the protozoan parasite trypanosoma 
 brucei is the etiological agent of african sleeping sickness , 
 also known as human african trypanosomiasis ( hat ) , which is transmitted 
 by the bite of an infected tsetse fly .
the disease is usually fatal 
 if left untreated and is estimated to be responsible for around 10000 
 deaths per annum in sub - saharan africa .
current treatments are expensive , toxic and difficult to administer , 
 leaving an urgent unmet need for new therapeutic agents.t .
brucei has a complex digenetic 
 lifecycle between an insect vector and a mammalian host , and its ability 
 to adapt its proteome to these disparate environments is essential 
 to its survival and virulence . during the early stages of an infection , 

the clinically relevant bloodstream form of the parasite proliferates 
 in the blood and lymph of the human host , then in the second stage 
 enters the cerebral - spinal fluid and brain , resulting in coma and 
 death . when a tsetse fly feeds on an infected host , the parasite is 
 ingested with the blood meal , triggering parasite differentiation 
 into the procyclic form to enable survival in its new environment .
both the procyclic form and bloodstream form of the parasite may 
 be cultured in vitro .
we have recently quantified 
 the changes in protein expression between the procyclic and bloodstream 
 form lifecycle stages using silac labeling of procyclic cells , demonstrating 
 that about 10% of the observed proteome is differentially regulated 
 by more than 5-fold .
the fold - change in 
 protein level showed a strong correlation ( pearson correlation 0.86 ) 
 to changes in mrna level recorded by transcriptomic studies , despite the atypical mechanism of gene expression 
 in trypanosomes , where genes are transcribed in large polycistronic 
 units that are trans - spliced into mature mrnas for translation , with 
 mrna abundance controlled by stability .
there is evidence that protein phosphorylation may be important 
 in t. brucei ; nett et al .
used sequential strong - cation 
 exchange ( scx ) and tio2 enrichment of phosphopeptides derived 
 from the cytosolic fraction of bloodstream form parasites to identify 
 1204 phosphorylation sites on 491 proteins , and immunoprecipitation with antiphosphotyrosine antibodies to identify 
 34 phosphotyrosine sites induced in response to the treatment of procyclic 
 form cells with hydrogen peroxide .
however , 
 to date , there has been no quantitative comparison of the phosphoproteomes 
 of the two lifecycle stages .
we set out to define the global protein 
 phosphorylation status of the two lifecycle stages to explore the 
 biological processes that might be modulated by phosphorylation , and 
 to highlight stage - specific phosphorylation changes .
here , we report 
 a global quantitative phosphoproteomic study of bloodstream and procyclic 
 form trypanosoma brucei using silac labeling of each 
 lifecycles stage that identifies 9314 previously unidentified phosphorylation 
 sites .
the results show that differential phosphorylation is widespread 
 between the procyclic and bloodstream form and that it adds significant 
 complexity to the changes in the proteome .
hmi9-t , a modification of the original imdm - based hmi-9 , uses 56 m 1-thioglycerol in place of 200 
 m 2-mercaptoethanol , 2 mm glutamax i ( invitrogen ) in place 
 of 4 mm l - glutamine , and contains 10% heat - inactivated fetal 
 bovine serum ( paa ) .
similarly , hmi11-t is a modification of the original 
 hmi11 ( hmi-9 lacking serum plus ) that 
 use 56 m 1-thioglycerol and 2 mm glutamax i with 10% heat - inactivated 
 fetal bovine serum .
rk was prepared in 
 the same way as hmi11-t but using imdm depleted of l - arginine , l - lysine ( sigma ) and 10% dialyzed heat - inactivated fetal bovine 
 serum ( 10 kda molecular weight cutoff , paa ) and was supplemented with 
 4 mg / l folic acid .
the hmi11-silac  rk was supplemented with 
 either normal l - arginine and l - lysine ( hmi11-silac 
 + r0k0 ) , or with l - arginine u  c6 and l - lysine 4,4,5,5-h4 ( hmi11-silac + r6k4 , cambridge isotope 
 laboratories ) at 30% of the original hmi11 concentration ( 120 and 
 240 m respectively ) unless otherwise stated .
original sdm-79 
 medium and silac sdm-79 medium ( sdm-79 
 + r6k6 ) were prepared as described previously .
procyclic form trypanosoma brucei lister 427 clone 29.13.6 cells were grown 
 at 28 c without co2 in sdm-79 in the presence of 
 15 g / ml g418 and 50 g / ml hygromycin .
culture adapted 
 strain lister 427 monomorphic bloodstream form t. brucei ( mitat 1.2 , expressing vsg221 ) were cultured in hmi-9 t containing 
 2.5 g / ml g418 at 37 c in a 5% co2 incubator . 

both cell lines have been genetically modified to express the t7 rna 
 polymerase and the tetracycline repressor protein , as described by 
 wirtz et al .
cell dimensions were measured 
 using a casy cell counter . for the growth curves ,
the t. brucei bloodstream form cells were washed twice in 10 
 ml hmi11-silac  rk and resuspended at 5  10 cells / ml in either hmi-9 t or hmi11-silac supplemented with r0k0 at 30% of the original hmi11 concentration . 

cells were counted using a neubauer chamber and phase contrast microscope , 
 and the cultures were diluted 100-fold every two days .
after 10 days 
 samples were collected for analysis by light microscopy . for 
 silac labeling ,
t. brucei bloodstream form log - phase 
 cells were diluted 10000-fold into hmi11-silac + r6k4 .
cells were harvested after 3 days growth at 2.5  10 cells / ml . silac labeling of t. brucei procyclic 
 form cells was performed in sdm-79 + r6k6 as 
 described previously .
cells were 
 harvested by centrifugation and hypotonically lysed at 5  10 cells / ml for 5 min on ice in the presence 0.1 m 1-chloro-3-tosylamido-7-amino-2-heptone 
 ( tlck ) , 1 mm benzamidine , 1 mm phenyl - methyl sulfonyl fluoride ( pmsf ) , 
 1 g / ml leupeptin , 1 g / ml aprotinin and phosphatase inhibitor 
 mixture ii ( calbiochem ) . the protein concentration was determined 
 by bca assay ( pierce ) to be 5 mg / ml from each cell type .
samples 
 were aliquoted , snap frozen , and stored at 80 c prior 
 to subsequent processing .
bloodstream form cells 
 at late log phase grown in either hmi9-t or hmi11-silac + r0k0 for 10 days were washed in 10 ml phosphate buffer saline 
 at 800  g at 4 c , fixed in 4% paraformaldehyde 
 in phosphate buffered saline at 4 c for 30 min , and placed on 
 a coverslip .
after air - drying the coverslips were washed in phosphate 
 buffered saline and mounted onto slides .
bloodstream form cells were grown in hmi11-silac 
 + r6k4 for 1112 cell divisions and hypotonically 
 lysed as described above . to reduce the sample complexity the proteins 
 were fractionated by sds - page , and a band corresponding to 2550 
 kda molecular weight range
was excised and subjected to in - gel tryptic 
 digest prior to analysis by lc 
ten peptides were chosen 
 at random and the relative abundance of the major isotopic peak of 
 heavy ( arginine - c6/lysine - h4 ) and light ( arginine - c6/lysine - h4 ) forms were measured using the extracted ion 
 chromatogram function in excalibur ( thermo scientific ) .
bloodstream form cells were grown in hmi9-t or hmi11-silac 
 + r0k0 for > 14 days prior to inoculation 
 of female balbc mice ( three per condition ) by inter peritoneal injection 
 of 1  10 , 1  10 or 1  10 cells in the same media .
daily tail bleeds were used to monitor 
 the number of parasites in the bloodstream using a heamocytometer , 
 and animals were euthanized when the parasite burden exceeded 5  
 10 cells / ml prior to overt distress .
a 1:1 mix of procyclic form cells grown 
 in light - silac media and bloodstream form cells grown in heavy - silac 
 media was solubilized with sds and tryptic peptides were generated 
 by fasp .
peptides were fractionated by scx prior to analysis by liquid 
 chromatography tandem mass spectrometry as described previously , except only 6 scx fractions were analyzed and 
 technical duplicates were not performed .
the previously reported the 
 analysis of a 1:1 mix of procyclic form cells grown in heavy - silac 
 media and bloodstream form cells grown in light - silac media , consisting 
 of technical duplicates of eight scx fractions and ten sds - page fractions , was reprocessed to ensure accurate comparison 
 with the current data .
peptide samples for analysis by mass spectrometry were prepared by 
 modification of the filter - aided sample preparation procedure ( fasp ) .
briefly , samples were defrosted and combined 
 according to the experiment design to give a total of 2.5  10 lysed cells ( 0.5 ml ) , solubilized with 4% sds and then reductively 
 alkylated in a 30000 molecular weight cutoff vertical spin filtration 
 unit ( vivascience ) using the fasp procedure adapted for the larger 
 volumes used here .
the sample was digested with 1:100 ratio ( w / w ) 
 of trypsin gold ( promega ) in the filtration unit for 18 h at 37 c , 
 tryptic peptides were eluted by centrifugation , and the filter washed 
 sequentially with 1 ml of 50 mm nh4hco3 and 
 1 ml of 0.5 m nacl .
the combined eluent was desalted using a 500 mg 
 c18 cartridge ( seppak , waters ) and lyophilized .
strong cation exchange ( scx ) 
 was performed on an agilent 1120 compact lc using a 3.0  200 
 mm 5 m polysulfethyl aspartamide column ( poly lc ) with a flow 
 rate of 350 l / min and detection at 220 nm .
dried peptides were dissolved in 200 l of solvent 
 a ( 5 mm khpo4 ph 2.7 , 30% mecn ) and separated by salt gradient 
 consisting of 5 min at 100% solvent a , a 15 min gradient to 15% solvent 
 b ( solvent a + 0.35 m kcl ) , 1 min gradient to 100% b , 15 min at 100% 
 b , and a 5 min gradient to 100% a. fractions of 0.7 ml were collected 
 throughout the run and combined into 8 fractions of equal peptide 
 content based on their absorbance at 220 nm . combined fractions were 
 lyophilized prior to tio2 enrichment .
phosphopeptides were enriched 
 using tio2 in batch mode in the presence of 5% tfa and 
 1 m glycolic acid .
briefly , lyophilized 
 peptides were resuspended in 80% mecn , 5% tfa , 1 m glycolic acid prior 
 to incubation for 15 min with tio2 beads ( gl sciences ) 
 that were prewashed in the same solution .
beads were recovered by 
 centrifugation at 700  g for 1 min , washed three 
 times in 80% mecn , 5% tfa and eluted twice in 0.6% nh3 ( aq ) . 

liquid chromatography tandem mass spectrometry was performed by the 
 fingerprints proteomic facility at the university of dundee .
liquid 
 chromatography was performed on a fully automated ultimate u3000 nano 
 lc system ( dionex ) fitted with a 1  5 mm pepmap c18 trap column and a 75 m  15 cm reverse phase pepmap 
 c18 nanocolumn ( lc packings , dionex ) .
samples were loaded 
 in 0.1% formic acid ( buffer a ) and separated using a binary gradient 
 consisting of buffer a ( 0.1% formic acid ) and buffer b ( 90% mecn , 
 0.08% formic acid ) .
peptides were eluted with a linear gradient from 
 5 to 40% buffer b over 65 min .
the hplc system was coupled to an ltq 
 orbitrap velos pro mass spectrometer ( thermo scientific ) equipped 
 with a proxeon nanospray ion source . for phosphoproteomic analysis
, 
 the mass spectrometer was operated in data dependent mode to perform 
 a survey scan over a range 3351800 m / z in the orbitrap analyzer ( r = 60000 ) , 
 with each ms scan triggering 15 ms acquisitions of the 
 15 most intense ions using multistage activation on the neutral loss 
 of 98 and 49 thomsons in the ltq ion trap .
the orbitrap mass analyzer was internally calibrated on the fly 
 using the lock mass of polydimethylcyclosiloxane at m / z 445.120025 . for proteomic analysis ,
the 
 hplc gradient was increased to 145 min and the mass spectrometer was 
 operated in data dependent mode with each ms scan triggering 15 ms acquisitions of the 15 most intense ions in the ltq ion trap .
proteins were identified 
 by searching a protein sequence database containing t. brucei 
 brucei 927 annotated proteins ( version 4.0 , downloaded from 
 tritrypdb , http://www.tritrypdb.org/ ) supplemented with the vsg221 sequence and frequently observed contaminants 
 ( porcine trypsin , bovine serum albumins and mammalian keratins ) that 
 contains a total of 10,081 protein sequences .
search parameters specified 
 an ms tolerance of 6 ppm , an ms / ms tolerance at 0.5 da and full trypsin 
 specificity , allowing for up to two missed cleavages .
carbamidomethylation 
 of cysteine was set as a fixed modification and oxidation of methionines , n - terminal protein acetylation and n - pyroglutamate 
 were allowed as variable modifications .
phosphoproteomic analysis 
 included phosophorylation of serine , threonine and tyrosine as additional 
 variable modifications .
peptides were required to be at least 7 amino 
 acids in length and a maxquant score > 5 , with false discovery rates 
 ( fdrs ) of 0.01 calculated at the levels of peptides , proteins and 
 modification sites based on the number of hits against the reversed 
 sequence database .
silac ratios were calculated using only peptides 
 that could be uniquely mapped to a given protein group , and required 
 a minimum of two silac pairs . to account for any errors in the counting 
 of the number of cells mixed , the distribution of silac ratios 
 was normalized within maxquant at the peptide level so that the median 
 of log2 ratios is zero , as described by cox et al . prior to statistical analysis
, the outputs 
 from maxquant were filtered to remove known contaminants and reverse 
 sequences , and phosphorylation sites with a maxquant localization 
 probability < 0.95 were discarded .
this localization probability 
 cut off was chosen following the manual inspection of the tandem mass 
 spectra showing the best localization evidence for phosphorylation 
 sites occurring on protein kinases .
quantitation of the changes 
 in abundance are derived from the two silac labeled experiments with 
 a 1:1 mix of procyclic form cells grown in heavy - silac media and bloodstream 
 cells grown in light - silac media , and the label swap experiment with 
 the silac labeling reversed .
silac ratios for phosphorylation sites 
 were calculated using only data from the phosphoproteomic experiments , 
 and silac ratios for proteins were calculated using only data from 
 the nonenriched proteomic experiments . when silac ratios were reported 
 in both label swap experiments ,
replicate trimming was applied by 
 calculating the mean and standard deviation  of the distribution 
 of the log2 ( h / l  l / h ) , and discarding data > 2 
 from the mean ( figure 3 ) . where silac ratios were reported in only one experiment , 

they were discarded if the percentage variation in the calculated 
 silac ratio ( calculated by maxquant ) was > 100% .
the mascot 
 delta score was calculated by determining the difference between the 
 best and second best mascot ion scores for alternative phosphorylation 
 site localizations on an otherwise identical peptide sequence , and appended to the maxquant outputs .
mascot 
 distiller was used to peak pick mass spectra and read the centroid 
 data from raw tandem mass spectra and submit searches to mascot ( server 
 version 2.2 ) using the same sequence database , enzyme specificity , 
 modifications and mass tolerance as described above .
data was 
 visualized using perseus 1.3.0.4 ( www.perseus-framework.org ) and further information on the identified proteins was obtained 
 from tritrypdb ( http://www.tritrypdb.org ) .
gene ontology ( go ) term enrichment was carried out using 
 model based gene set analysis or term for term enrichment in ontologiser using a t. brucei specific 
 go set containing 16765 terms for 4340 genes obtained from genedb 
 ( http://www.genedb.org ) .
to 
 make our data accessible to the scientific community , we uploaded 
 our study to tritrypdb and deposited the thermo raw files and search 
 engine output into proteomexchange ( http://www.proteomexchange.org ) accession number pxd000049 , enabling researchers to access the 
 data presented here .
to enable silac 
 labeling of bloodstream form t. brucei cells , a modified 
 medium was created based on hmi11-t ( hmi9-t lacking serum plus ) , where l - arginine and l - lysine 
 could be replaced by stable heavy isotope forms ( hmi11-silac ) .
as 
 bloodstream form cells are usually only cultured to < 2  10 cells / ml , a 10-fold lower density than procyclic cells , we 
 first set out to establish labeling conditions that would reduce the 
 amount of label required and maximize the number of cells obtained . 

we found that the growth of bloodstream form t. brucei cells in hmi11-silac containing only 30% of the normal concentration 
 of l - arginine and l - lysine was equivalent to cell 
 growth in the hmi9-t medium generally used for bloodstream form cell 
 culture ( figure 1a ) .
as expected , the hmi11-silac 
 medium failed to support cell growth in the absence of l - arginine 
 and l - lysine whereas cells were readily able to grow to 5 
  10 cells / ml in the log  linear phase in the 
 presence of l - arginine and l - lysine ( figure 1b ) .
furthermore , the gross morphology of the cells 
 was unaffected after ten days in culture in hmi11-silac + 30% l - arginine and l - lysine , as judged by light microscopy 
 ( figure 1c ) and measurement of cell dimensions 
 by light scattering ( supplemental table 1 , supporting 
 information ) .
importantly , cells cultured in hmi11-silac + 
 30% l - arginine and l - lysine for > 14 days maintained 
 infectivity in mice ( supplemental figure 1 , supporting 
 information ) . taking all the above results into consideration
, 
 we concluded that culture of bloodstream form t. brucei in hmi11-silac + 30% l - arginine and l - lysine is 
 equivalent to culture in hmi9-t media .
cells were 
 grown in conventional hmi9-t medium or in the hmi11-silac medium developed 
 in this paper .
( a ) cumulative growth over 10 days ( with subculturing 
 every 23 days ) .
( c ) light 
 microscopy of cells after 10 days in culture . to experimentally assess the efficiency of heavy isotope 
 incorporation in hmi11-silac , bloodstream form cells were grown in 
 hmi11-silac + r6k4 for 1112 cell divisions 
 and extracted proteins subjected to analysis by lc 
if 
 heavy isotope incorporation occurred only by dilution ( neglecting 
 protein turnover ) , then 1112 cell divisions should produce 
 > 99.9% incorporation , but in reality will be limited by the isotopic 
 purity of the labeled amino acids .
the heavy isotope incorporation 
 at steady state was estimated to be 98.5  1.5% by comparing 
 the relative abundance of the major isotopic peak of the heavy ( arginine - c6/lysine - h4 ) and light 
 forms of ten peptides chosen at random .
no significant incorporation 
 of proline - c5 ( by conversion of arginine - c6 ) was observed ( supplemental figure 2 , supporting information ) , as noted previously 
 for the procyclic form .
a prerequisite for comparative quantitative 
 phosphoproteomic analyses is access to a relevant comparative quantitative 
 proteomic data with which to distinguish the different types of quantitative 
 phosphorylation differences ( i.e. , those that simply track changes 
 in protein levels , those that do not correlate with changes in protein 
 level and those that are unique to one or other lifecycle stage ) . 

our previous comparative silac proteomic analysis of bloodstream grown 
 in light medium and procyclic form t. brucei grown 
 in heavy medium gave us ratios for 3553 proteins , and demonstrated 
 that large changes in specific protein abundance occur between the 
 lifecycle stages . to improve the quality 
 of this comparative quantitative proteome data set
, we exploited the 
 development of silac labeling of bloodstream form trypanosomes , described 
 above , to perform a 
label - swap  experiment using a 
 1:1 mix of procyclic form cells grown in light - silac medium and bloodstream 
 form cells grown in heavy - silac medium .
the combined cells were solubilized 
 with sds , and the tryptic peptides generated by fasp were fractionated 
 by scx prior to analysis as described previously , except that six scx factions were used and a longer gradient 
 was applied for the lc 
a total of 3780 proteins were identified , with silac ratios 
 determined for 3313 of them .
the new label - swap data set showed very 
 good agreement with our published silac data , with a comparable depth of coverage and a pearson correlation of 
 0.92 ( figure 3a ) . given the good correlation 
 between the two data sets , we combined them to provide a definitive 
 comparative quantitative proteome with which to interpret our phosphoproteomic 
 data .
the objective 
 of this study was : ( i ) to define the phosphoproteome of procyclic 
 form t. brucei for the first time ; ( ii ) to enhance 
 the existing phosphoproteome of bloodstream form t. brucei .
( iii ) to quantify the changes in phosphorylation 
 state which occur between the lifecycle stages using silac methodology . 

four separate phosphoproteomic experiments were conducted ( figure 2 ) starting from : ( i ) procyclic form cells grown 
 in light - silac medium .
( ii ) a 1:1 mix of procyclic form cells grown 
 in heavy - silac medium and bloodstream form cells grown in light - silac 
 medium . (
 experiment consisting of 
 a 1:1 mix of bloodstream form cells grown in light - silac medium and 
 procyclic cells grown in heavy - silac medium .
to maximize coverage of membrane 
 and structural proteins , total protein extracts were prepared with 
 4% sds using the filter - aided sample preparation ( fasp ) procedure , resulting in near - complete solubilization of 
 the parasites .
resulting phosphopeptides 
 were enriched and fractionated into eight samples by scx chromatography prior to further enrichment using tio2 chromatography in batch mode . for each 
 phosphoproteomic experiment ,
the eight fractions were analyzed by 
 liquid chromatography tandem mass spectrometry in technical triplicates 
 on a ltq oribtrap velos using multistage acquisition .
differentially labeled 
 ( heavy and light ) bloodstream and procyclic form trypanosomes were 
 lysed in sds , and total proteins s - alkylated and digested with trypsin 
 using the fasp method .
phosphopeptides were separated into eight fractions 
 by scx hplc , enriched with tio2 , and analyzed by lc 
ms / ms 
 runs , with 96 runs for samples enriched for phosphopeptides and 88 
 for samples with silac labeling .
altogether 589101 ms / ms spectra 
 were identified , corresponding to 58087 nonredundant peptide sequences 
 belonging to 5478 protein groups with a false discovery rate of 1% 
 ( supplementary tables s2 and s3 , supporting information ) .
a total of 10096 phosphorylation sites were identified on 2551 
 proteins ( supplementary tables s4 and s5 , supporting 
 information ) , of which silac ratios could be determined for 
 8275 phosphorylation sites on 2233 protein groups , with a pearson 
 correlation of 0.78 between the label - swap experiments .
a total of 
 8120 phosphorylation sites on 2137 protein groups were also identified 
 using mascot software , of which 6196 phosphorylation sites on 1933 
 protein groups had a mascot delta score 10 , reported to give 
 a 1% false localization rate ( flr ) for orbitrap - msa data .
of the 10096 phosphorylation sites identified 
 by maxquant , 9314 ( 92% ) have not previously been reported .
the data 
 presented here therefore represents a major advance in the depth of 
 the trypanosome phosphoproteome .
black squares < 2  , gray square > 2 . the distribution of phosphorylation between residues 
 was found to be 8087 phosphoserine ( ps ) sites ( 80% ) , 1,720 phosphothreonine 
 ( pt ) sites ( 17% ) , and 289 phosphotyrosine ( py ) sites ( 3% ) , in reasonable 
 agreement with the t. brucei bloodstream form cytosolic 
 phosphoproteome observations of 75% ps , 21.5% pt , 3.5% py . of the 10096 phosphorylation sites observed , 
 6493 occurred on 1491 proteins annotated as hypothetical or hypothetical 
 conserved , reflecting the high proportion of genes of unknown function 
 in the trypanosome genome .
gene ontology terms are associated with 
 only 1310 out of the 2552 phosphoproteins we observed , limiting the 
 power of go term enrichment analysis . performing model based gene 
 set analysis , a method designed to give 
 a high - level summarized view of the data , identified only three terms : 
 cilliary or flagella motility ( go:001539 , pep 1 ) , protein tyrosine 
 kinase activity ( go:004713 , pep 0.7 ) , and regulation of gene expression 
 ( go:0010468 , pep 0.5 ) .
this is an intriguing outcome as the t. brucei genome lacks any identifiable dedicated tyrosine 
 kinases , yet has the associated go terms , illustrating 
 the pitfalls of such analyses .
performing term - for - term enrichment 
 generated 45 significantly enriched go terms ( p < 
 0.01 , figure 4 ) confirming the predominance 
 of terms associated with flagella motility , protein kinase activity 
 and regulation of gene expression , and revealing additional terms 
 for rna , zinc and calmodulin binding ( go:0005488 , go:0008270 , go:0005516 ) , 
 and peptidase activity ( go:0004181 , go:0004197 ) .
greater biological 
 insight from the data is likely to come from the community inspecting 
 the phosphorylation patterns , and their changes between lifecycle 
 stages , of proteins of specific interest to them , a few examples of 
 which are provided below .
term - for - term enrichment , p < 
 0.01 ; white box , biological process ; light gray box , cellular component ; 
 dark gray box , molecular function . for clarity
parental terms are 
 omitted ; the complete graph can be found in the supporting information ( figure s3 ) .
silac labeling allows the fold - changes 
 that occur between the bloodstream and procyclic form of t. 
 brucei ( log2 pcf / bsf ) to be quantified at both 
 the protein and phosphorylation site level .
as the phosphoproteomic 
 workflow includes selective enrichment for phosphorylated peptides , 
 this data can not be used in the calculation of protein silac ratios , 
 which are instead derived from separate proteomic experiments .
we 
 were able to determine fold - changes for a total of 8275 phosphorylation 
 sites on 2233 protein groups , with 5768 ( 70% ) of the phosphorylation 
 sites on 1385 protein groups having ratios at both the protein- and 
 site - level .
in general , the changes in phosphorylation site occupancy 
 between the lifecycle stages are greater than the changes in protein 
 levels ; for example , 318 ( 5.5% ) of the phosphorylation sites change 10-fold 
 ( log2 |3.32| ) between lifecycle stages , while only 
 33 ( 0.87% ) of protein groups change 10-fold .
indeed , plotting 
 the fold - changes in protein ratio against the fold - changes in phosphorylation 
 site ratio ( figure 5a ) reveals that the observed 
 changes are relatively weakly correlated ( pearson correlation 0.49 ) . 

however , there are numerous examples where : ( i ) the change in phosphorylation 
 correlates with the change in protein abundance , suggesting that phosphorylation 
 stoichiometry is maintained between lifecycle stages in these cases . 

( ii ) the change in phosphorylation correlates negatively with the 
 change in protein abundance .
( iii ) protein abundance is maintained 
 but phosphorylation stoichiometry changes significantly ( both up and 
 down ) .
( iv ) different phosphorylation sites on the same protein show 
 a wide range of fold - changes ( figure 5b and 
 figure 6 ) .
these data demonstrate that changes 
 in protein phosphorylation status between the lifecycle stages of t. brucei add significant complexity to the differential 
 expression of the proteome .
correlation of fold - changes in protein and fold - changes 
 in phosphorylation sites .
( b ) correlation of average fold - change in phosphorylation sites for 
 each phosphoprotein , with bars indicating the upper and lower limits 
 of the range . in order to judge the quality 
 of the phosphorylation site assignments , and to empirically assess 
 the false localization rate ,
a subset of the automated phosphorylation 
 site assignments were manually inspected ( figure 6 and supporting information ) .
the subset chosen was the t. brucei kinome consisting of 176 protein kinases as they are expected to occur at relatively 
 low abundance and to show physiologically relevant phosphorylation . 

using a maxquant localization score of 0.75 as an initial cutoff , 
 we identified 641 specific phosphorylation sites on 127 protein kinases 
 ( for comparison a cutoff of 0.95 gave 493 sites on 124 kinases ) . 

of these 641 automatically annotated sites on 127 kinases , 549 sites 
 on 122 kinases were validated by manual inspection ( supplemental table 
 s6 , supporting information ) .
overall , the 
 manual validation correlated well with the sites chosen using the 
 maxquant localization score and the mascot delta score ( mds ) methods .
however , we noted that only 42% of sites in 
 the maxquant localization score range 0.750.95 were manually 
 validated , whereas over 94% of the sites maxquant localization score 
 > 0.95 were manually validated , increasing to over 98% at a localization 
 score of > 0.99 .
further , at an mds of 10 , reported to give a global 
 1% false localization rate for orbitrap velos 
msa data , we were able to manually confirm 98% of the 
 automatically annotated sites .
however , it is worth noting that 81% 
 of sites with mds < 10 and 66% of sitesidentified by maxquant 
 but not by mascot were manually validated .
based on this manual survey 
 of a significant number of phosphorylation site assignments , we have 
 chosen to report all automatically annotated phosphorylation sites 
 with maxquant localization scores > 0.95 ( with an estimated false 
 localization rate of < 6% ) .
( a ) ste11 family protein 
 kinase ( tb11.46.003 ) with ps357 and ps358 with maxquant localization 
 score of 1.0 and mascot delta score of 57.8 .
( b ) camk family protein 
 kinase tb11.01.2290 with ps5 with maxquant localization score of 1.0 
 and mascot delta score of 27.1 , and ps8 with maxquant localization 
 of 1.0 but which was not identified by mascot .
data acquired on a 
 ltq orbitrap velos using msa , * designates loss of phosphate ( 98 
 da ) , * * loss of two phosphates ( 196 da ) .
interestingly , the variation in the phosphorylation of the 
 kinome between the two lifecycles stages is lower than the general 
 data set , with only 15 phosphorylation sites showing a 10-fold or 
 greater change .
the protein kinase a catalytic subunit isoform 1 ( tb09.211.2410 ) 
 showed strongly up - regulated phosphorylation in the bloodstream form 
 parasite at three sites ( log2 range 5.0 to 4.3 ) , 
 although the assignment of increased phosphorylation in the putative 
 activation loop at t179 ( log2 2.6 ) is slightly 
 ambiguous as an identical ( phospho)peptide is shared by protein kinase 
 a catalytic subunit isoform 2 ( tb09.211.2360 ) .
similarly , the protein 
 kinase a regulatory subunit ( tb11.02.2210 ) has 5 sites which are also 
 up - regulated in the bloodstream form , though to a lesser extent ( log2 range 4.4 to 0.8 ) .
protein kinases with phosphorylation 
 up - regulated in the procyclic form include a nek kinase ( tb11.01.6650 ) 
 with two sites with a log2 range 5.6 to 4.7 , and two clk 
 dual specificity kinases tb927.2.4200 ( 5 sites , log2 range 
 3.3 to 0.6 ) and tb927.10.15020 ( 4 sites , log2 range 3.2 
 to 2.4 ) .
two  other  group protein kinases have phosphorylation 
 sites that are up - regulated in the procyclic and bloodstream form , 
 respectively , ( tb927.7.3210 , 5 sites , range log2 3.7 
 to 2.8 ; tb927.7.3220 , 5 sites , range log2 3.2 to 
 1.6 ) . of the 15 mapks present in the t. brucei kinome , 9 ( eck1 , kfr1 , mok , rck , mapk2 , mapk9 , mapk10 , mapk12 , and 
 an unnamed mapk )
are shown by the data presented here to have dual 
 phosphorylation in the txy motif required for activation .
in addition , 
 multiple phosphorylation sites are observed on three potential upstream 
 ste7 map2k and nine ste11 map3k protein kinases , suggesting the presence 
 of active mapk signaling cascades .
the observation of differential 
 phosphorylation on certain mapks , map2ks and map3ks between the two 
 lifecycle stages suggests that mapk signaling may play distinct roles 
 in each lifecycle stage ( figure 7 ) .
heatmap showing the 
 change in abundance between the procyclic to bloodstream ( log2 ) at the protein and phosphorylation site level for the mapk , 
 ste7 and ste11 kinase quantified in this study .
a total of 203 phosphorylation sites on 119 protein groups were 
 up - regulated 10-fold ( log2  3.32 ) 
 in bloodstream form t. brucei , with a distribution 
 of these up - regulated phosphorylation sites of 74% ps , 15% pt , 11% 
 py , showing a significant enrichment in py compared to the total observations . 

an additional 429 sites with no silac ratio ( on 319 protein groups ) 
 were observed exclusively in the bloodstream form , with the distribution 
 of 64% ps , 27.5% pt , 8.5% py showing a similar trend .
these data suggest 
 that tyrosine phosphorylation is significantly over represented in 
 sites differentially regulated between the lifecycle stages . in total ,
439 proteins have 631 individual phosphorylation sites 
 that are either 10-fold up - regulated in the bloodstream form 
 or are bloodstream form specific ( supplemental table s7 , supporting information ) .
this group contains 
 a large number of proteins annotated as hypothetical or hypothetical 
 conserved , including 13 of the 15 proteins with > 40-fold change 
 ( log2 < 5.32 ) at the site level .
go term enrichment 
 of the bloodstream form up - regulated group ( of the 191 annotated proteins 
 out this total 439 ) produced a very similar set of enriched terms 
 to the global phosphoproteome ( flagella , protein kinase , and gene 
 expression ) , with the addition of terms for glycolysis and energy 
 metabolism , although this may be due , at least in part , to large changes 
 at the protein level in some of these proteins .
nevertheless , researchers interested in the changes in 
 parasite metabolism between the procyclic and bloodstream form parasites 
 are likely to find clues to physiologically relevant phosphorylation 
 changes in the data provided .
significantly , there are examples where 
 protein up - regulation in bloodstream form parasites may be further 
  amplified  by the coordinate up - regulation of phosphorylation
. 
 notable examples of this include the rna recognition motif protein 
 rbp10 ( tb927.8.2780 ) that has been reported to promote a bloodstream 
 form mrna expression pattern , which has 
 4 phosphorylation sites changing by an average of log2 4.8 
 ( from log2 5.4 to 4.2 ) , potentially amplifying 
 a change in protein abundance of log2 3.3 , and 
 the receptor - type adenylate cyclase gresag4 ( tb927.8.7940 ) which has 
 6 phosphorylation sites changing by an average of log2 3.9 
 ( from log2 4.7 to 3.1 ) , potentially amplifying 
 a change in protein abundance of log2 2.4 .
a total of 115 phosphorylation sites on 83 protein groups were 
 10-fold up - regulated ( log2  3.32 ) in procyclic 
 form t. brucei with a residue distribution of 70% 
 ps , 22% pt , 9% py , and an additional 499 sites with no silac ratio 
 on 376 protein groups were observed exclusively in the procyclic form 
 with a residue distribution of 77% ps , 15.5% pt , 7.5% py .
the significant 
 enrichment in py compared to the total observations mirrors that seen 
 in the bloodstream form up - regulated phosphorylation sites , reinforcing 
 the importance of changes in the phosphorylation of tyrosine between 
 the lifecycle stages . in total ,
459 proteins have 614 individual 
 phosphorylation sites that are either 10-fold up - regulated 
 in the procyclic form or are procyclic form specific ( supplemental 
 table s8 , supporting information ) .
this 
 procyclic up - regulated group contains a large number of proteins annotated 
 as hypothetical or hypothetical conserved , as observed for the bloodstream 
 form up - regulated group .
go term enrichment of the procyclic up - regulated 
 group ( of 207/460 proteins ) again produced a very similar set of enriched 
 terms to the global and bloodstream form up - regulated phosphoproteome 
 ( flagella , protein kinase , and gene expression ) with no significant 
 new terms .
a total of 9 proteins annotated as transporters have > 10-fold 
 change ( log2 > 3.32 ) at the site level , potentially 
 amplifying the corresponding changes in protein abundance .
interesting 
 specific examples include : the pad2 carboxylic acid transporter ( tb927.7.5940 ) , 
 associated with slender to stumpy bloodstream form transformation 
 in pleomorphic cell lines , where 3 sites 
 increase in phosphorylation with an average log2 of 3.3 
 ( range log2 1.5 to 6.2 ) in addition to a protein level 
 increase of log2 2.9 . the ubiquitin carboxy - terminal hydrolase 
 ( tb927.10.2210 ) , which has 10 phosphorylation sites with an average 
 log2 of 3.6 ( range log2 1.6 to 5.6 ) , while the protein has almost no change in level ( log2 0.41 ) . the c-14 sterol reductase ( tb11.01.7170 ) , 
 involved in the biosynthesis of ergosterol and its isomers that are 
 the principal sterols in the procyclic parasites , has 2 phosphorylation sites with an average log2 of 3.8 ( range log2 3.7 to 3.9 ) , 
 potentially amplifying the protein fold change of log2 2.2 .
a significant 
 number of proteins that are multiply phosphorylated exhibit phosphorylation 
 sites that are differentially regulated within the protein between 
 the lifecycle stages ( figure 8) .
there are 
 234 proteins ( supplementary table s9 , supporting 
 information ) where the range of fold - changes of phosphorylation 
 sites within the protein is greater than 10-fold ( range log2 >
|3.32| ) , with an average of 10 phosphorylation sites per protein . 
 interestingly , of the 18 proteins where the change in site occupancy 
 is greater than 80-fold ( range log2 >
go term 
 enrichment analysis ( of 113/234 proteins ) produced significantly enriched 
 terms ( p < 0.01 ) including flagellum , gene regulation , 
 and protein phosphatase activity , but that did not include protein 
 kinase activity .
three protein phosphatases are found in the intraprotein 
 differentially regulated group : ( i ) a kinetoplastid - specific protein 
 phosphatase ( tb11.01.4320 ) with a differential range log2 of |6.6| ( 10 sites , range log2 4.8 to 1.8 ) .
( ii ) 
 an atypical dual specificity phosphatase ( tb11.01.5870 ) with a differential 
 range log2 of |5.6| ( 3 sites , range log2 3.8 
 to 1.7 ) .
( iii ) the tfiif - stimulated ctd phosphatase ( tb927.10.11450 ) 
 with a differential range log2 of |4.1| ( 4 sites , range 
 log2 2.1 to 2.0 ) .
distribution of intraprotein differentially 
 regulated phosphorylation sites . only proteins with more than one 
 phosphorylation site are shown .
we have previously developed silac labeling 
 in procyclic form t. brucei,(3 ) and here we report the development of silac labeling in the clinically 
 relevant bloodstream form and demonstrate that growth , morphology 
 and infectivity are unchanged .
both lifecycle stages are rapidly dividing , 
 enabling efficient isotope incorporation > 98% , and show no propensity 
 to convert arginine to proline .
two recent proteomic analysis using 
 silac labeling in either procyclic or 
 procyclic and bloodstream formt .
the quantitation of changes in the global phosphoproteome 
 between procyclic and bloodstream form lifecycle stages of trypanosoma brucei , described here , reveals that differential 
 phosphorylation is widespread , is not strongly correlated to change 
 in protein abundance and adds significant complexity to the differential 
 expression of the proteome .
the observation of intraprotein differential 
 phosphorylation suggests that phosphorylation may play distinct functional 
 roles in each lifecycle stage , although how this effect is mediated 
 is at present unclear and , because of the lack of comparative phosphorylation 
 data to date , there are few existing hypotheses to examine .
however , 
 one process where protein phosphorylation is suggested to play a central 
 role is the differentiation of the parasite from bloodstream to procyclic 
 form in a coordinated , programmed event . while we have not studied the actual process of differentiation 
 in this study , we have examined the phosphoproteomes of the initial 
 and final states of the process , and observe phosphorylation changes 
 in many of the proteins involved in differentiation .
for example , 
 tor4 ( tb927.1.1930 ) has 10 sites with 
 log2 changes in the range 0.42.7 , mapk5 ( tb927.6.4220 ) has a protein change log2 of 0.5 
 and pt207 site change log2 of 0.8 ; zfk ( tb11.01.1030 ) has a ps520 log2 of 1.3 ; 
 nopp44/46 ( tb927.8.760 ) has a protein 
 log2 of 1.7 and py5 observed uniquely in procyclic parasites ; 
 pad2 ( tb927.7.5940 ) has a protein log2 of 2.9 and 3 sites with log2 in the range 1.5 
 to 6.2 .
the absence of detected phosphorylation on pip39 ( tb09.160.4480 , 
 protein log2 of 0.9 ) , and ptp1 ( tb927.10.6690 , not observed 
 in our data set ) may be due to up - regulation or transient phosphorylation 
 during differentiation , such as in the division arrested stumpy form 
 that was not studied here .
alternatively , the cocktail of phosphatase 
 inhibitors used in this study may have limited effect against the 
 activity of one or more trypanosome phosphatase .
clearly , further 
 detailed studies of the process are required before firm conclusions 
 can be drawn on the precise rules(s ) of protein phosphorylation in 
 trypanosome differentiation . in this study
we have used culture 
 adapted lister 427 bloodstream form ( mitat 1.2 ) and procyclic form 
 ( 29.13.6 ) cell lines that have been made amenable to genetic manipulation 
 by wirtz et al . by the insertion of exogenous genes coding for the 
 t7 rna polymerase and tetracyclic repressor protein using either one 
 ( bloodstream form ) or two ( procyclic form ) selectable drug resistance 
 markers .
the two cell lines used are 
 independent clones that are not genetically identical , and this may 
 lead to some differences in protein expression and phosphorylation 
 status , although such differences are likely to be minor in comparison 
 to the developmentally regulated changes between the lifecycle stages . 
 in addition , the culture adapted lister 427 strains are neither genetically 
 nor morphologically identical to field isolates , and it is not possible 
 to account for the impact this will have on protein expression and 
 phosphorylation status .
a significant proportion of the t. brucei kinome of 176 protein kinases are phosphorylated , 
 with individual examples of phosphorylation sites up - regulated in 
 either procyclic or bloodstream form , and examples of intraprotein 
 differential phosphorylation .
the abundance of active map kinases , 
 as judged by the presence of dual txy motif phosphorylation , suggests 
 that mapk pathways are functional in both lifecycle stages of t. brucei , although how or whether the pathways are regulated 
 remains unclear .
efforts to determine the detailed biological role 
 of t. brucei protein kinases are ongoing and knock - down 
 by rna interference has provided evidence of the essential nature 
 of a significant number of protein kinases .
we have recently used chemical proteomic profiling to establish 
 that t. brucei protein kinases are sensitive to typical 
 kinase inhibitors with nanomolar potency , demonstrating the potential 
 for the development of species - specific inhibitors .
response studies , using the data published 
 here to anchor them , may reveal the pinch - points that are most suitable 
 for therapeutic intervention .
often , eukaryotic phosphorylation 
 signaling cascades terminate in the modulation of transcription factors 
 to mediate changes in gene expression .
however , classical transcription 
 factors are missing in t. brucei , where the processing 
 and stability of polycistronically transcribed mrna precursors appears 
 to regulate gene expression .
one potential 
 mechanism to modulate gene expression in t. brucei is the use of rna binding zinc finger proteins to influence mrna 
 processing and stability , such as such as zfp3 ( tb927.3.720 ) and rbp10 ( tb927.8.2780 ) .
a significant number of zfps ( 31 out of 49 ) and rbps ( 13 
 out of 47 ) are present in the phosphoproteomic data reported here , 
 with a subset of these ( 11 and 5 , respectively ) showing lifecycle 
 specific regulation of phosphorylation status ( 5-fold change ) . 

since the dynamic phosphorylation / dephosphorylation of these proteins 
 has the potential to modulate gene expression , they are reasonable 
 candidates to be the ultimate effector molecules of at least some 
 of the trypanosome signaling cascades . despite the lack of identifiable 
 dedicated tyrosine kinases in t. brucei
, 234 phosphotyrosine 
 residues were identified , and tyrosine phosphorylation was 34 
 fold over - represented in the phosphorylation sites that change between 
 the two lifecycle stages .
tyrosine phosphorylation is proposed to 
 arise from putative dual - specificity protein kinases such as dyrk , 
 mek and clk , but there has been no experimental verification of the 
 dual specificities of the trypanosome kinases to date .
go term 
 enrichment analysis has revealed that the flagellum is consistently 
 over - represented among the phosphorylated proteins .
flagella can play 
 both motility and sensory roles , and while it is clear that the trypanosome 
 flagellum is highly motile , its potential sensory role is less well - defined .
the dynamic phosphorylation of dynein motors 
 is proposed to control flagella motility in other organisms , and in 
 trypanosomes flagella motility is dispensable in the procyclic form 
 but essential for cytokinesis in the bloodstream form . interestingly ,
a number of flagella proteins 
 show extensive intraprotein differential phosphorylation , including : 
 ( i ) dynein heavy chain ( tb927.7.920 ) with 7 sites with log2 changes in the range 2.8 to 3.2 and protein log2 changes of 0.5 ; ( ii ) a paraflagella rod component ( tb927.8.1550 ) 
 with 14 sites with log2 changes in the range 3.1 
 to 2.0 and a protein log2 change of 0.4 ; and ( iii ) 
 an outer dynein docking complex ( tb11.01.7750 ) with 3 sites with log2 changes in the range 3.6 to 1.8 and a protein log2 change of 0.6 .
in summary , we have 
 presented a global quantitative phosphoproteomic analysis of the bloodstream 
 and procyclic form of trypanosoma brucei , which represents 
 both the largest set of phosphorylation sites and the first report 
 of genome wide quantitative phosphorylation data for any kinetoplastid . 

the establishment of silac labeling in both culture - amenable lifecycle 
 stages of the parasite provides the community with the tools required 
 to examine signaling events at the molecular level .
this data set 
 reveals the breadth of protein expression and phosphorylation in t. brucei , and provides a resource for the community to 
 begin to examine the effect of phosphorylation upon biological processes . 

the present study of rapidly dividing nonsynchronized cells provides 
 a static , averaged snapshot of the basal level of protein phosphorylation , 
 and valuable information will be gained from future studies of the 
 dynamics of stimulus - response events such as differentiation .
 we 
 report a global quantitative phosphoproteomic study of bloodstream 
 and procyclic form trypanosoma brucei using silac 
 labeling of each lifecycle stage . 
 phosphopeptide enrichment by scx 
 and tio2 led to the identification of a total of 10096 
 phosphorylation sites on 2551 protein groups and quantified the ratios 
 of 8275 phosphorylation sites between the two lifecycle stages . more 
 than 9300 of these sites ( 92% ) have not previously been reported . 
 
 model - based gene enrichment analysis identified over representation 
 of gene ontology terms relating to the flagella , protein kinase activity , 
 and the regulation of gene expression . 
 the quantitative data reveal 
 that differential protein phosphorylation is widespread between bloodstream 
 and procyclic form trypanosomes , with significant intraprotein differential 
 phosphorylation . despite a lack of dedicated tyrosine kinases , 
 234 
 phosphotyrosine residues were identified , and these were 34 
 fold over - represented among site changing > 10-fold between the 
 two lifecycle stages . 
 a significant proportion of the t. brucei kinome was phosphorylated , with evidence that mapk pathways are 
 functional in both lifecycle stages . 
 regulation of gene expression 
 in t. brucei is exclusively post - transcriptional , 
 and the extensive phosphorylation of rna binding proteins observed 
 may be relevant to the control of mrna stability in this organism . 
Introduction
Experimental Section
Results
Discussion
Conclusions