depletion of glycerol across conditions, emphasizing the role of pbs in controlling glycerol accumulation via the hog pathway even under non-stress conditions. additionally, the gpd out, gut out, sko out and lys out mutants associated with themselves across conditions. these mutants are cases without strong effects under any tested condition, but with subtle condition-independent changes, which suggest a possible fine-tuning function of the phosphosites. the respective metabolites driving condition-independent correlations were diverse, e.g. affecting nucleotide metabolism in gut out and lysine in lys out. as expected, we also recovered pda in that has effects throughout all conditions. to systematically interpret the evidence for functionality of a given mutated phosphosite, we ranked the functionality evidence based on a score that reflects the number of changing ions, enriched pathways, correlation with other mutants, and correlation with itself for each phosphosite. the number of changing ions was scored between and the changes were classified as either little, affecting equal or less ions than in the wild-type, where ions were changing on average, intermediate, or many changes, affecting a broad part of metabolism with changes in > 10% of all averagely detected ions. the growth condition with the most changes was considered for scoring. the three other aspects were scored between and according to p-values. a score was attributed based on the most significant p-value obtained in any growth condition. a p-value of at least 10− was scored with a while a p-value below 10− was scored with a supporting our approach, most of the already characterized phosphosites scored very high. the three characterized cases with low scores can be explained by incomplete mutation of all relevant phosphosites or lack of protein relevance under the tested condition. our data suggests that the reported phosphoregulation of pfk at position s resulted, at least in part, from a non-endogenous increase of pfk levels from the employed plasmid construct. since we cannot exclude that the reported phenotype of this pfk variant originated from increased protein abundance, we did not consider pfk s as a known functional phosphosite and found only weak functionality evidence in the pfk in mutant. overall, the metabolomics data identified most of the already characterized phosphosites as functional with a total score of or higher, one novel regulatory phosphosite with high confidence, and several other novel functional phosphosites with lower confidence. the much higher number of phosphosite functionality identification by metabolomics compared to growth phenotype analysis underlines the value of metabolomics in assessing at least metabolic functionality. in the next sections, we exploit the metabolomics data to hypothesize specific functions for phosphosites in the glycerol dehydrogenases gpd and gpd and the mak kinase pbs fig. phosphomutant functionality scoring based on metabolic profiles. the number of changing ions was scored between and according to the following scheme: for no change; for 1– changes; for 5– changes; for >  changes. the remaining three criteria were scored between and based to on the strongest p-value: for p-value > 10−2; for p-value < 10−2; for p-value < 10− mutants exhibiting a growth phenotype in the growth analysis are highlighted in blue phosphorylation of pbs at s negatively regulates hog activity phosphorylation at position s and t activates the mapk kinase pbs which in turn activates the hog kinase in response to osmotic stress. besides these two well-studies phosphosites, the here investigated s phosphosite was suggested to be important for the inactivation of pbs after adaptation to osmotic stress. our approach scored both pbs in and its target hog out as regulated by phosphorylation. the strongest metabolic responses of both mutants occurred during growth on pyruvate, presumably due to the osmotic stress of increased sodium concentration in this medium. the striking similarity of the hog out and pbs in profiles and the very different pbs out profiles strongly support the hypothesis that phosphorylation at position s deactivates pbs which causes reduced hog activity.fig. metabolic profiles, hog signaling activation and phenotype of pbs phosphomutants. a log fold changes of all ions corresponding to deprotonated metabolites of hog out compared to pbs in in pyruvate/nh4 + media. b log fold changes of all ions corresponding to deprotonated metabolites of hog out compared to pbs out in pyruvate/nh4 + media. c time course western blot of hog activity in wild-type and pbs phosphomutants, measured as phosphorylated hog upon osmotic stress. total hog was measured as control. d phenotype analysis upon osmotic stress and exposure to different temperatures. spotting assay of pbs phosphomutants and hog out in synthetic complete -glucose, sc-glucose supplemented with  m nacl, sc-glucose at 42 °c and sc-glucose at 16 °c. the assay was done three times, the results of one exemplary experiment are shown we further investigated the presumably inactivating role of s by exposing the pbs mutants to elevated concentrations of nacl and monitored hog pathway activity by following hog phosphorylation over time. consistent with the above reasoning, pbs in failed to phosphorylate and activate hog upon osmotic stress. however, no delay in inactivation of hog was observed for pbs out compared to wild-type dynamics, suggesting that deactivation of pbs was not impaired in the pbs out mutant. since the hog signaling pathway is known to cross-talk with other signaling pathways, we wondered whether phosphorylation of s had other physiological roles besides the deactivation of pbs after adaptation to osmotic stress. one such interconnected pathway is the cell wall integrity pathway. to test a link of pbs s phosphorylation to cell wall integrity, we grew the mutants at different temperatures to impose different cell wall stress levels. we found that growth of pbs out was exclusively impaired at elevated temperatures, while pbs in and hog out showed growth defect most strongly when growing at 16 °c. a change in cell wall stress leading to conditional phenotypes of the pbs phosphomutants suggests that phosphorylation of s is important for proper activation of hog signaling under specific cell wall stress conditions and plays a critical role in the cross-talk between the two signaling pathways. phosphorylation of gpd and gpd influences arginine metabolism the glycerol dehydrogenases gpd and gpd catalyze the conversion of dihydroxyacetone phosphate to glycerol-3-phosphate, the first step of glycerol synthesis. although gpd and gpd have distinct roles, they can at least partially compensate for each other. in addition to transcriptional regulation, both enzymes are subject to phosphoregulation. in our screen, both glycerol dehydrogenases were detected as regulated phosphoproteins, with gpd out ranking slightly higher in the metabolic profile analysis than gpd out. although we did not detect changes in the direct reactants of either of the mutated isoenzymes, both out mutants exhibited specific metabolic responses throughout most conditions with an enrichment in arginine and proline metabolism, in particular when growing on proline as nitrogen source. unexpectedly, we detected the strongest changes in citrulline and ornithine, along with some less pronounced changes in arginine that we confirmed by targeted lc-ms/ms measurements. the striking correlation of gpd out with itself across all conditions demonstrates that this phosphoregulation is not specific to the proline condition.fig. metabolite responses in arginine biosynthesis of the gpd out and gpd out mutants. a metabolite response in arginine biosynthesis under all tested conditions. coloring indicates strength and direction of fold changes. metabolites in grey were not detected in the respective conditions. metabolites without color boxes were not detected. b quantification of arginine and citrulline levels by lc-ms/ms for gpd out and wild-type in glucose/nh4 + medium. c possible metabolic routes for g3p how glycerol metabolism influences arginine metabolism remains unclear. the out mutants have more active glycerol dehydrogenases and hence generate more g3p than the wild-type, but neither of the known routes from g3p connects to arginine metabolism. although the observed metabolite changes suggest a possible blockage at arg or arg in the glycerol dehydrogenases phosphomutants, the influence is more likely to occur indirectly via a regulatory interaction. previously, transcriptional and translational regulation of arginine metabolism has been suggested to be implicated in adaptation to osmotic stress. in our study, pbs in and hog out, which have impaired hog signaling and cannot activate gpd by dephosphorylation, showed strong changes mostly in arginine but also citrulline upon osmotic stress. these metabolites change in opposite directions compared to the gdp out and gpd out mutants where arginine is decreased while citrulline is increased, supporting a phosphorylation-dependent influence of the glycerol dehydrogenases on arginine metabolism. discussion the large networks of kinases and phosphatases represent possibly the most complex post-translational regulation system and play key roles in essentially all cellular processes. given the rapidly growing collection of reported phosphosites, a major limitation is assessing phosphosite functionality and generating hypotheses on their precise roles, at a scale and speed that matches the high-throughput of modern mapping methods. here we demonstrate that untargeted metabolomics of point mutants with phospho in or out modifications has the potential to address this functionality problem. from phosphosite mutants of proteins involved in metabolism, tor and hog signaling as a proof-of-concept, we were able to recover of the positive controls by metabolomics, compared to only three when relying on quantitative growth assays. importantly, metabolomics was able to detect also more subtle metabolic effects that were phenotypically silent. the other three known phosphoproteins were presumably not detected because we did not mutate all relevant phosphosites or did not chose the appropriate conditions that required activity of these proteins. of the remaining so far uncharacterized phosphosites, metabolomics provides evidence that those on cys gln gut nbp pfk tco and tip are generally functional. at least gln and tip had been suspected to be regulated by phosphorylation, although no phosphosites were identified so far. the strongest evidence in our data was for s on tip an essential regulatory protein in tor signaling whose role upon activation of tor is not yet clearly established. the exclusively strong effect of tip out during growth on a poor nitrogen source suggests that s is phosphorylated when the tor complex is inactive and becomes dephosphorylated upon activation of tor. since our recovery of positive controls was fairly good, we believe that the newly tested phosphosites for which we could not obtain functionality evidence are indeed mostly without functional relevance. the employed, metabolomics-based scoring system allowed classification of functionality, but does not directly identify the regulatory consequences of phosphorylation. nevertheless, in several cases the metabolite profiles provided starting hypotheses for the specific biological function without prior knowledge. for instance accumulation of pathway end products suggested that phosphorylation of lys the first enzyme in lysine biosynthesis, specifically regulates lysine pathway flux. the correlation of pbs in and hog out metabolite profiles strongly suggested that the two phosphorylation events have a similar function in mediating hog signal flow. however, changes in metabolism were not always intuitive and possibly not direct consequences of the mutated phosphosites, as observed in the gpd and gpd mutants. the main benefit of the metabolomics data in the process of understanding phosphosite functionality is to point out relevant cases efficiently, which can then be investigated in more depth by other methods or in combination with proteomics and transcriptomics data. even though about half of the tested phosphosites appeared to be functional, most phosphomutants had no detectable growth phenotype and exhibited relatively modest metabolic changes, both in number and magnitude. this apparent resilience to point mutations does not come entirely unexpected given that the much more drastic intervention of kinase and phosphatase deletions in s. cerevisiae resulted in few detectable growth phenotypes where only 26% of the mutants exhibited a significant transcriptional response and about 40% had no detectable metabolite response. several reasons are likely to contribute to this robustness, including the large overlap and redundancy both in kinase targets and phosphosites on a given protein. additionally, adaptation to a new steady state in our experiments will mask many direct regulatory effects and our population-based measurements will have missed, for example, cell cycle specific regulation events.  in this study, we investigated the potential of untargeted metabolomics along with growth analysis to identify functionality of phosphosites in a higher throughput. while growth analysis was suitable to detect functionality of a small number of phosphosites, metabolomics allowed recovering most of the previously characterized sites, along with some novel functional phosphosites and aided in hypothesis generation on biological roles of functionally relevant sites. our results demonstrate the potential of untargeted metabolomics to experimentally identify functionality and conditional relevance of post-translational modifications at high throughput to complement current computational approaches. 
coordinated through a complex network of kinases and phosphatases, protein phosphorylation regulates essentially all cellular processes in eukaryotes. we show that metabolomics largely outperforms growth analysis and recovers out of the previously characterized phosphosites and suggests functionality for several novel sites, including s on the tor regulatory protein tip we analyze metabolic profiles to identify consequences underlying regulatory phosphorylation events and detecting glycerol metabolism to have a so far unknown influence on arginine metabolism via phosphoregulation of the glycerol dehydrogenases. we demonstrate that metabolic profiles can be exploited for gaining insight into regulatory consequences and biological roles of phosphosites. however, functionality of the vast majority of these sites remains unclear and we lack suitable approaches to evaluate functional relevance at a pace that matches their detection. recent advances in proteomics enable detection of thousands of phosphorylation sites in single experiments.
 coordinated through a complex network of kinases and phosphatases, protein phosphorylation regulates essentially all cellular processes in eukaryotes. recent advances in proteomics enable detection of thousands of phosphorylation sites in single experiments. however, functionality of the vast majority of these sites remains unclear and we lack suitable approaches to evaluate functional relevance at a pace that matches their detection.  here, we assess functionality of phosphosites by introducing phosphodeletion and phosphomimic mutations in metabolic enzymes and regulators from the tor and hog signaling pathway in saccharomyces cerevisiae by phenotypic analysis and untargeted metabolomics. we show that metabolomics largely outperforms growth analysis and recovers out of the previously characterized phosphosites and suggests functionality for several novel sites, including s on the tor regulatory protein tip we analyze metabolic profiles to identify consequences underlying regulatory phosphorylation events and detecting glycerol metabolism to have a so far unknown influence on arginine metabolism via phosphoregulation of the glycerol dehydrogenases. further, we also find s in the mapkk pbs as a potential link for cross-talking between hog signaling and the cell wall integrity pathway.  we demonstrate that metabolic profiles can be exploited for gaining insight into regulatory consequences and biological roles of phosphosites. altogether, untargeted metabolomics is a fast, sensitive and informative approach appropriate for future large-scale functional analyses of phosphosites. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords phosphorylationmetabolismpost-translational modificationtor signalingosmotic stresshttp://dx.doi.org/ /501100001711schweizerischer nationalfonds zur förderung der wissenschaftlichen forschungsignalxp300p3_147895raguz nakic zrinka seisenbacher gerhard spanish ministry of economy and competitivenessbfu2015-64437-pfederposas francesc catalan government sgr 599posas francesc http://dx.doi.org/ /501100006373fundación botínhttp://dx.doi.org/ /501100002809generalitat de catalunyaicrea acadèmiaposas francesc issue-copyright-statement© the author 2016 