eukaryotic cells possess 
 membrane - bound organelles called peroxisomes , 
 defined by the presence of the pex proteins responsible for their 
 biogenesis . peroxisomes generally contain enzymes to deal with reactive 
 oxygen species and enzymes of fatty acid -oxidation . beyond these common functions ,
the peroxisomes of the trypanosomatids 
 are called glycosomes because , uniquely , they contain most of the 
 enzymes of glycolysis .
ether lipid and isoprenoid / sterol 
 biosynthesis are relatively common peroxisomal functions , and these 
 too have been demonstrated and suggested , respectively , to be present 
 in trypanosomatids .
other metabolic processes that 
 are atypically compartmentalized in glycosomes include purine salvage , 
 the latter stages of pyrimidine biosynthesis , and 
 much of sugar nucleotide biosynthesis .
an inventory of an organelle s contents is highly desirable 
 to help understand its functional significance , its mechanism(s ) of 
 biogenesis , and its metabolite transport systems . given the parasitic 
 nature of the trypanosomatids and the serious human and animal diseases 
 inflicted by them , glycosome metabolism and organization are of considerable 
 interest with respect to identifying therapeutic opportunities related 
 to their unusual metabolic compartmentalization and requisite metabolite 
 transport . here
this organism causes human african trypanosomiasis 
 ( african sleeping sickness ) and nagana in cattle and is transmitted 
 by the tsetse fly in much of sub - saharan africa .
the procyclic form 
 of the parasite normally resides in the gut of the tsetse fly vector 
 and is the life cycle stage most readily grown to moderate densities 
 in cell culture .
given that subcellular fractionation can only 
 provide an enrichment 
 of a given organelle , the principle problem in obtaining high - confidence 
 organelle proteomes is in deciding which of the many hundreds or thousands 
 of protein  hits  are genuine components of the organelle 
 rather than contaminants from other locations .
we therefore decided 
 to approach this problem for the trypanosome glycosomes by using epitope - tagged 
 glycosomes and stable isotope labeling in cell culture ( silac ) , such 
 that in antiepitope pull - out experiments we can distinguish genuine 
 glycosomal components from contaminants according to peptide isotopic 
 signatures .
the general approach is summarized in figure 1 , and we compare our results to previously published 
 glycosome proteomes .
overview of the approach taken to obtain a high - confidence 
 glycosome 
 proteome .
wild - type procyclic form trypanosomes were grown in normal 
 ( r0k0 , light ) medium , and cells transfected to express gfp - tagged 
 pex13.1 were labeled with heavy ( r6k4 ) lys and arg .
the cells were 
 mixed 1:1 , and glycosomes were enriched by centrifugation , followed 
 by affinity - selection on anti - gfp magnetic beads .
peptides from genuine 
 glycosome components have high heavy / light isotope ratios , whereas 
 those from contaminants have isotope ratios close to 1:1 .
procyclic 
 form t. brucei 427 strain 
 containing t7 rna polymerase and tet repressor protein , respectively , 
 under control of g418 and hygromycin ( clone 29.13.6 cells , kindly 
 provided by george cross and referred to from hereon as wild - type 
 cells ) were grown at 28 c without 
 co2 in original sdm-79 medium , containing 15% ( v / v ) heat inactivated fetal bovine serum ( fbs ) , 
 2 g / l sodium bicarbonate , 2 mm glutamax i ( invitrogen ) , 22.5 g / ml 
 hemin ( added from a 10 mg / ml stock in 50 mm naoh ) , and ph adjusted 
 to 7.3 .
hygromycin b ( roche ) at 50 g / ml and g418 ( invitrogen ) 
 at 15 g / ml were also added .
these cells were electroporated 
 with 10 g / cuvette of not i linearized plasmid pgc1 containing 
 an orf encoding the full length t. brucei pex13.1 
 protein sequence ( tb927.10.14720 ) fused to an n - terminal green fluorescent 
 protein ( egfp ) tag , a kind gift from paul michels .
electroporation conditions were as previously described , and selection was done using 10 g / ml 
 of blasticidin ( invitrogen ) .
the resistant cells were cloned by dilution 
 to a single cell / ml in 96-well plates using sdm-79 containing all 
 reagents previously described but with 20% ( v / v ) heat - inactivated 
 fbs and 4
g / l sodium bicarbonate and grown in a co2 incubator 
 at 28 c .
thirty clones were selected and activated with tetracycline 
 added from a freshly prepared 1 mg / ml stock in 70% ethanol .
clones 
 were tested by western blot and by fluorescence microscopy to confirm 
 the appropriate gfp - tagging of pex13.1 and its glycosomal localization .
the silac labeling of t. brucei procyclic 
 form cells was performed as described recently . in brief ,
log phase parasites were washed three times with 
 10 ml of sdm-79 depleted of l - lysine and l - arginine 
 ( sdm-79-rk ) and used to seed cultures where the sdm-79-rk medium was 
 supplemented with either normal isotopic abundance l - arginine 
 and l - lysine ( sdm-79 + r0k0 , referred to as light label ) 
 or with [ u  c]-l - argininehcl and 
 [ 4,4,5,5-h]-l - lysine2hcl ( sdm-79 + r6k4 , 
 referred to as heavy label ) .
the stable isotope - labeled amino acids 
 were obtained from cambridge isotope laboratories , u.k .
the heavy 
 and light labels were used at the same concentration , as described 
 in the original sdm-79 formulation . in 
 general , apart from the label - swap ( table 1 , experiment 4 ) , wild - type cells were suspended at 2.5  10 cells / ml in sdm-79 + r0k0 , and gfp - pex13.1 expressing cells 
 were suspended in sdm-79 + r6k4 at the same concentration .
cultures 
 were diluted about 7.7-fold ( back to 2.5  10 cells / ml ) every 2 days with fresh media to allow for eight cell 
 divisions under the labeling conditions and to enlarge cultures to 
 about 500 ml at final concentration of 2.5  10 cells / ml . the expression of gfp - pex13.1 was induced by adding 40 
 ng / ml fresh tetracycline 16 h prior to harvesting .
cells were counted , 
 and r0k0- and r6k4-labeled cells were mixed 1:1 , lysed , immunoprecipitated 
 using anti - gfp magnetic beads , eluted , and processed for proteomics , 
 as described later .
biological replicate 4 was a label - swap 
 experiment , performed in parallel with replicate 3 .
trypanosome procyclic forms were 
 labeled with unlabeled l - arg and l - lys , designated 
 as r0k0 or with [ c6 ] -l - arg and [ h4]-l - lys , designated as r6k4 .
tryptic peptides were generated 
 either by trypsin in - gel digestion of protein bands from sds - page 
 or by filter aided sample preparation ( fasp ) and further fractionation 
 using strong cation exchange chromatography ( scx ) .
the silac label - chase experiment 
 was performed similarly , except 
 that after steady - state labeling for eight cell divisions both cultures 
 were split in half .
one half of each culture was washed twice with , 
 and resuspended in , r0k0 media to perform a chase for 5 h at 28 c , 
 while the other halves of the steady - state labeled cultures were simply 
 harvested at the end of the labeling period and processed as described 
 later .
light and heavy amino - acid - labeled cells were harvested separately 
 by centrifugation at 600 g for 10 min at 4 c , 
 washed twice , and resuspended in 250 mm sucrose , 25 mm tris - hcl ph 
 7.4 , and 1 mm edta ( ste buffer ) , counted using a casy innovatis counter 
 ( scharfe ) , and mixed 1:1 to obtain a final cell suspension of 2  
 10 cells / ml in ste containing 0.1 mm 1-chloro-3-tosylamido-7-amino-2-heptone 
 ( tlck ) , 1 g / ml leupeptin , 1 g / ml aprotinin , and complete 
 protease inhibitor cocktail without edta ( roche ) .
the combined cells 
 were lysed with 2 shots of 10  000 psi using a one shot ( constant 
 cell disruption system ; northants , u.k . ) .
these conditions of cell 
 density and pressure had previously been optimized to give the best 
 yield of glycosomes through a conventional density centrifugation 
 procedure .
the lysates were centrifuged 
 twice at 2000 g for 15 min at 4 c to remove 
 any remaining live cells , cell ghosts . and nuclei .
the supernatant 
 was then centrifuged twice at 5000 g for 15 min to 
 remove material enriched in mitochondria , known as large granular 
 fraction .
( note : further subcellular fractionation , by pelleting the 
 small granular fraction , resuspending , and performing density gradient 
 centrifugation , was avoided because previous 
 experiments had shown that anti - gfp immuno - affinity enrichment of 
 the tagged pex13.1-gfp glycosomes was compromised when this was performed , 
 possibly due to glycosome aggregation or membrane fusion during the 
 high - speed pelleting step . )
the supernatant from the 5000 g spin , enriched in glycosomes and endoplasmic reticulum ( er ) and 
 containing cytosol , was precleared by rotating with 0.2 ml of magnetic 
 beads without antibody for 1 h at 4 c .
the preclearing beads 
 were removed on a magnet , and the supernatant was immunoprecipitated 
 using magnetic beads coupled to anti - gfp , as described in table 1 , using either 16 g of immunopure anti - gfp 
 mouse monoclonal antibody ( roche ) and 0.2 ml magnetic protein g dynabeads 
 at 30 mg / ml ( invitrogen ) for 1 h at 4 c or camelid anti - gfp 
 ( chromoteck , germany ) precoupled to nhs - magnetic beads according to 
 the manufacturers instructions ( pierce , thermo scientific ) for 1 h 
 at 4 c .
beads were washed three times with 1 ml of ste then 
 once with tris - buffered saline ( tbs ) and resuspended in 35 l 
 of tbs .
samples for analysis 
 by mass spectrometry were prepared either by sds - page separation followed 
 by in - gel trypsin digestion or by fasp followed by strong cation exchange 
 ( scx ) chromatography . for sds - page ,
an equal volume of 2 sds - sample 
 buffer and 0.1 m fresh dithiothreitol ( cleland s reagent , ultrol 
 grade , calbiochem ) was added and heated to 50 c for 20 min . 

samples were run in 10% nupage gels with mops running buffer and stained 
 with simply blue ( invitrogen ) .
gel lanes were cut in slices , as shown 
 in figure s1 in the supporting information , and washed sequentially in 50% ( v / v ) acetonitrile , 0.1 m nh4hco3 , 0.1 m nh4hco3/acetonitrile 
 ( 1:1 v / v ) , and acetonitrile .
gel pieces were dried in speed - vac ( thermo 
 scientific ) in - gel reduced with 0.01 m dithiothreitol , alkylated with 
 0.05 m iodoacetamide ( sigma ) for 30 min in dark , washed in 0.1 m nh4hco3 , and digested with 12.5 g / ml modified 
 sequence grade trypsin ( roche ) in 0.02 m nh4hco3 for 16 h at 30 c .
tryptic peptides were recovered in 50% acetonitrile 
 and 2.5% formic acid and lyophilized prior to analysis by lc  ms / ms . for filter - aided sample preparation ( fasp ) ,
an equal volume of 4% sds in 0.1 m tris - hcl ph 7.2 containing 
 0.1 m dithiothreitol was added , and fasp protocol ii was followed 
 but using 10  000 mwco vivacon 500 filters ( sartorius ) .
tryptic 
 peptides obtained from fasp were desalted using c18 empore cartridges 
 ( agilent technologies ) and freeze - dried .
prior to scx fractionation , 
 samples were dissolved in 200 l of scx solvent a ( 10 mm kh2po4 ph 3.0 , 30% acetonitrile ) and chromatographed 
 as previously described .
fractions were 
 combined in 10 pools according to their uv absorbance at 210 nm and 
 desalted using c18 microspin silica columns ( nest group , usa ) , freeze - dried , 
 and submitted to mass spectrometry .
liquid 
 chromatography tandem mass spectrometry was performed by the fingerprints 
 proteomics facility at the university of dundee . for experiments 
 1 and 2 ,
liquid chromatography was performed on a fully automated 
 ultimate u3000 nano lc system ( dionex ) fitted with a 300 m 
 i d  5 mm acclaim pepmap 100 c18 trap column and a 
 75 m  15 cm acclaim pepmap c18 nanocolumn 
 ( thermo scientific ) .
samples were loaded in 0.1% formic acid ( buffer 
 a ) and separated using a binary gradient consisting of buffer a and 
 buffer b ( 90% acetonitrile , 0.08% formic acid ) .
peptides were eluted 
 with a linear gradient from 2 to 35% buffer b over 65 min . for experiments 
 3 and 4 ,
liquid chromatography was performed on a fully automated 
 ultimate 3000 rslcnano system ( thermo scientific ) fitted with a 100 
 m  2 cm acclaim pepmap100 trap column ( 5 m particle 
 size ) and a 75 m  50 cm pepmaprslc c18 nanocolumn 
 ( 2 m particle size ) ( thermo scientific ) .
samples were loaded 
 in 0.1% formic acid ( buffer a ) and separated using a binary gradient 
 consisting of buffer a and buffer b ( 80% mecn , 0.08% formic acid ) . 

peptides were eluted with a linear gradient from 2 to 40% buffer b 
 over 125 min and a column temperature of 50 c .
the hplc 
 systems were coupled to an ltq orbitrap velos pro mass 
 spectrometer ( thermo scientific ) equipped with a proxeon easy - spray 
 nanosource .
the mass spectrometer was operated in data - dependent mode 
 to perform a survey scan over a range of m / z 3351800 in the orbitrap analyzer ( resolution = 
 60  000 ) , with each ms scan triggering 15 ms acquisitions 
 of the 15 most intense ions .
the orbitrap mass analyzer was internally 
 calibrated on the fly using the lock mass of polydimethylcyclosiloxane 
 at m / z 445.120024 .
data were 
 processed using maxquant version 1.3.0.5 , which incorporates the andromeda search engine .
proteins were identified by searching a protein 
 sequence database containing t. brucei brucei 927 
 annotated proteins ( version 4 downloaded from tritrypdb , http://www.tritrypdb.org/ ) supplemented 
 with the vsg221 sequence and those of frequently observed contaminants 
 ( porcine trypsin , bovine serum albumin , and human keratins ) .
search 
 parameters specified an ms tolerance of 10 ppm , an ms / ms tolerance 
 at 0.6 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 . 

peptides were required to be at least six amino acids in length , with 
 a maxquant score > 5 and 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 2 silac 
 pairs .
the distribution of silac was normalized within maxquant at 
 the peptide level so that the median of log2 ratio is zero 
 to account for any errors in cell counting . prior to statistical analysis ,
the outputs from maxquant were 
 filtered 
 to remove known contaminants and reverse sequences .
quantitation of 
 the fold - enrichment of glycosomal proteins by gfp - pex13.1 pull down 
 versus the untagged control is derived from four silac - labeled biological 
 replicates , including one label swap experiment .
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 . 
 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% . because 
 the log2(h / l ) ratios 
 were not normally distributed , with a distribution of high log2(h / l ) ratios ( glycosomal 
 proteins ) superimposed onto the normal distribution ( nonspecific proteins ) 
 centered about zero , an intensity - weighted local standard deviation of the normally distributed background ( log2h / l <
data were 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 ) .
transmembrane domain prediction for protein hits was performed 
 using thmm analysis , and we searched 
 for pts1 and pts2 ( peroxisome targeting sequences ) using the following 
 motif searches : [ stagcn][rkh][livmafy]$ and ^m.{0,20}[rk][lvi] ..... [hq][ilaf ] , 
 respectively , in the tritrypdb t. brucei strain 927 
 predicted protein database .
proteins separated by 
 sds - page ( nupage , invitrogen ) were transferred onto nitrocellulose 
 using iblot system ( invitrogen ) .
after 7 min transfer in program 3 , 
 the nitrocellulose was recovered and set up on a snapid system ( millipore ) 
 to develop with antibodies , as recommended by manufacturer .
the blocking 
 buffer used was 50 mm tris - hcl ph 7.4 , 0.15 m nacl , 0.25% bsa , 0.05% 
 ( w / v ) tween-20 , 0.05% nan3 , and 2% ( w / v ) fish skin gelatin , 
 filtered ( 0.2 m ) to remove particles .
mouse antiphosphomannose 
 isomerase ( pmi ) serum was raised against recombinant t. brucei pmi , and rabbit antialdolase serum 
 was a generous gift from paul michels .
purified mouse anti - gfp 
 monoclonal antibody ( roche ) was diluted to 0.4 g / ml in blocking 
 buffer .
secondary antibodies for the licor odyssey imager were diluted 
 in blocking buffer containing 0.01% sds ( w / v ) at 1:15  000 for 
 infrared green antimouse ( irdye 800 ) and 1:20  000 for infrared 
 red ( irdye 680 ) antirabbit .
gfp - pex13.1 
 mutant procyclic 
 cells grown in sdm-79 were incubated with and without 40 ng / ml tetracycline 
 for 16 h and pelleted by centrifugation at 600 g for 
 10 min .
cells were washed in phosphate - buffered saline ( pbs ) , fixed 
 in 4% ( w / v ) paraformaldehyde in pbs at 4 c for 30 min , and placed 
 on a coverslip .
after air - drying , coverslips were washed in pbs and 
 mounted onto slides using prolong gold with dapi ( invitrogen ) .
gfp 
 fluorescence images were collected in a zeiss lsm 710 meta confocal 
 microscope , setting the red channel for cell autofluorescence .
electron microscopy was performed on immunopurified gfp - tagged glycosomes 
 still attached to protein g dynabeads after fixation in 4% paraformaldehyde , 
 0.25% glutaraldehyde in pbs overnight at 4 c . for scanning electron 
 microscopy ( sem ) ,
immunopurified gfp - tagged glycosome beads were retained 
 on track - etch membrane filters ( nuclepore , whatman ) and processed 
 as previously described , except that 
 8 nm gold 
palladium coating was used . for immune - transmission 
 electron microscopy ( tem ) ,
beads containing immunopurified gfp - tagged 
 glycosomes were fixed as previously described and blocked overnight 
 in 10% normal goat serum and 1% ( w / v ) fish skin gelatin ( sigma ) in 
 tris buffered saline .
the beads were washed in blocking buffer and 
 incubated for 1 h at room temperature with 1:200 rabbit anti - peroxin 
 11 ( a kind gift from christine clayton ) in blocking buffer , washed , 
 and developed for 1 h at room temperature with 1:500 protein a conjugated 
 to 10 nm gold ( bbi solutions ) .
the beads were collected using a magnet 
 and resuspended in 1% aqueous osmium tetroxide , dehydrated , and set 
 in durcupan epoxy resin ( sigma ) .
sections were cut using a leica ultracut 
 uct system and analyzed using a philips tecnai 12 tem instrument . 

the wild - type 
 procyclic cell line was genetically manipulated to introduce a tetracycline - inducible 
 ectopic copy of a gene encoding the pex13.1 protein with a gfp tag 
 fused to its n - terminus ( gfp - pex13.1 ) . because pex13.1 is a transmembrane 
 protein that spans the glycosome membrane twice , with both the n- 
 and c - terminus on the surface of the glycosome ( figure 1 ) ,
anti - gfp magnetic beads can 
 be used to capture and enrich whole glycosomes made by transgenic 
 parasites expressing the gfp - pex13.1 construct .
the inducible gfp - pex13.1-transformed 
 cells were cloned by limiting dilution , and the resulting 30 clones 
 were induced with a range of tetracycline concentrations .
cell lysates 
 were analyzed by western blot for the presence of the gfp - pex13.1 
 fusion protein and by fluorescence microscopy for the presence of 
 gfp fluorescent glycosomes .
we observed that full induction with 0.5 g / ml 
 tetracycline lead to unstable expression of gfp - pex13.1 , such that 
 following a burst of gfp - pex13.1 expression the cells became unresponsive 
 to tetracycline . this may indicate that overexpression of gfp - pex13.1 
 is detrimental to trypanosomes .
however , we were able to select a 
 clone that , when induced with 0.04 g / ml tetracycline for 16 
 h , showed reproducible and stable expression of the gfp - pex13.1 fusion 
 protein ( figure 2a ) .
this clone was analyzed 
 by fluorescence microscopy and showed the presence of gfp tagged glycosomes 
 in the presence but not the absence of 0.04 g / ml tetracycline 
 induction for 16 h ( figure 2b ) . under these 
 induction conditions
, the growth of this gfp - pex13.1 expressing clone 
 is very similar to the parental cell line ( figure 2c ) , allowing us to grow the transgenic and parental cell lines 
 in parallel for silac labeling .
expression of gfp - pex13.1 in procyclic 
 form t. brucei . ( a ) cell lysate from 1.2  10 gfp - pex13.1 transgenic 
 parasites induced with 40 ng / ml tetracycline for 16 h was immunoprecipitated 
 with gfp - trap magnetic beads , and the proteins eluted with boiling 
 sds - sample buffer were separated by sds - page and subjected to western 
 blotting with a mouse anti - gfp antibody , revealing the transgene product . 

the positions of ponceau red - stained molecular weight markers are 
 shown on the left .
( b ) fluorescence imaging of the same cells for 
 gfp ( green ) and dna ( blue ) .
( c ) growth curves of wild type ( open squares ) 
 and the gfp - pex13.1 clone induced with 40 ng / ml tetracycline ( open 
 circles ) .
the wild - type and gfp - pex13.1 expressing cells were grown 
 in sdm-79+r0k0 and sdm-79+r6k4 media , respectively .
cells were passaged 
 every 2 days , and the cumulative growth is plotted against time . to get a visual impression of 
 our ability to enrich gfp - pex13.1-tagged 
 glycosomes from trypanosome lysates with anti - gfp magnetic bead preparations , 
 we performed electron microscopy on the sample obtained using mouse 
 anti - gfp and protein - g dynabeads .
tem showed the beads associated 
 with round organelle structures with dimensions consistent with those 
 of glycosomes ( figure 3a ) that could be decorated 
 with rabbit anti - pex11 antibody to a glycosome membrane peroxin and detected with 10 nm gold - conjugated protein 
 a ( figure 3b ) .
in addition , sem of the beads 
 showed several such organelles associated with each magnetic bead 
 ( figure 3c ) compared with control beads that 
 had not been incubated with trypanosome lysate ( figure 3d ) .
transmission and scanning electron microscopy of purified gfp - tagged 
 glycosomes attached to magnetic beads .
( a ) transmission electron micrograph 
 showing a magnetic bead ( left ) attached to a spherical organelle ( glycosome ) . 

( b ) transmission immunoelectron micrograph of a magnetic bead ( bottom 
 left ) and attached gfp - tagged glycosome labeled with rabbit anti - pex11 
 antibody and antirabbit antibody conjugated to 10 nm gold ( arrow ) . 

( c ) scanning electron micrograph of magnetic beads with attached gfp - tagged 
 glycosomes .
( d ) scanning electron micrograph of a magnetic bead prior 
 to the capture of gfp - tagged glycosomes .
the scale bars correspond 
 to 0.1 m in panels a and b and 1 m in panels c and d. four separate biological replicate silac 
 experiments were performed in total , and these are summarized in table 1 . for three of these experiments , wild - type 
 and transgenic parasites expressing gfp - pex13.1 were grown for eight 
 cell divisions in parallel and under identical conditions , except 
 that the transgenic parasites expressing gfp - pex13.1 were grown in 
  heavy medium  containing stable isotope - labeled lys 
 and arg ( r6k4 ) , whereas the wild - type cells were grown in 
light 
 medium  containing unlabeled lys and arg ( r0k0 ) . in each experiment , 
 the transgenic parasites expressing gfp - pex13.1 and the wild - type 
 cells were harvested , washed , counted , mixed together in a 1:1 ratio , 
 and lysed .
these lysates , containing equal amounts of labeled and 
 unlabeled proteins , were used to capture the heavy - labeled gfp - pex13.1 
 tagged glycosomes on anti - gfp magnetic beads for proteomic analysis . 
 using this protocol
, specifically enriched glycosomal proteins can 
 be distinguished from nonspecific contaminant ( nonglycosomal ) proteins 
 by the isotope ratios of their tryptic peptides .
thus , true glycosomal 
 protein peptides will have high heavy / light isotope ratios , whereas 
 contaminant proteins will have heavy / light isotope ratios close to 
 1 . for these three experiments ,
the beads were recovered magnetically 
 and washed prior to either ( i ) processing by sds - page into gel - slices 
 ( see figure s1 in the supporting information ) and performing in - gel reduction , s - alkylation , and tryptic digestion 
 for lc 
ms / ms ( table 1 , experiments 1 
 and 2 ) or ( ii ) boiling in sds containing buffer and performing the 
 fasp method , with subsequent fractionation 
 into subfractions by scx hplc prior to lc 
, 
 whereby the transgenic parasites expressing gfp - pex13.1 were grown 
 in light medium , and the wild - type cells were grown in the heavy medium . 

otherwise , this experiment ( table 1 , experiment 
 4 ) was identical to that described in table 1 , experiment 3 .
for all four experiments , the peptide fractions 
 were run in triplicate 
 by lc 
ms / ms ( technical replicates ) . in total , data from 111 
 lc 
ms / ms runs were used for data analysis . the data from each individual 
 experiment ( table 1 , experiments 14 ) 
 and the combined data for all four experiments were used to search 
 a t. brucei predicted protein database using maxquant software . for the proteins identified in the combined data set , we 
 performed a pairwise comparisons of the log2(h / l ) ratios with those for the proteins identified 
 in the four individual data sets .
these showed good correlations ( pearson 
 coefficients of 0.916 , 0.872 , 0.906 , and 0.879 ; figure s2 in the supporting information ) .
consequently , we used 
 the combined data ( after inverting the h / l ratios for experiment 4 , the label swap ) for all further 
 analyses .
a total of 3182 proteins were identified , at a false discovery 
 rate of 1% , using the combined data ( table s1 in the supporting information ) .
each protein identification 
 was displayed on a plot ( figure 4 ) of the log10 value of the combined intensities of the unique peptides 
 of that protein ( y axis ) and the log2 value 
 of the mean heavy to light isotope ratios of the same peptides ( x axis ) . because of the significant number of glycosomal 
 proteins selectively enriched by the anti - gfp bead pull - out procedure , 
 the log2-transformed h / l ratios were not normally distributed around zero .
log2(h / l ) ratio glycosomal proteins superimposed onto a normal distribution 
 of nonspecific , contaminant , proteins with log2(h / l ) ratios centered about zero ,
we used the intensity - weighted local standard deviation of the normally 
 distributed contaminant proteins ( log2(h / l ) < |1.2| ) to account for the fact that the h / l ratio of more intense ions can be measured 
 with greater certainty .
we have then 
 used the calculated local standard deviation ( sd ) to divide the proteins 
 into three populations ( table s2 in the supporting 
 information ) .
group ( i ) : a large group of 2876 proteins centered 
 around a log2(h / l ) ratio 
 of 0 , corresponding to proteins that were not enriched by the anti - gfp 
 pull - out , that is , contaminant proteins .
group ( ii ) : a group of 177 
 proteins that are significantly enriched in log2(h / l ) ratio over the first group by > 3 
 sd 
 and < 8 sd .
group ( iii ) : a group of 129 proteins that are very highly 
 enriched in log2(h / l ) 
 ratio over the first group by > 8 sd plot of the combined 
 glycosome proteomics data .
each protein identification 
 is represented by a point plotted as the log2 of their 
 heavy - to - light isotope ratio ( x axis ) versus the 
 log10 value of the combined intensities of the peptides 
 belonging to each protein ( y axis ) .
the black and 
 blue curves represent three and eight standard deviations ( sd ) from 
 the mean .
proteins plotted in red within the three sd curves are designated 
 group ( i ) ( contaminants ) , those plotted in amber between the 3 and 
 8 sd curves are group ( ii ) , and those beyond the 8 sd curves are designated 
 group ( iii ) .
the 129 highly enriched group ( iii ) proteins include 
 9 members of the pex proteins , involved in glycosome biogenesis , and 
 74 enzyme or enzyme - complex proteins , covering 65 different enzyme 
 activities .
the latter ( table s3 in the supporting 
 information ) include the seven glycolytic enzymes from hexokinase 
 ( hk ) to phosphoglycerate kinase ( pgk ) as well as the nad - dependent 
 glycerol-3-phosphate dehydrogenase ( g3pdh ) and glycerol kinase ( gk ) . 

the presence of the pex and glycolytic pathway components confirms 
 that this group of proteins includes the contents of mature glycosomes . 

this is further supported by the presence of all of the enzymes of 
 purine salvage and pyrimidine biosynthesis , with the exceptions of 
 bifunctional orotate phosphoribosyltransferase / orotidine 5-phosphate 
 decarboxylase ( oprt / opdc ) and phosphoribosyl pyrophosphate synthase 
 ( prps ) , although the latter appears in the group ( ii ) proteins .
also 
 consistent with known glycosome biochemistry is the presence of many 
 components of the peroxide and superoxide inactivation system , that 
 is , trypanothione reductase ( tr ) , tryparedoxins ( tpn ) , tryparedoxin 
 peroxidase ( tpx ) , and isocitrate dehydrogenase ( icdh ) , which provides 
 the nadph necessary to provide the reducing power for the system . 

in addition , l - galactonolactone / d - arabinolactone 
 oxidase ( glo ) , involved in the synthesis of the antioxidant ascorbate , 
 was found , consistent with its reported glycosomal location .
most peroxisomes are synonymous with long - chain 
 fatty acid beta - oxidation , and , appropriately , the trifunctional enoyl - coa 
 hydratase / enoyl - coa isomerase / hydroxyacyl - coa dehydrogenase ( ecoah / i / hacoadh ) 
 was found in the group ( iii ) glycosomal proteins .
glycosomes ( like 
 many peroxisomes ) are also known for ether lipid synthesis , and dihydroxyacetone-3-phosphate acyltransterase ( dhapat ) and alkyl - dihydroxyacetone-3-phosphate 
 synthase ( dhps ) were both found .
finally , the presqualene segement 
 of isoprenoid / sterol synthesis has been described in mammalian peroxisomes and suggested to be glycosomal based on the 
 presence of pts sequences on some of the components . here
we find two enzymes on the pathway from mevalonate 
 to squalene , mevalonate kinase ( mk ) and isopentyl - diphosphate delta 
 isomerase ( ipdi ) , as well as some other postsqualene enzymes of sterol 
 biosynthesis .
there are several proteins in group ( ii ) that 
 are known to belong to the er , for example , components of the protein 
 n - glycosylation , er quality control , and gpi anchor addition machineries . 

it is likely that pex13.1 , like many peroxisomal membrane proteins , 
 is made in the er , and that , therefore , using a 
 gfp - pex13.1 fusion protein as bait for the pull - out experiments will 
 not only enrich glycosomal proteins but also some er proteins that 
 are physically close to gfp - pex13.1 the moment of cell lysis .
discriminating 
 bone fide glycosomal proteins from er proteins in group ( ii ) is therefore 
 complex .
the prevailing model of peroxisome biogenesis is that they 
 multiply by growing and budding .
while the newly budded organelles 
 directly take up matrix proteins and some membrane proteins ( via the 
 pex19/pex16 pathway ) they also fuse with er - derived vesicles that 
 provide membrane lipids and other membrane proteins . on the basis of this
, we performed a label and chase experiment , 
 whereby we heavy - labeled trypanosome proteins to steady - state with 
 r6k4 , inducing the expression of gfp - pex13.1 
 for the last 16 h of that labeling and then chased that label by incubating 
 the cells in light medium containing r0ko for 
 5 h. we then enriched for glycosomes using anti - gfp beads and performed 
 quantitative proteomics , as before .
as expected , most peptides , especially 
 those from the group ( iii ) proteins , showed a significant reduction 
 in their heavy / light ratios ( by about 1 log2 unit ; table 
 s4 in the supporting information ) .
however , 
 the group ( ii ) proteins could now be separated into subgroups : 50 
 with decreasing heavy / light ratios , which we suggest are er proteins 
 that were simply in the vicinity gfp - pex13.1 the moment of cell rupture ; 
 29 with increasing heavy / light ratios , which we suggest are bone fide 
 er - derived glycosomal proteins that , for whatever reason , take longer 
 to reach the mature glycosomes than the group ( iii ) proteins ; and 
 42 with relatively unchanged heavy / light ratios , which we would not 
 like to call either way .
finally , there are an additional 56 group 
 ( ii ) proteins in table s2 in the supporting information that were not featured in this particular experiment .
this brings 
 our list of high - confidence glycosomal proteins to 158 ( indicated 
 in green in the glycosome status column ; table s2 in the supporting information ) , with a further 98 ( i.e. , 
 42 + 56 ) in the 
possible  category ( indicated by gray 
 and white , respectively , in the glycosome status column ; table s2 
 in the supporting information ) and 50 excluded 
 ( indicated in red in the glycosome status column ; table s2 in the supporting information ) .
some of the group ( ii ) 
 proteins elevated to the status of high - confidence glycosomal proteins 
 by this chase experiment include : pex6 , part of the peroxisome biosynthetic 
 machinery ; monoglyceride lipase ( mgl ) and acyl - coa binding protein 
 ( acbp ) , which most likely play a role in beta - oxidation by generating 
 free fatty acid and chaperoning acyl - coa molecules , respectively ; 
 glyoxylase ii ( glxii ) , which presumably plays a role in the redox 
 system , returning trypanothione thioesters to free trypanothione ; the enzyme udp - glucose 4-epimerase ( gale ) , 
 part of sugar nucleotide metabolism and previously shown to be glycosomal ; and the caax protease , a membrane protein that 
 is part of the protein prenylation system , presumably with its active site facing the cytosol .
other components , 
 discussed further later , are associated with post - translational modification 
 and protein folding , such as a heat - shock protein ( hsp70 ) , a protein 
 phosphatase 2c ( pp2c-2 ) , a protein kinase a catalytic subunit ( pka ) 
 as well as a  nucleotide phosphate linked to x hydrolase 
 ( nudix-2 ) , and a methyltransferase of 
 unknown function ( drevmt ) .
many of the well - annotated 
 proteins identified in our analysis are indicated in figure 5a , b with those belonging to group ( i )  non - glycosomal  
 in red , those belonging to the  possibly glycosomal  

category from group ( ii ) in amber , and those belonging to group ( iii ) 
 plus the elevated group ( ii ) proteins belonging to  the high - confidence 
 glycosome proteome  in green .
proteins that are expected to 
 be glycosomal but that were either not detected or for which the log2(h / l ) ratio variance was 
 too great are indicated in gray .
all proteins that , so far as we are 
 aware , have not been previously described to be glycosomal , are boxed . 
 a list of the enzymes and their abbreviations shown in figure 5a , b is provided in table s3 in the supporting information .
glycosome metabolism and function according 
 to the glycosome proteome 
 data reported in this paper .
the blue lines indicate the glycosome 
 membrane , and the blue arrows indicate transmembrane transport of 
 metabolites .
( a ) areas of known or suggested glycosomal metabolism 
 and ( b ) the glycosomal protein import machinery ( pex proteins ) and 
 areas of glycosomal metabolism that have not been previously described . 

the enzyme abbreviations are in italics 
 and appear in boxes if they have not been previously demonstrated 
 or suggested to be glycosomal .
the key to their full names appears 
 in table s3 in the supporting information , and the numbers 1 and 2 in brackets following the enzyme abbreviations 
 indicate the presence of pts1 and pts2 targeting sequences . enzyme 
 and protein abbreviations appear green if they belong to the  high - confidence 
 glycosome proteome  , amber if they belong to the  possibly 
 glycosomal protein category , red if their silac ratios suggest 
 they are without the glycosome , and gray if they were either not detected 
 or if data variance was too great to assign location
. while the group ( iii ) and elevated group ( ii ) proteins 
 are highly 
 consistent with the known or suggested functions of t. brucei glycosomes , a significant number of the enzymes of the pentose phosphate 
 pathway , generally considered to be both cytosolic and glycosomal , are missing .
thus , whereas glucose-6-phosphate 
 dehydrogenase ( g6pdh ) and 6-phosphogluconolactonase ( 6pgl ) are clearly 
 present , as previously reported , along with 
 sedheptulose 1,7-bisphosphatase ( sbp ) , others ( enzymes in gray ) were either not detected , like ribulose 
 5-phosphate isomerase ( r5i ) , or statistically excluded due to high 
 variance between replicates , like transketolase ( tk ) .
furthermore , 
 some pentose phosphate pathway enzymes were identified in group ( i ) , 
 suggesting they are contaminants rather than bone fide glycosome components 
 ( enzymes in red ) .
these include transaldolase ( ta ) , ribulose 5-phosphate 
 epimerase ( r5e ) and 6-phosphogluconate dehydrogenase ( 6pgdh ) .
while 
 these data do not formally exclude the presence of a complete pentose 
 phosphate pathway in procyclic form glycosomes , they suggest that 
 direct experimental localization studies are perhaps warranted before 
 this is taken for granted .
indeed , the majority of transketolase has 
 been previously reported to be in the cytosol .
with respect to the glycolytic pathway downstream 
 of glycerate 
 1,3 bisphosphate ( 1,3bpga ) , phosphoenolpyruvate carboxykinase ( pepck ) , 
 malate dehydrogenase ( md ) , and nadh - dependent fumarate reductase ( fr ) 
 are all clearly present in the group ( iii ) proteins , but fumarate 
 hydratase ( fh ) is , like enolase ( eno ) and malic enzyme ( me ) , a group 
 ( i ) protein , suggesting that all are located without the glycosome
. 
 this may be significant because the presence of fh in the glycosomes 
 as well as in the cytosol has been previously suggested .
the absence of fh in the glycosomes would mean 
 that the transmembrane transport of malate and fumarate would be essential 
 for the production of glycosomal succinate , required for nad / nadh balance inside the procyclic glycosome .
interestingly , 
 in addition to the abc transporter gat1 that transports 
 oleoyl - coa into the glycosome , a putative 
 carnitine o - acyltransferase ( act ) was also found , 
 suggesting that there may be an acyl carnitine translocase / carnitine o - acyltransferase shuttle to bring acyl - carnitines into 
 the glycosome and convert them to acyl - coa species as substrates for 
 beta - oxidation and other processes like ether lipid synthesis .
other 
 possible sources of acyl - coa substrates are the phospholipase a1 ( pla1 ) , lyso - phospholipase ( lysopl ) , and monoglyceride lipase ( mgl ) 
 enzymes that could provide free fatty acids to the acyl - coa synthase 
 ( acs ) .
in addition , a putative thioesterase could be involved in regulating 
 the intraglycosomal acyl - coa to free fatty acid balance .
several of the enzymes in the 158 confidently 
 assigned glycosomal proteins list do not obviously or directly fall 
 into conventional glycosomal metabolic pathways , and these are mostly 
 shown in figure 5b .
the presence of nudix enzymes 
 in the glycosome ( in the case of tb927.5.4350 , consistent with having 
 a pts2 signal sequence and with one of the colasante proteomes ) probably makes sense with respect to the relatively 
 high concentrations of nucleotides , sugar nucleotides , ( acyl-)coa , 
 and nad(h ) , which will be present in the organelle .
thus , nudix hydrolases 
 can help to destroy nucleotides damaged by reactive oxygen species , and mammalian and yeast peroxisomal nudix proteins 
 have been shown to act as coa and acyl - coa diphosphatases , preventing 
 the buildup of coa cofactors .
similarly , 
 the presence of some protein quality control / folding machinery in 
 such a metabolically active compartment ( i.e. , hsp40 , hsp70 , and peptidyl - prolyl 
 isomerase ( ppi ) ) is , in retrospect , not particularly surprising .
perhaps 
 the most unexpected glycosomal components are members of protein phosphatase 
 families , which include serine / threonine phosphatases and a pseudotyrosine 
 phosphatase . by cross - referencing our 
 glycosomal proteome and phosphoproteome data
, we see that although the glycosomes contain several phosphoproteins 
 and protein phosphatases ( table s2 in the supporting 
 information ) , they do not contain detectable protein kinases 
 other than pka , which together with its putative neurobeachin membrane 
 anchoring protein most likely faces the 
 outside of the organelle .
this suggests that information ( e.g. , relating 
 to metabolic or cell - cycle status ) might be transmitted from the cytosol 
 to the glycosome to exert metabolic control through the import of 
 phosphorylated cargo and that this  information  might 
 naturally decay once in the glycosome through dephosphorylation .
furthermore , one might speculate that the glycosomal 
 catalytically inactive tyrosine phosphatase could be present to  read  
 the tyrosine phosphorylation status of imported phosphoproteins .
some 
 of the phosphorylated glycosomal proteins include enzymes of carbohydrate 
 metabolism , such as phosphofructokinase ( pfk ) , triosephosphate isomerase 
 ( tpi ) , aldolase ( ald ) , pepck , fr , hk , pgk , and gk , as well as redox 
 and other components .
many of the phosphorylation site occupancies 
 vary considerably between bloodstream form and procyclic form trypanosomes 
 for these enzymes and other glycosomal components ( table s2 in the supporting information ) ; however , whether phosphorylation modulates any of these activities 
 remains to be determined .
glycosome 
 proteomes have been previously published using density 
 gradient centrifugation enrichment prior to proteomic analyses .
while the organelle enrichment in these studies may be superior 
 to that described here by epitope - tagging and magnetic bead pull - out , 
 only the latter is compatible with silac methodology , summarized in 
 figure 1 , which greatly assists the discrimination 
 of genuine components from contaminants .
preselected known and suggested 
 glycosome proteins from the data sets and made semiquantitative comparisons , 
 by spectral counting , between the bloodstream and procyclic forms 
 of the parasite .
this successfully highlighted metabolic changes between 
 the two lifecycle stages but did not claim to automatically distinguish 
 glycosomal from nonglycosomal proteins .
studies also fully acknowledged the limitations of glycosome enrichment 
 alone to unambiguously identify the glycosome proteome because contamination 
 from other compartments , particularly the mitochondrion , is inevitable . 

this becomes apparent if their protein identifications are superimposed 
 on our silac data set , when the extent of group ( i ) ( likely contaminant ) 
 protein identifications in these proteomes becomes evident ( figure 6a , b ) .
correlation of the combined glycosome proteomic 
 data in this study 
 with other experimental and theoretical data .
the plots are the same 
 as that shown in figure 4 , except that only 
 the three sd limit is shown ( black curves ) and all data points are 
 plotted in gray unless they appear in the following data sets : ( a ) 
 red points indicate those proteins identified in the total glycosome 
 proteome of colasante et al .
( b ) 
 red points indicate those proteins identified in the total glycosome 
 proteome of colasante et al .
( c ) 
 light blue points indicate those proteins containing a pts1 signal 
 peptide , black points indicate those proteins containing a pts2 signal 
 peptide , and red points indicate those proteins containing a pts1 
 and pts2 signal peptide .
( d ) red points indicate those proteins predicted 
 to contain one transmembrane domain and blue points indicate those 
 proteins predicted to contain more than one transmembrane domain . a convenient way to predict potential 
 glycosomal proteins is to 
 search for peroxisome targeting sequences ( pts ) in the predicted amino 
 acid sequences of trypanosome orfs .
this 
 approach recognizes that false positive and negative assignments are 
 made in searching for c - terminal pts1 and n - terminal pts2 sequences , 
 and the extent of the problem is evident when proteins containing 
 one or both of these signals is superimposed on our data set ( figure 6c ) .
overall , 60 pts - containing proteins were found 
 in the 158 proteins of the high - confidence glycosome proteome , and 
 77 were found among the group ( i ) protein contaminants , suggesting 
 that use of pts signal sequence search in t. brucei , while useful , has a sensitivity of < 40% and a specificity of 
 < 50% . finally , we , and others , are interested 
 in glycosome 
 membrane proteins that might be involved in metabolite transport into 
 and out of these organelles .
proteins containing one or more predicted 
 transmembrane domains are well distributed across all three protein 
 groups , as indicated in figure 6d .
of the 35 
 transmembrane proteins in the high - confidence glycosome proteome , 
 13 are of known glycosome location , such as eight of the pex proteins 
 ( pex2 , 10 , 11 , 12 , 13.1 , 13.2 , 14 , 22 ) , the pex11-related proteins 
 gim5a and gim5b , and the glycosome abc 
 transporters ( gat1 , 2 , and 3 ) .
it is worth 
 noting that gat2 was previously thought to be bloodstream form specific . in summary , we have used organelle epitope tagging , magnetic 
 bead 
 enrichment , and silac proteomics to obtain a high - confidence procyclic 
 form t. brucei glycosome proteome of 158 proteins 
 with a further 98 proteins in a possibly glycosomal category .
 the 
 glycosome of the pathogenic african trypanosome trypanosoma 
 brucei is a specialized peroxisome that contains most of 
 the enzymes of glycolysis and several other metabolic and catabolic 
 pathways . 
 the contents and transporters of this membrane - bounded organelle 
 are of considerable interest as potential drug targets . here 
 we use 
 epitope tagging , magnetic bead enrichment , and silac quantitative 
 proteomics to determine a high - confidence glycosome proteome for the 
 procyclic life cycle stage of the parasite using isotope ratios to 
 discriminate glycosomal from mitochondrial and other contaminating 
 proteins . 
 the data confirm the presence of several previously demonstrated 
 and suggested pathways in the organelle and identify previously unanticipated 
 activities , such as protein phosphatases . 
 the implications of the 
 findings are discussed . 
Introduction
Experimental Procedures
Results
Discussion