the phosphatidylinositol-3-kinase ( pi3-k ) pathway , one of the signaling pathways downstream of receptor tyrosine kinases , controls a wide range of biological processes including survival , cell proliferation , metabolism and cell mobility.(3 ) activation of pi3-k pathway involves growth factor binding to a receptor tyrosine kinase and activation of pi3-k , which phosphorylates membrane - bound phosphatidylinositol biphosphate , ( pip2 ) to generate phosphatidylinositol trisphosphate ( pip3 ) . it is believed that , upon binding of pip3 to the pleckstrin homology ( ph ) domain , the phosphoinositide dependent protein kinases pdk-1 is recruited to the plasma membrane and become activated although the degree of recruitment remains controversial.(5 ) pdk1 was discovered a decade ago(6 ) and is known to transduce signals by phosphorylating and activating a number of downstream protein kinases such as akt / pkb , sgk , pkczeta , pkcdelta , p70 s6 kinase , p90 ribosomal s6 kinase ( p90 rsk ) and p21-activated kinase ( see ref ( 7 ) for review ) . the identity of pdk2 has been controversial with various kinases proposed to act as the pdk-2 for akt , such as ilk and pkc . however , recent biochemical studies have strongly suggested that pdk-2 is the mtor / rictor complex.(8 ) the activity of the pi3-k pathway is negatively regulated by the lipid phosphatases pten and ship . akt / pkb is a critical component in the pi3-k pathway residing directly downstream of both pdk-1 and -2.(5 ) there are three akt protein kinase isoforms ( akt 1 , 2 , 3 ) showing a high degree of sequence homology , but differences in location of expression in mammals . akt1 is the predominant isoform in most tissues , akt2 is highly expressed in the insulin - responsive tissues , and akt3 is abundant in brain.(13 ) each akt isoform has three functionally distinct domains : an n - terminal ph domain providing a lipid - binding module to pip3 , a central catalytic domain , and a c - terminal hydrophobic domain . activation of akt is regulated through two distinct phosphorylation events at thr308 and ser473 in akt1 , 309 in akt2 and 305 in akt3 . phosphorylation of thr308 , which precedes ser473 , is mediated by pdk-1 and is sufficient to activate akt . phosphorylation of thr308 by pdk-1 is believed to render the ser473 site accessible to pdk-2.(14 ) these two phosphorylation events are often used as indicators for pi3-k pathway activation . the pi3-k pathway is also found to be deregulated in many diseases such as cancer and diabetes . for example , overactivated pi3-k is found in 32% of colon , 27% of glioblastoma , 25% of gastric and 8% of breast cancers.(15 ) the overactivation of such a pathway could be caused by mutations , amplification / overexpression of key components in the pathway or through inactivation of tumor suppressor protein pten . in diabetes , the compromised insulin signaling pathway which includes pi3-k / mtor pathway has been implicated in causing insulin resistance in tissues and  cell dysfunction in pancreas . because of its importance in physiological and pathological conditions , measurement of pi3-k activation has became an essential task for scientists in cell biology and biomedical research . conventionally , detection of pi3-k pathway activation is performed by measuring phosphorylation at thr308 and ser473 on akt using immunoassays such as western blot , or less frequently by a kinase activity assay ( either pi3-k itself or its downstream kinase such as akt ) . the latter provides most direct activity measurements but requires fresh samples making it less applicable to clinical samples . the former estimates akt phosphorylation by the ratio of signals from two immunoassays : one using phospho - specific antibody and the other total akt antibody . despite being very sensitive , this method provides only arbitrary ratios and a reference control sample to which the comparison of the levels of phosphorylation in experimental samples is required . without absolute stoichiometry readings , critical information is missed . in the case of pten mutation , although it is known that pten knockout causes tumorigenesis via activation of akt ( for review , see ref ( 20 ) ) , the quantity of akt and the stoichiometries of phosphorylation at thr308 and ser478 that are required to trigger such an event are unknown . quantitative information about the total amount of akt and its phosphorylation stoichiometry could be used not only to understand the biological roles of akt , but also to assess the effectiveness and/or appropriate dosing of pi3-k inhibitors in cancer patients . the other advantage of absolute quantification of phosphorylation stoichiometry is that data from different studies can be directly compared , and thus , population studies with large sample sizes can be conducted . a relatively new strategy of quantifying phosphorylation using lc - ms has been proposed by gygi and his colleagues.(21 ) the procedure consists of immunoprecipitation , sds - page , protease digestion , internal standard spike - in , and lc - ms / ms . this method utilizes a synthetic peptide with incorporated stable isotopes as ideal internal standards to mimic native peptides formed by proteolysis . the internal standard peptide is then used to quantitatively measure the absolute levels of proteins and post - translationally modified proteins after proteolysis by using triple quadrupole tandem mass spectrometer in multiple reaction monitoring ( mrm ) mode . this approach has been used to monitor phosphorylation events in human separase protein,(21 ) focal adhesion kinase , cyclin - dependent kinases,(24 ) erk and s6 kinase.(25 ) in this study , we adapted the same approach to measure and monitor the phosphorylation stoichiometry of akt in both t and u-87 mg cells grown in various conditions . human cd8 activated t - cells were isolated and purified from the peripheral blood mononuclear leucocytes.(26 ) isolated t - cells were activated with 2 g / ml phytohemagglutinin and cultured in rpmi medium for 3 days . the cells were then washed and subcultured in rpmi supplemented with 10% fetal calf serum ( fcs ) in presence of il2 ( 20 ng / ml ) every 3 days until reaching confluency ( 2  10 cell / ml ) . prior to treatments , cells were washed with pbs , resuspended in fresh rpmi at 10 cell / ml and then divided equally into four separate treatment groups consisting of the control , pi3-k inhibitor alone ( treatment with 10 m of pi3-k inhibitor ly294002 for 35 min ) , pi3-k inhibitor ly294002 and pervanadate ( 100 m ) , and pervanadate ( 100 m ) alone . t - cells were subsequently harvested by centrifugation ( 10 cells , for each phosphorylation site ) , washed twice with pbs and then lysed in 2 ml of lysis buffer ( 50 mm tris - hcl , ph 7.4 , 2 mm edta , 1% np40 , 5 mm sodium pyrophosphate decahydrate ( sigma ) , 5 mm -glycerol 2-phosphate disodium salt hydrate ( sigma ) , 5 mm sodium pervanadate , protease inhibitor cocktail ( roche ) , and phosphatase inhibitor cocktail ( calbiochem ) ) . lysed cells were left on ice for 10 min with regular vortexing for 10 s every 2 min . seven micrograms of rabbit polyclonal anti - akt1 antibody ( millipore ) bound to protein g agarose beads ( 60 l of 50% slurry ) was added to the clear lysate . the mixture was left at 4 c for 2 h on a shaking platform and beads were subsequently washed 5 times in 500 l of wash buffer ( lysis buffer supplemented with 200 mm nacl ) to remove nonspecifically bound proteins . ten microliters of 4 loading buffer ( invitrogen ) and 5 l of 50 mm dtt were then added to the beads followed by incubation at 70 c to reduce cysteine residues . alkylation was performed by adding 5 l of 300 mm iodoacetamide ( sigma ) and incubation at room temperature in the dark for 30 min . proteins were separated on a nupage bis - tris 412% gradient gel ( invitrogen ) at 200 v for 45 min and gels were washed for 5 min in milli - q water before staining with 20 ml of simply blue ( invitrogen ) to visualize proteins . the gel was subsequently washed with milli - q water four times over 10 h. for the u-87 mg ( american type culture collection ) experiments , cells were grown in dmem medium supplemented with 10% fcs in a humidified atmosphere of 95% air and 5% co2 at 37 c . the medium was changed every second day and cells were subcultured every 3 to 4 days when they reached 8090% confluency . u-87 mg cells were grown in the presence or absence of 7 nm wortmannin for 2 h to evaluate the effect of the pi-3k inhibitor on akt phosphorylation . about 3  10 cells in each group cell lysis , immunoprecipitation and sds - page were performed as described above for t cells . the akt protein band was cut with a clean scalpel blade , transferred to a microcentrifuge tube and cut into small pieces . gel pieces are washed for 15 min sequentially with 200 l of milli - q water , 200 l of acetonitrile , 200 l of 100 mm ammonium bicarbonate , 200 l 20 mm ammonium bicarbonate and 50% acetonitrile and finally 100 l of acetonitrile . the gel pieces were then air - dried in a laminar flow . in - gel digestion was carried out with trypsin ( measurement of phosphorylation at ser473 site ) or chymotrypsin ( measurement of phosphorylation at thr308 ) at a final concentration of 12 g / ml ( in 30 mm ammonium bicarbonate ) . heavy peptides ( cambridge research bioscience , cambridge , u.k . ) corresponding to the phospopeptide and nonphosphopeptide of both sites were spiked - in at a final amount of 37 fmol . after overnight incubation ( 16 h ) on a shaker at 37 c , an equal volume of acetonitrile ( i.e. , 30 l ) was added and the peptide mixtures were extracted by shaking ( 1000 rpm ) at room temperature for 10 min . peptides in the gel pieces were further extracted first by adding 50 l of 5% formic acid and shaking for 20 min then 50 l of 100% acetonitrile for another 20 min . the gel pieces were finally washed with 50 l of acetonitrile for 10 min and the three pooled fractions were dried in speed - vac and stored at 70 c until further processing . the peptides used for quantification of the phosphorylation site thr308 contain a cysteine residue which required reduction and alkylation . this was achieved by adding 10 l of 10 mm dtt to 10 l of light or heavy peptides ( both phospho- or nonphosphopetides , 1 nm ) and incubation at room temperature for 45 min with regular sonication every 15 min . a total of 2.5 l of ammonium bicarbonate ( 1.0 m ) was then added to the sample prior to the incubation at 60 c for 1 h. after cooling to room temperature , 4.5 l of iodoacetamide ( 300 mm ) was added to the samples and the mixture was incubated at room temperature in the dark on an orbital shaker for 1 h. three microliters dtt ( 100 mm ) was added to the samples followed by the addition of 20 l of 5% formic acid and 140 l of milli - q water . quantification of akt peptides ( and phospho - peptides ) was performed by lc - ms on a q - trap 4000 ( abi - mds - sciex ) mass spectrometer coupled with a dionex 3000 nano - lc system . lc buffers were the following : buffer a ( 2% acetonitrile and 0.1% formic acid in milli - q water ( v / v ) ) and buffer b ( 90% acetonitrile and 0.08% formic acid in milli - q water ( v / v ) . peptide mixtures were reconstituted in 54 l of 0.5% formic acid and then pipetted into microvials containing 1 l of 25 mm edta . aliquots of 16 l of each sample were loaded at 20 l / min onto a trap column ( 300 m  5 mm , pepmap c18 column , 5 m , 100  , lc packing ) equilibrated in 99% buffer a. the trap column was washed for 4 min at the same flow rate and then the trap column was switched in - line with an lc - packings , resolving c18 column ( 300 m  15 mm , pepmap c18 column , 3 m , 100  , lc - packings ) . the peptides were eluted from the column at a constant flow rate of 300 nl / min with a linear gradient from 99% buffer a to 45% buffer b in 20 min , then to 90% buffer b by 22 min . the column was then washed with 90% buffer b for 10 min and re - equilibrated in 99% buffer a. the hplc was interfaced to the mass spectrometer with an fs3602010 picotip ( new objectives ) fitted to a nanospray ii source and interface kit ( applied biosystems ) . the q - trap 4000 was operated in the positive mode using mrm mode with analyte and source dependent parameters that were optimized for each peptide and mrm transition ( table 1 ) . the same amount of proteins ( e.g. , 20 g ) from each group of cultured cells and 12.2 ng of recombinant akt ( millipore ) as control were subjected to gel - electrophoresis ( nupage bis - tris 412% gradient gel ) . after electrophoresis , proteins were transferred onto pvdf membrane ( invitrogen ) at 25 ma for 2 h at room temperature using x cell sure lock and blot module ( invitrogen ) . after blocking overnight in buffer c ( pbs containing 4% bsa and 0.5% tween 20 ) at 4 c , membranes were incubated with primary antibody ( anti - phospho - akt , thr308 ( cell signaling ) diluted 1/500 , anti - phospho - akt ser473 ( cell signaling ) diluted 1/1000 or total anti - akt antibody ( millipore ) diluted 1/1000 in buffer c for 4 h at room temperature . loading was confirmed using anti - actin antibody ( santa cruz ) diluted 1/1000 in buffer c. membranes were washed three times for 5 min in buffer c and then incubated for 1 h at room temperature with secondary antibody ( stabilized goat anti - rabbit or anti - mouse conjugated to horseradish peroxidase ( hrp ) , pierce ) diluted 1/600 in buffer c. visualization was by enhanced chemiluminescence ( ge biosciences ) . data analysis was performed using microsoft excel ( version 2003 , microsoft ) or analyst software ( version 1.4 , applied biosystems ) . peak areas for the phospho- and nonphospho peptides from both phosphorylation sites were calculated for the dominant mrm transition of each peptide . quantity of peptide of interest = ( peak area of transition representing peptide of interest)/(peak area of transition representing the internal standard ( is ) )  the quantity of is . percentage of phosphorylation was calculated as ( phospho peptide)/(phospho peptide + nonphospho peptide )  100 . the procedure of mrm - based quantitative analysis is shown in figure 1a and is primarily based on the workflow published by gygi and colleagues.(21 ) in brief , after cells or tissues lysis , akt was immunoprecipitated from the lysate and the precipitated proteins were then reduced and alkylated ( to eliminate disulfide bridges and hence cross - linked peptides ) before sds - page protein separation . the akt band was excised , washed and mixed with heavy isotope internal standards corresponding to the peptides and phosphopeptides of interest . enzymatic digestion was then carried out prior to lc - ms / ms using the mrm mode . before the levels of phosphorylated and nonphosphorylated peptides can be measured in samples , several assay development steps including selection of protein digestion method and immunoprecipitation were optimized . the former is particularly important as it also determines the characteristics ( i.e. , length and amino acid composition ) of the resultant peptides to be analyzed and quantified . assay procedure of lc - ms - based quantification of akt phosphorylation stoichiometry at thr308 and thr473 . ( a ) the procedure for lc - ms based phosphorylation measurement contains six steps including lysis of cell / tissue , immunoprecipitation , reduction / alkylation , proteolytic digestion , and lc - ms / ms . ( b ) representative mrm extracted ion chromatogram for thr308 , phospho - thr308 , ser473 and phospho - ser473 peptides . phosphopeptides are shown in red . in silico digestion of the akt amino acid sequence with various proteolytic enzymes suggested that asp - n might generate suitable peptide fragments for measuring both the thr308 and ser473 phosphorylation sites . however , subsequent trials using recombinant akt , asp - n and lc - ms / ms analysis showed that only the peptide containing ser473 , and not that containing thr308 , was detected ( data not shown ) . as an alternative , trypsin and chymotrysin were chosen to digest akt into peptide fragments for monitoring phosphorylation at the ser473 and thr308 sites , respectively . both phospho- and nonphospho - peptides ( phospho - thr308 and thr308 ; phospho - ser473 and ser473 ) generated , respectively , by chymotrypsin and trypsin digestion of recombinant akt , can be detected ( figure 1b ) . to quantify the levels of thr308 , phospho - thr308 , ser473 and phospho - ser473 peptides , a total of eight peptides ( isotopic heavy and light peptide for each site ) were synthesized . these peptides were also used to optimize conditions for both lc separation and ms detection . the retention time , mrm transition , and major settings in lc - ms are summarized in table 1 . phosphopeptides are retained longer on the column than their unmodified counterparts ( figure 1b ) . the standard curve of phospho - ser473 peptide showed linearity in the range of 0.05500 fmol ( figure 2a ) . the lower limits of detection for the eight peptides were around 40120 amol , as defined by signal - to - noise ratios > 5 ( figure 2b ) . the lower limits of quantification were between 90250 amol , as determined by intra- and interassay imprecision and inaccuracy < 25% ( figure 2b ) . analytical performance of lc - ms based quantification of akt phosphorylation stoichiometry at thr308 and thr473 . ( a ) an example of calibration curves for phospho - ser473 peptide indicates a good linearity over a range between 50 amol and 500 fmol . the detection limit ( defined as signal - to - noise ratio = 5 ) is 50 amol for this peptide ( bottom left ) . limit of quantification ( loq ) is defined as 25% cv and bias for all intraday and interday precision and accuracy . an immunoprecipitation step is normally required to detect low - abundance proteins such as akt . the immunoprecipitation enriches protein - of - interest and improves the sensitivity of lc - ms / ms methods . 466480 , rabbit polyclonal anti - akt1 , ( millipore ) ) of akt1 were tested for their ability to immunoprecipitate 0.2 g of the active form of recombinant akt1 . 140480 and a.a . 466480 gave the best recovery ( 80% ) ( supplementary figure s1 ) . 140480 contained an interfering protein that migrated close to the recombinant akt1 band on sds - page and therefore was not considered for further use . the efficiency of immunoprecipitation using this antibody was also tested on t cells cultured in the presence or absence of pervanadate in order to examine the ability of pool - down for both phosphorylated and unphosphorylated forms of akt . the results showed that this antibody pooled down quantitatively both phosphorylated and unphoshorylated ( or low phosphorylated ) forms of akt1 ( supplementary figure s2 ) . the thr308 and phospho - thr308 peptides contain a methionine residue at position 306 prone to oxidation by free radicals , and a cysteine residue requiring alkylation to prevent cross - linking ( table 1 ) . lc - ms / ms analysis of chymotrypsin digested recombinant akt after sds - page indicates that both unoxidized and oxidized forms were observed for the phospho - thr308 peptide and the thr308 peptides using a data dependent ms / ms acquisition method and also using mrm mode taking into account the extra 16 da mass shift resulting from methionine oxidation . both peptides elute 2 to 3 min earlier than their native counterparts in accordance with their polar character ( figure 3a ) . methionine oxidation of both thr308 and phospho - thr308 was also confirmed by mascot database search ( figure 3b ) . oxidation of the methionine residues of the phospho - thr308 and thr308 peptides during sample processing . recombinant akt ( 0.2 g ) was processed through the assay procedures as described in figure 1 and the resulting peptide mixture was subjected to lc - ms / ms using an extra transition that takes into account the 16 da of methionine oxidation . ( a ) the potential oxidized form ( + 8 , doubly charged ion ) of the thr308 peptide can be observed ( upper panel , blue trace ) . the oxidized peptide was eluted 2 to 3 min earlier than its native counterpart in accordance with its polar character . ( b ) mascot database search indicates that the oxidized forms of both thr308 and phospho - thr308 can be observed . to examine the influence of potential oxidation of methionine and the alkylation step on the measurement of akt phosphoryaltion at thr308 , the assay was performed 5 times ( 3 replicates each time ) on different days using recombinant akt protein ( 0.2 g ) and the interday imprecision was estimated . we found that the imprecisions for the absolute quantification of unoxidized thr308 peptide and phospho - thr308 peptide were 28% and 32% , respectively , which are significantly higher than those of ser473 and phospho - ser473 ( 14.2% and 14.0% , respectively , table 2 ) . these results imply that oxidation of methionine and the alkylation step increase imprecision for measuring the absolute quantities of the thr308 and phosphothr308 peptides . nevertheless , the ratio of phospho - thr308 to thr308 was reliable , as indicated by the interday imprecision of only 3.8% . the active form of human recombinant akt1 was used as a quality control sample for all measurements . the levels of phosphorylation at thr308 and ser473 sites in the recombinant akt1 were found to be 78.1  3.0% and 82.2  1.9% , respectively ( mean  sd ) ( table 2 ) . these ratios did not change when other buffer systems ( e.g. , 80% acetonitrile and 50% methanol ) were used , suggesting that the phospho- and nonphospho - peptides behave similarly during protein digestion and extraction . measurements are in femtomole of peptide . a human t - cell model with pervanadate and /or pi-3 kinase inhibitor treatments was used to test if the lc - ms / ms method can be used to measure akt phosphorylation stoichiometry in biological samples . pervanadate , an extremely potent protein - tyrosine phosphatase inhibitor,(28 ) is known to activate the pi-3k pathway and stimulate phosphorylation of akt . human cd8 t - cells ( 2  10 cells ) from healthy volunteers were cultured in the absence or presence of pervanadate for 15 min with and without a pi3-k inhibitor ( ly294002 ) . the stoichiometry of akt1 phosphorylation was monitored using both the lc - ms / ms method and traditional western blotting . western blotting analysis indicated that pervanadate stimulated substantial , but undefined , akt phosphorylation at thr308 and ser473 phosphorylation sites compared to the unstimulated cells ( figure 4a ) . however , the presence of ly294002 decreased phosphorylation levels in both pervanadate stimulated cells and unstimulated cells ( figure 4a ) . the levels of total akt1 , which did not show any significant difference in all various treatments , provide a reference point to normalize the phosphorylation signals . useful , but ultimately arbitrary , semiquantitative data can be extracted by expressing the antiphosphorylation western blot signals as a percentage of the signal for total akt . in this case , the phosphorylation status for thr308 and ser473 in the unstimulated and ly294002 treated cells were undetectable , making any estimates of fold - change upon pervanadate treatment impossible ( figure 4a ) . in the presence of pervanadate , the pi-3k inhibitor ly294002 decreased akt phosphorylation by approximately 55% and 32% for thr308 and ser473 , respectively ( figure 4a ) . the same samples from these experiments were also analyzed by the lc - ms based method . we found that in the untreated cells the basal stoichiometry of thr308 and ser473 phosphorylation was just 0.92% and 0.62% , respectively , and that pervanadate treatment provoked a 39- and 18-fold increase of phosphorylation at thr308 and ser473 , respectively ( figure 4b , c ) . the presence of the pi-3 kinase inhibitor ly294002 decreased phosphorylation at both sites by 30% in the pervanadate stimulated t - cells ( figure 4c ) . in the untreated t - cells , the presence of ly294002 also decreased the phosphorylation of thr308 and ser473 by 22% and 47% , respectively ( figure 4c ) . in these experiments , the lc - ms method showed significant advantages over western blotting procedure . specifically , the lc - ms method uniquely allowed the accurate measurement of basal akt phosphorylation stoichiometries of the two key phosphorylation sites , their precise fold - change upon stimulation and the precise effects of the pharmacological agent ly294002 on the stoichiometries of phoshphorylation in resting and stimulated t - cells . cd8 t - cells ( 2  10 cells ) derived from a healthy volunteer ( see experimental procedures for the details ) were cultured in the absence or presence of the stimulant ( 100 m pervanadate ) with / without 10 m ly294002 and the phosphorylation levels of akt were monitored using traditional western blotting method and the lc - ms / ms method . ( a ) western blotting analysis of t - cells treated with pervanadate or ly294002 using phospho - thr308 , phospho - ser473 , total akt and actin antibodies . a total of 60 g of protein were loaded in each well and recombinant akt ( 12.2 ng ) was used as a positive control ( shown as r ) . ( b ) extracted ion chromatogram of phospho - thr308 and thr308 peptides in control and pervanadate stimulated t - cells . ( c ) stoichiometry of akt phosphorylation ( thr308 and ser473 ) in t - cells cultured in different conditions . we also compared the lc - ms method with western blot in u-87 mg cells where the phosphorylation of akt is known to be elevated due to the loss of pten.(31 ) cells were treated either with or without ptdlns-3-kinase inhibitor ( wortmannin ) for 2 h prior to lysis . the basal phosphorylation levels of akt at thr308 and ser473 in u-87 mg cells were 4.80% and 1.49% , respectively , using the lc - ms / ms method ( figure 5a ) . the presence of wortmannin resulted in an average reduction of 28% and 59% in the phosphorylation levels at thr308 and ser473 , respectively ( figure 5a ) . similarly , western blots showed a 60% decrease of phosphorylation at ser473 . however , the decrease was higher at thr308 ( 60% ) compared to the reading derived from the lc - ms / ms method ( figure 5b ) . measurements of phosphorylation stoichiometry of akt in u-87 mg cells . cells ( 20 million cells ) were cultured in the presence or absence of 7 nm wortmannin for 2 h prior to cell lysis . stoichiometry of akt phosphorylation ( thr308 and ser473 ) were analyzed using lc - ms / ms ( a ) and western blotting ( b ) . measurement of protein phosphorylation is typically qualitative or semiquantitative by western blot using phospho - specific antibodies . consequently , one usually only knows whether the phosphorylation status of a specific site in a given protein component of a signaling cascade has gone up or down in response to extracellular stimuli . this approach usually does not indicate the absolute quantity of the protein or the precise stiochiometry of phosphorylation at specific sites . this missing quantitative information is important for understanding how the cells utilize intracellular machinery to propagate signals . with the lc - ms based quantification method , we are able to monitor phosphorylation events in a much accurate manner . in this study , we have demonstrated that phosphorylation stoichiometry of akt phosphorylation at thr308 and ser473 can be measured accurately in cells . our results indicate , for the first time , that at the basal state , only 0.92% of thr308 and 0.62% of ser473 of akt were phosphorylated in human t - cells as opposed to the subjective claim of  very low  traditionally quoted by cell biologists . this result suggests that , in resting t - cells , less than 1% of akt is activated for executing normal cellular function , while 99% of the akt pool is inactive , presumably reserved to respond to potential extracellular stimulation . treatment for 12 min with pervanadate , a potent tyrosine phosphatase inhibitor , provoked 39- and 18-fold increase in phosphorylation at thr308 and ser473 to 55% and 35% , respectively , suggesting that even under this extreme condition only about 3055% of t - cell akt ( but not all ) is activated in the phosophorylation cascade downstream to receptor tyrosine kinases . interestingly , the level of phosphorylation at thr308 was higher than that at ser473 with pervanadate treatment . it is generally believed that phosphorylation of akt by pdk-2 at ser473 is required to fully activate akt and stabilize its activation state.(14 ) thus , our results indicate that only a maximum of 35% of akt molecules are in a stable , fully activated , state after 12 min treatment with pervanadate in human t - cells . the method was also tested on u-87 mg cells which are known to be lacking pten . inactivation of pten leads to accumulation of pip3 which results in an increase in akt phosphorylation.(31 ) our result demonstrated , for the first time , that even without pten , the majority of akt remains unphosphoryated at thr308 and ser473 and such a low level of phosphorylation is sufficient to stimulate abnormal cell proliferation contributing to tumorigenesis . for both western blotting and the lc - ms / ms method , the decrease of phosphorylation caused by the pi3-k inhibitor was similar at ser473 ( 60% ) , while at thr308 , the decrease was less pronounced using the lc - ms / ms method . this discrepancy may be linked to oxidation of the methionine residue present in the phospho - thr308 and thr308 peptides ( figure 3 ) . the chymotryptic peptide containing thr308 also contains two reactive amino acid residues : methionine and cysteine . however , s - alkyl cysteine is known to be susceptible to oxidation during sample processing , especially during gel electrophoresis.(32 ) similarly , and more acutely , methionine oxidation occurs in vivo and/or during sample preparation and electrophoresis and methionine sulfoxide can be further oxidized irreversibly to methionine sulfone.(32 ) we have evaluated these effects by estimating the intraday imprecision in five independent experiments . as expected , a lower yield and higher imprecision for thr308 and phospho - thr308 peptides , compared to those of ser473 and phospho - ser473 peptides , were apparent ( table 2 and figure 4c ) . for this reason , we only recommend using the ser473 figures for absolute quantification of akt . however , although significant variation was observed at the individual peptide level , the imprecision in the ratio of phospho - thr308 to thr308 was acceptable . thus , it seems that the oxidation and phosphorylation status of akt prior to chymotryptic digestion does not distort the ratio of the phospho - thr308 and thr308 peptides . it also appears that the two peptides , once generated , behave similarly with regard to methionine oxidation . from these data , we conclude that the phosphorylation stoichiometry measurements for thr308 using this method are robust . one potential application of this lc - ms method is for patient selection and for pharmaco - dynamic measurements for pi-3k / mtor pathway inhibition . it is often very difficult to compare results from different studies ( or from different laboratories ) due to variation in methods and references used . this method could potentially provide more definitive answer as to whether or not a patient should receive pi-3k / mtor inhibitor , and could also offer crucial information on drug dosage and inhibition of the targets . our preliminary data using this procedure allow measurement of akt phosphorylation status using approximately 20 mg of breast tumor tissue ( a. thompson , a. atrih , and j. huang , unpublished data ) . although this is more than what is normally required for immunoassays , one can foresee that this issue could be improved by new generation of mass spectrometers with better sensitivity . as the mrm approach allows monitoring multiple sites ( up to 100 transitions at the same time ) , this approach can in principle be extended to monitor multiple phosphorylation sites from multiple proteins in a single lc - ms / ms run . once developed , this could open up a new era for quantitative proteomic analysis in systems biology by providing phosphorylation stoichiometry at the pathway level , we have developed a reliable lc - ms method for stoichiometric quantification of akt phosphorylation of thr308 and ser473 sites . compared to western blot , the lc - ms based method provides similar sensitivity but significantly more in - depth information . akt , protein kinase b ; pi3-k , phosphoinositide 3-kinases ; pip2 , phosphatidylinositol biphosphate ; pip3 , phosphatidylinositol trisphosphate ; ph , pleckstrin homology ; pdk-1 , 3-phosphoinositide - dependent protein kinase ; sgk , serum and glucocorticoid inducible kinase , pkc ; protein kinase c ; ilk , integrin - linked kinase ; mtor , the mammalian target of rapamycin ; pten , phosphatase and tensin homologue ; ship , sh2 domain containing inositol phosphatase ; mrm multiple reaction monitoring ; erk , extracellular signal - regulated kinase ; il2 , interleukin 2 . akt , protein kinase b ; pi3-k , phosphoinositide 3-kinases ; pip2 , phosphatidylinositol biphosphate ; pip3 , phosphatidylinositol trisphosphate ; ph , pleckstrin homology ; pdk-1 , 3-phosphoinositide - dependent protein kinase ; sgk , serum and glucocorticoid inducible kinase , pkc ; protein kinase c ; ilk , integrin - linked kinase ; mtor , the mammalian target of rapamycin ; pten , phosphatase and tensin homologue ; ship , sh2 domain containing inositol phosphatase ; mrm multiple reaction monitoring ; erk , extracellular signal - regulated kinase ; il2 , interleukin 2 .
the ptdlns-3-kinase ( pi3-k ) signaling pathway plays a vital role in cell survival , proliferation , apoptosis and differentiation in normal cells , as well as in diseases such as cancer and diabetes . 
 quantification of phospho - akt is a standard way of assessing the activity of the pi3-k signaling pathway in both cells and tumors . 
 this measurement is traditionally performed semiquantitatively using immunoassays such as western blot . here 
 we report an lc - ms method to accurately measure the stoichiometry of akt phosphorylation in biological samples . 
 the procedure includes immunoprecipitation , gel electrophoresis , in - gel digestion , addition of isotopicaly labeled internal standards and lc - ms / ms . 
 two proteolytic enzymes , chymotrypsin and trypsin , were used to generate suitable peptide fragments for measuring thr308 and ser473 phosphorylation , respectively . 
 the interday imprecision was estimated to be 3.8% and 2.3% for thr308 and ser473 , respectively . 
 this method has been tested on human t - cells grown in presence and absence of pervanadate and with or without a pi3-k inhibitor and on human glioblastoma cells ( u-87 mg ) grown in presence and absence of wortmannin ( pi3-k inhibitor).the results of t cells suggest that the levels of akt phosphorylation in untreated cells were below 1% for both phosphorylation sites . 
 pervanadate treatment provoked an 18-fold increase in phosphorylation of thr308 and the pi3-k inhibitor partially reversed the increase . 
 a comparison between lc - ms / ms and western blotting suggests that the lc - ms based method is of comparable sensitivity and provides a more accurate phosphorylation stoichiometry , a wider dynamic range and more in - depth information . 
 the application of the new method and its utility to providing predictive markers of response to targeted therapies is discussed .