In recent years, several groups have performed comprehensive tissue phosphoproteome analyses 
. The animal tissue most often used for phosphoproteome analysis has been liver 
. The first such study, performed by Jin et al. 
, utilized iron IMAC for the enrichment of phosphorylated peptides and conventional linear ion-trap mass spectrometry (LTQ) for the phosphopeptide analysis. These investigators identified 26 nonredundant phosphorylation sites. A subsequent study 
utilized high capacity iron IMAC and a higher mass accuracy MS Q-TOF instrument to identify 339 non-redundant phosphorylation sites from over 200 proteins. The analyses performed by Villen et al. in 2007 
was a breakthrough study. These investigators identified 5,635 nonredundant phosphorylation sites from 2,149 proteins, signifying the first in-depth global analysis of phosphopeptides from liver. Notably, these investigators used more selective iron IMAC beads and MS instruments of higher mass accuracy (the LTQ-FT and the LTQ-Orbitrap) as compared to previous studies. However, it is likely that the SCX pre-fractionation of tryptic peptides was a critical factor accounting for the increase in phosphopeptide identification.
Lack of reproducibility has been an issue with large scale MS-based phosphoproteomic profiling. Moser and White 
tested the reproducibility of their methodology by performing three replicate analyses of the same rat liver homogenate. They observed that 56–63% (131 out of 207–234) of the peptides from each analysis were observed in all three analyses. Another issue is method of analysis. Alcolea et al. 
found that analyzing the same phosphopeptide enriched murine NIH/3T3 fibroblast lysate by two different LC-MS/MS platforms based on Q-TOF and LTQ-Orbitrap mass spectrometers led to identification of partially overlapping, but also distinct, phosphoproteome profiles. The Q-TOF based platform resulted in 1,485 non-redundant phosphopeptide identifications, whereas the LTQ-Orbitrap based platform identified 4,308 non-redundant phosphopeptides. Only 1,077 of the total population of phosphopeptides were detected by both platforms. Analyzing duplicate samples by LC-MS/MS on the LTQ-Orbitrap platform showed that ~70% of the identified phosphopeptides were identical. In a study comparing the peptides identified using two workflows, TiO2
-SCX and SCX-TiO2
, the overlap was 58 and 51% for the two methods, respectively 
. A similar overlap of 60% was observed, when they performed replicate LC-MS/MS analyses of the same TiO2
-SCX sample by an LTQ-Orbitrap.
These previous studies profiling the liver phosphoproteome have not been aimed at characterizing signaling events in a physiological context. Accomplishing this required improved sensitivity and accuracy, and a demonstration of reproducibility. We found that peptide abundance affects reproducibility, but that reproducibility could be enhanced by the performance of technical replicates. There are several indications that our methods were sufficient to detect mTORC1-mediated protein phosphorylation. These included the identification of known mTORC1 targets, the significant enrichment for mTOR signaling pathway constituents as indicated by pathway analysis, and the kinase prediction results.
In their seminal studies, Hunter and Sefton reported the relative abundances of pSer, pThr, and pTyr to be 90%, 10%, and 0.05%, respectively 
. The order of magnitude higher tyrosine phosphorylation frequency in our results may be a function of the tendency for pTyr-containing peptides to yield better quality MS/MS spectra with collision-induced dissociation fragmentation. In contrast, phosphoserine and phosphothreonine containing peptides may produce low scores due to fragmentation patterns dominated by neutral loss of phosphate 
. We are left to conclude that the actual proportion of protein phosphorylation accounted for by pTyr is between 0.05% and 2%, but likely toward the lower end of this range.
Two groups have recently reported large-scale, MS-based analysis of TORC1-dependent protein phosphorylation events 
. Chen et al. 
used epidermal growth factor-induced HeLa cell cultures and employed the use of stable isotope labeling in cell culture (SILAC) to achieve quantitative analyses. They identified 250 rapamycin-sensitive phosphorylation sites from 161 cellular proteins. Their main finding was the identification of CDC25B (Ser375) as the key phosphorylation event in mediating rapamycin-induced oncogenic Akt activation 
. Huber et al. 
used Saccharomyces cerevisiae with various genetic backgrounds for label-free quantitative phosphoproteomic screens. This study reported 41 rapamycin-sensitive yeast proteins and revealed that rapamycin-regulated Sch9 (a homolog of mammalian kinases Akt and p70S6K1) is a central coordinator of protein synthesis.
To our knowledge, our work represents the first in-depth, global analysis of rapamycin-dependent phosphoproteomics performed on whole tissue samples. One noteworthy result is the identification of several rapamycin-sensitive candidates that are related to translation. eIF3a is the largest component of the eIF3 complex, which is required for several steps in the initiation of protein synthesis 
. The eIF3 complex interacts with p70S6K under conditions of nutrient depletion or starvation 
. We found that rapamycin administration was associated with a marked reduction in phosphorylation of a component of eIF3, eIF3a, at Ser584 in all three animal sets. Although the function of this site has not been defined, a search of the Minimotif Miner (http://mnm.engr.uconn.edu/
) database reveals that the [KR]xRxx[ST] consensus motif is also present in ribosomal protein S6, a substrate for p70S6K. Our observation is consistent with the hypothesis that refeeding of rats after starvation causes the activation of mTORC1, leading to phosphorylation and release of p70S6K from the eIF3 complex.
We observed rapamycin-sensitive phosphorylation events involving several other proteins associated with translation. Ribonuclease UK114, also identified as “translational inhibitor protein p14.5”, has been shown in previous studies to be related to inhibition of cell proliferation 
. We found that rapamycin administration was associated with a >5-fold increase in phosphorylation of this protein at Thr10 and Ser11 in all three paired analyses. These sites have not been previously identified or characterized.
We also identified several known direct interactors of mTOR as rapamycin-sensitive phosphoproteins. Among these were the mTORC1 component raptor on Ser863 
and PRAS40, a novel mTOR binding partner (53). Rapamycin has been shown to decrease the association of PRAS40 with mTORC1 proteins (53), an event for which the mechanism has not been elucidated. The exact mechanism by which PRAS40 inhibits mTORC1 activity is not well understood. We found that rapamycin treatment was associated with reduced phosphorylation of rat PRAS40 at Ser203 and Ser213 in all three paired analyses. A recent phosphopeptide mapping study identified Ser183, Ser212 and Ser221 as mTOR-dependent phosphorylation sites in human PRAS40 
. Ser212 of human PRAS40, which is homologous to Ser213 in rat PRAS40, was not identified as sensitive to rapamycin treatment by these investigators.
AMPK is a critical sensor of metabolic stress that can turn off biosynthetic pathways when cellular ATP/AMP ratios decline 
. The two AMP kinase isoforms, which generally function in the same manner (53), can inhibit mTORC1 by phosphorylating and activating TSC2. We detected a rapamycin-associated reduction in phosphorylation of AMPK2 at Ser377 in all three paired analyses. The Kinexus' kinase predictor software indicated that mTOR is a candidate kinase for this particular site. Our results may indicate a feedback mechanism between mTOR and AMPK through this rapamycin-sensitive phosphorylation event.
Gephyrin is a microtubule-associated protein involved in membrane protein-cytoskeleton interactions that is purported to directly interact with mTOR in a manner that is required for rapamycin-sensitive signaling 
. The underlying mechanism has not been identified. We found that rapamycin administration was associated with a >12-fold increase in phosphorylation of gephyrin at Ser200, a site that has not been assigned a biological function.
Chen and coworkers detected more transcription-related proteins (9.3%) than translation-related proteins (5.4%) among their rapamycin-sensitive proteins 
, just as we did. However, the only rapamycin-sensitive phosphorylation events common to our dataset and theirs were the well-characterized ribosomal protein S6 sites. An RNA binding protein, the La ribonucleoprotein (LARP), was identified as a rapamycin-sensitive candidate in both studies. Phosphorylation of human LARP at Ser849, which is homologous to Ser648 of rat LARP, was reduced upon rapamycin exposure in the study by Chen et al. 
, while we observed an increase in the phosphorylation of rat LARP at the Thr644 and Ser648 sites in association with rapamycin administration. A comparison of our data to those of Huber and coworkers 
shows that in both cases Maf1 and S6 were the only common rapamycin-sensitive proteins previously reported in the literature.
While our approach allowed us to identify what may be novel, physiologically relevant rapamycin-sensitive sites, our study has some important limitations. One is incomplete coverage of any given protein due to a suboptimal density of tryptic sites. For example, lysine and arginine content within eIF4G1 is very high, so digestion with trypsin results in very short peptide fragments. This likely accounted for our inability to identify peptides containing the rapamycin-sensitive Ser1108 phosphorylation site in eIF4G1 
. Other established rapamycin-sensitive phosphorylation sites include p70S6K (Thr389) and 4E-BP1 (Thr37) and (Thr46) 
. We do not have a definitive explanation for the absence of p70S6K Thr389 in our analyses, though low abundance of this phosphoprotein is likely. While we detected the Thr37 and Thr46 phosphorylation sites in 4E-BP1, their phosphorylation was not affected by rapamycin in either our study or another published study 
. This may be consistent with recent biochemical studies indicating a complex mechanism behind the effect of rapamycin on 4E-BP1 phosphorylation 
There are other limitations inherent in our approach. We began with a preparation of proteins that were soluble under aqueous, non-detergent-containing conditions. Since the presence of detergents in samples complicates MS analysis 
, extension of our methods to lipid-soluble proteins may be challenging. Very low abundance proteins or proteins with a low stoichiometry of phosphorylation would not have been detected in our MS analysis due to sensitivity limits of the LTQ-FTICR classic mass spectrometer and the suppression of ionization efficiency with the detection of phosphorylated peptides in the positive ion mode 
. These issues notwithstanding, we made another observation of potential physiological significance. That was the high frequency with which previously unidentified phosphorylation sites were up-regulated in association with rapamycin administration. It is, of course, the case that rapamycin-sensitive phosphoproteins may not be direct targets of mTOR kinase. The up-regulation of phosphorylation in response to rapamycin can be accounted for by a downstream kinase that is activated upon its own dephosphorylation. An example of such a mechanism is eukaryotic elongation factor 2 kinase (eEF2K), which is activated in response to its dephosphorylation 
. Alternatively, such changes may reflect modulation of phosphatase activity.
Identification of potential rapamycin-sensitive phosphorylation sites is only the first step in characterizing the signaling events we have identified. The assignment of functional significance to newly identified sites will require a traditional biochemical approach. Nonetheless, through the application of a phosphoproteomics approach to an in vivo model of mTORC1 signaling we have identified 67 rapamycin-sensitive candidate phosphorylation events. Identification of known rapamycin-sensitive phosphorylation sites supported the reliability of our analysis. We identified a high number of novel rapamycin-sensitive candidate phosphorylation sites on proteins that are related to transcription, translation or cell growth, or are known to interact with mTOR kinase. While contributing to the understanding of mTOR action, our results indicate the potential utility of phosphoproteomic profiling of in vivo tissues in identifying new targets for drug therapies.