Subcellular localization by immunofluorescence microscopy

In previous work, we have purified the following soluble, enzymatically active, recombinant nucleotide sugar biosynthetic enzymes: TbUGP, TbGALE, TbGNA, TbPAGM, TbUAP, TbPMI and TbPMM (Table 1). With these, we have prepared either rabbit (TbGALE) or mouse polyclonal antibodies for immunofluorescence microscopy (IFM) localization studies against bsf trypanosomes (Table 1). To these we have added new polyclonal mono-specific mouse antibodies to recombinant TbGALE, TbGNA, TbUAP, TbMPGT and TbGMER (see Methods). In all cases, the mouse antibodies were affinity-purified against immobilized immunogen and/or demonstrated to be mono-specific by Western blotting (Table 2; S2 Fig). These antibodies were used in immunofluorescence microscopy against fixed and permeabilized pcf trypanosomes, along with rabbit antibodies to definitive glycosomal (TbGAPDH) and cytosolic (TbEnolase) markers (Figs 2, 3, and 4). Wider field images containing several cells are available in (10.6084/m9.figshare.13124909). In all cases, except for TbPAGM (Fig 2, middle panels), the mono-specific mouse antibodies produced punctate staining that was coincident, at least in part, with the rabbit anti-TbGAPDH antibody staining, suggesting glycosomal locations. In contrast, there was no overlap with cytoplasmic anti-TbEnolase immunostaining. In the case of TbPAGM, the immunostaining suggested both glycosomal and cytosolic localization. Interestingly, the punctate co-staining with anti-NS biosynthetic enzyme and anti-TbGAPDH antibodies was imperfect, in so far as some puncta were more red or green than yellow. This implies heterogeneity in glycosomal contents, which is discussed later. However, although the ratios of TbGAPDH (red) to NS enzyme (green) signals vary widely, quantitative analysis (Table 3) shows that the majority of the NS enzyme signals colocalize with punctate TbGAPDH signals, with the exception of TbPAGM.

Fig 2

Immunofluorescence microscopy localisation of UDP-GlcNAc biosynthetic enzymes in procyclic form T. brucei.

Enzymes of UDP-GlcNAc biosynthesis were localised with mono-specific mouse antibodies (green channels). Authentic glycosome and cytosol markers TbGAPDH and TbEnolase, respectively, were localised with specific rabbit antisera (red channels). Merged green and red channels and phase-contrast images are also shown.

Fig 3

Immunofluorescence microscopy localisation of GDP-Man biosynthetic enzymes in procyclic form T. brucei.

Enzymes of GDP-Man biosynthesis were localised with mono-specific mouse antibodies (green channels). Authentic glycosome and cytosol markers TbGAPDH and TbEnolase, respectively, were localised with specific rabbit antisera (red channels). Merged green and red channels and phase-contrast images are also shown.

Fig 4

Immunofluorescence microscopy localisation of UDP-Glc, UDP-Gal and GDP-Fuc biosynthetic enzymes in procyclic form T. brucei.

Enzymes of UDP-Glc, UDP-Gal and GDP-Fuc biosynthesis were localised with mono-specific mouse antibodies (green channels). Authentic glycosome and cytosol markers TbGAPDH and TbEnolase, respectively, were localised with specific rabbit antisera (red channels). Merged green and red channels and phase-contrast images are also shown.

Table 2

[TP] = this paper.

Antibodies to nucleotide sugar biosynthetic enzymes used in this study.

Enzyme	Antibody species	Affinity purified	Mono-specific by Western	Reference
TbUGP	Mouse	Yes	Yes	[45]
TbGALE	Mouse	No	Yes	[TP]
TbGNA	Mouse	Yes	Yes	[TP]
TbPAGM	Mouse	No	Yes	[42]
TbUAP	Mouse	Yes	Yes	[TP]
TbPMI	Mouse	No	Yes	[46]
TbPMM	Mouse	No	Yes	[42]
TbMPGT	Mouse	No	Yes	[TP}
TbGMER	Mouse	No	Yes	[TP]

Table 3

Quantitative analysis of anti-NS enzyme and anti-TbGAPDH colocalization by IFM in pcf cells.

NS Enzyme	na	Mander’s colocalization coefficientb
TbGNA	6	0.85 ± 0.05
TbPAGM	5	0.33 ± 0.06
TbUAP	5	0.89 ± 0.02
TbPMI	9	0.96 ± 0.01
TbPMM	4	0.73 ± 0.06
TbMPGT	4	0.89 ± 0.04
TbUGP	8	0.89 ± 0.03
TbGalE	4	0.88 ± 0.05
TbGMER	7	0.88 ± 0.05

a n is number of cells uses in the analysis.

b The Mander’s colocalization coefficient (± standard deviation of the mean) is a measure of the amount of green (anti-NS enzyme) signal above background that colocalizes with red (anti-TbGAPDH) stained compartments.

Subcellular localization by digitonin latency

Digitonin latency is a powerful adjunct technique to assess whether soluble proteins are either cytosolic or sequestered in membrane-bound intracellular organelles. Digitonin is a steroidal-saponin detergent-like natural product that first permeabilizes and subsequently solubilizes biological membranes. Digitonin has a preference for high sterol content membranes. Thus, cytosolic soluble proteins are liberated from cells at very low digitonin concentrations, as the sterol-rich plasma membrane is the first affected, and organellar contents are subsequently liberated at significantly higher digitonin concentrations. The requirement for a higher digitonin concentration to effect protein release is known as ‘digitonin latency’. This method has proved extremely useful in discriminating cytosolic versus glycosomal proteins in trypanosomatids, for example [42,58–60,61,69]. In our studies, we used the aforementioned mono-specific polyclonal mouse antibodies to eight nucleotide biosynthetic enzymes, along with rabbit antibodies to authentic glycosome lumen (TbGAPDH or TbAldolase), glycosome membrane (TbPEX13.1) and soluble cytosolic (TbEnolase) markers to assess digitonin latency of these enzymes in pcf and bsf trypanosomes (Fig 5A and 5B, respectively). As expected, the cytosolic marker, TbEnolase, was released from both lifecycle stages at very low digitonin concentrations whereas TbAldolase and TbGAPDH were not quantitatively released until 0.1% TX100 was used. This was not because the glycosome membrane was not being gradually solubilized by the digitonin, since the gradual release of membrane protein TbPEX13 was evident in these experiments, but because of the possibly aggregated nature of these very abundant proteins in what has been proposed to be a dense crystalloid core inside of glycosomes [56]. In contrast, TbPAGM showed a biphasic release pattern, consistent with dual cytoplasmic and glycosomal location. All the other nucleotide sugar biosynthetic enzymes showed release starting around 0.04 mg digitonin / mg protein, consistent with them being predominantly or exclusively glycosomal proteins. Interestingly, in all cases, the proteins seem to be slightly harder to release with digitonin from pcf cells than from bsf cells.

Fig 5

Digitonin latencies of nucleotide sugar biosynthetic enzymes in bloodstream and procyclic forms of T. brucei.

Trypanosomes (bsf, panel A; pcf, panel B) were treated with increasing concentrations of digitonin (0.01 to 0.1 mg digitonin per mg trypanosome protein) and with no detergent and 0.1% TX100 as controls for no cell permeabilization and total cell and glycosome membrane permeabilization, respectively. The release of the cytoplasmic marker TbEnolase at the lowest digitonin concentration is consistent with the selective permeabilization of the plasma membrane over the glycosome membrane. The release of TbAldolase (panel A) or TbGAPDH (panel B) at higher digitonin concentrations, or only with 0.1% TX100, i.e., showing latency, is consistent with their known glycosome lumen locations. From the patterns of digitonin latency of the nucleotide sugar biosynthetic enzymes, we conclude that all shown, except TbPAGM, are principally or exclusively protected against release by digitonin by being sequestered in a low-sterol intracellular membrane-bound compartment.

Subcellular localization by subcellular fractionation and evidence for glycosome heterogeneity by density gradient centrifugation and proteomics

Procyclic cells were lysed using a high-pressure homogenizer [54] and the lysate was subjected to differential centrifugation according to [70]. This procedure results in a nuclear fraction, a large granular fraction (LG), a small granular fraction (SG) and a cytoplasmic fraction (C), with the latter also containing microsomes of the plasma membrane, Golgi apparatus and endoplasmic reticulum (ER). These fractions were subjected to SDS-PAGE and Western blotting and the blots were probed with antibodies to authentic glycosomal, mitochondrial, cytoplasmic and ER markers (TbGAPDH, TbGAP1 [71], TbENO and calreticulin, respectively) which confirmed that the majority of mitochondrial TbGAP1 was in the LG fraction, that the majority of cytoplasmic TbENO and ER calreticulin was in the C fraction and that the majority of glycosomal TbGAPDH was in the SG fraction, as expected [70] (Fig 6A). Antibodies to three representative NS biosynthetic enzymes (TbPMI, TbMPGT and TbUGP) showed that these enzymes are found exclusively to the glycosome-containing SG fraction (Fig 6A, Table 1), consistent with the immunofluorescence microscopy and digitonin latency data described above.

Fig 6

Subcellular localization by subcellular fractionation and evidence for glycosome heterogeneity by density gradient centrifugation and proteomics.

Panel A: Pcf trypanosome lysate was submitted to differential centrifugation [70] and aliquots of the large granular (LG), small granular (SG) and cytosolic plus microsomal fractions (C) were subjected to SDS-PAGE and Western blotting with antibodies to authentic glycosomal (TbGAPDH), mitochondrial (TbGAP1), cytoplasmic (TbENO) and ER (calreticulin) markers and to NS biosynthetic enzymes (TbPMI, TbMPGT and TbUGP), as indicated. Panel B: The SG fraction was further fractionated by iodixanol density gradient centrifugation and two opalescent bands (fractions 1 and 2) were isolated. Panel 3: Aliquots of fractions 1 and 2 from panel B were subjected to proteomic analysis by LC-MS/MS. The relative abundances (iBAQ values over three scales, y-axes) are plotted against the identified proteins (TriTrypDB identifiers, x-axes).

The same SG fraction was further subjected to iodixanol density gradient centrifugation. Two opalescent bands (fractions 1 and 2) were observed (Fig 6B) at densities of 1.15 and 1.18 g/mL, similar to that reported for rat liver peroxisomes using iodixanol (1.175 g/mL) [72]. Each was isolated and, following reduction, alkylation and trypsin digestion of the protein content, subjected to proteomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The protein groups identified in both fractions were highly-enriched for glycosomal proteins, as expected. Intensity-based absolute quantitation (iBAQ) values were extracted for the protein groups belonging to a reference high-confidence glycosomal proteome [54]. These protein groups (x axis) are plotted against three iBAQ scales, from low abundance (iBAQ values <1 x10⁴) to high-abundance (iBAQ values >3 x 10⁶), in (Fig 6C). Consistent with its higher density, fraction 2 contains more glycosomal protein than fraction 1. However, the relative abundances of glycsomal proteins between the two fractions are different, suggesting compositional heterogeneity between them.

Cell culture

Procyclic form T. brucei 427 strain containing T7 RNA polymerase and Tet repressor protein genes, respectively under control of G418 and hygromycin (clone 29.13.6 cells, kindly provided by George Cross) were grown at 28°C without CO₂ in original SDM-79 medium [82] containing hygromycin B (Roche) at 50 μg/ mL, G418 (Invitrogen) at 15 μg/ mL, 15% (v/v) heat inactivated fetal bovine serum (FBS), 2 g/L sodium bicarbonate, fresh 2 mM Glutamax I (Invitrogen) and 22.5 mg / ml haemin (added from a 0.05 M stock in NaOH), adjusted to pH 7.3. Bloodstream form T. brucei 427 strain containing T7 RNA polymerase and Tet repressor protein, under control of G418, known as single markers cells, kindly provided by George Cross, were grown at 37°C with 5% CO₂ in HMI-9T medium [83] containing fresh 2mM Glutamax I (Invitrogen) and 2.5 μg/ mL G418 (Invitrogen).

Production of recombinant TbMPGT

The TbMPGT ORF (gene Tb927.8.2050) was amplified from genomic DNA by PCR and cloned into pET28a, containing a 6-His N-terminal tag with a thrombin cleavage site, and overexpressed in BL21 (DE 3) Gold E. coli grown at 37°C in LB medium supplemented with kanamycin (50 μg / ml) and tetracycline (10 μg / ml). When the OD₆₀₀ reached about 2.0, the temperature was lowered to 16°C and protein expression was induced with 1 mM IPTG (Formedium) for 22 h. Cells were harvested, resuspended in lysis buffer (20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 8.0), supplemented with DNAse I and EDTA-free protease inhibitors (Roche), disrupted using a French press and centrifuged at 30,000 g for 30 min at 4°. The supernatant was filtered (0.2 μm) and applied to a 5 ml HisTrap HP chelating column (GE healthcare), pre-equilibrated in lysis buffer, and eluted with a linear imidazole gradient from 96% buffer A (20 mM Tris, 500 mM NaCl, pH 8) to 100% buffer B (20 mM Tris, 500 mM NaCl, 500 mM imidazole, pH 8). Fractions containing TbMPGT (which eluted around 300 mM imidazole) were pooled, concentrated, digested with thrombin (NEB) to remove the His-tag and subsequently purified on a Superose 12 10/300 column (GE healthcare) equilibrated with 10 mM Hepes, 100mM NaCl, pH 7.4. The yield, estimated by BCA assay, was about 10 mg of soluble, enzymatically active and highly purified TbMPGT per litre of culture (S1 Fig). The identity of the recombinant TbMPGT was confirmed by proteomics.

Production of mouse antibodies against recombinant nucleotide sugar biosynthetic enzymes

Aliquots of recombinant purified TbMPGT (S1 Fig), TbGALE [41], TbGNA [49], TbUAP [50] and TbGMER [52] were submitted to David’s Biotech (Germany) for the immunization of two mice per protein, according to their recommended protocol using Freund’s complete adjuvant for the primary immunisation with 0.1 mg protein per mouse and two secondary boosts with 0.05 mg per mouse in Freund’s incomplete adjuvant. Pooled mouse anti-TbMPGT, anti-TbUAP and anti-TbGNA sera were immunopurified by affinity chromatography on Sepharose4B beads coupled to recombinant TbMPGT, TbUAP and TbGNA, respectively, at 3–5 mg / ml gel. The affinity chromatography beads were prepared using CNBr-Sepharose 4B (GE Healthcare) according to the manufacturer’s instructions. The coupled beads were stored in phosphate buffered saline (PBS) containing 0.05% (w/v) sodium azide. For affinity purification, pooled mouse sera were centrifuged at 16,000 g for 15 min at 4°C and the supernatants mixed with an equal volume of PBS. These were incubated with the corresponding affinity chromatography beads for 2 h with rotation at 4°C. The beads were washed with PBS until the optical density at 280 nm (OD280) of the supernatant was below 0.05 and the specifically bound antibodies were then eluted with 50 mM sodium citrate pH 2.8. The eluates were immediately neutralized with 1M Tris-HCl pH 8.8 and quantitated by OD280.

Immunoprecipitation

T. brucei bsf cells were washed three times with trypanosome dilution buffer (TBD; 20 mM Na₂HPO₄, 2 mM NaH₂PO₄, 80 mM NaCl, 5 mM KCl, 1 mM MgSO₄, 20 mM glucose pH 7.7) and lysed at 1 x 10⁹ cells/ml in 1% (w/v) SDS in 20 mM Tris-HCl pH 6.8 containing 0.1 M DTT and heated at 50°C for 15 min. The SDS lysate was diluted to 0.03% SDS with 1% (w/v) Triton X-100 in 20 mM Tris-HCl pH 6.8, 0.15 M NaCl, 0.1 mM TLCK, 1 μg / ml leupeptin, 1 μg / ml aprotinin and 1 mM PMSF. Insoluble material was removed by centrifugation at 16,000 g for 10 min at 4°C and an aliquot of the supernatant (equivalent to 2 x 10⁸ cells) was incubated with 5 μL of rabbit anti-TbGalE antiserum [44] for 1 h at 4°C and immunoprecipitated with 50μL of Protein A magnetic beads (Invitrogen). A similar procedure was used for TbGMER immunoprecipitation, but instead mouse antisera anti-GMER and protein G magnetic beads (Invitrogen) were used. Samples were run in SDS-PAGE for Western blotting.

Western blotting

Samples were run on 4–12% gradient NuPAGE gels (Invitrogen) and Western blotted onto nitrocellulose using an iBlot system (Invitrogen). The pieces of nitrocellulose were either stained with Ponceau red for protein or blocked with 30 ml pre-filtered (20 μm) blocking buffer (50 mM Tris-HCl pH 7.4 containing 0.15 M NaCl, 0.25% BSA (w/v), 2% (w/v) fish skin gelatin and 0.05% (w/v) Tween 20) using a SNAPid system (Millipore). The latter were incubated with 0.5–1 μg / ml affinity purified antibody or with 1:1,000 diluted mouse antiserum (both diluted in blocking buffer) against the respective nucleotide sugar biosynthetic enzymes for 1 h at room temperature. Using the SNAPid system, the blots were washed 3 times 30 ml PBS, 0.1% Tween 20 and then incubated with 5 ml anti-mouse HRP (Invitrogen) diluted 1:10,000 in blocking buffer without sodium azide. The blots were subsequently washed with PBS, 0.1% Tween 20, removed from the SNAPid system and developed with ECL (ThermoFisher). Rabbit anti-TbGAPDH, rabbit anti-TbEnolase, rabbit anti-TbPEX13 and rabbit anti-TbAldolase (kind gifts from Prof. Paul Michels, Univ. of Edinburgh, UK) were used at 1:5,000, 1:5,000, 1:10,000 and 1:5,000 dilutions in blocking buffer, respectively. In addition, developments with mitochondrial marker [rabbit anti-TbGAP1 (a kind gift from Prof. Julius Luke, Institute of Parasitology, Czech Republic)] were performed at a dilution of 1:1,500 and developments with ER marker [immunopurified rabbit anti-calreticulin (Stressgen, Enzo LifeSci, USA)] were performed at a dilution of 1:1,000. Blots were developed with 1:10,000 anti-rabbit HRP (Invitrogen) and ECL reagents according to the manufacturer’s instructions (ThermoFisher). The blots were exposed onto ECL film (GE Healthcare Amersham), developed and scanned in an Epson office scanner.

Immunofluorescence microscopy

Trypanosomes were washed in ice-cold phosphate buffer saline (PBS) for pcf cells or ice-cold trypanosome dilution buffer (TDB; 20 mM Na₂HPO₄, 2 mM NaH₂PO₄, 80 mM NaCl, 5 mM KCl, 1 mM MgSO₄, 20 mM glucose, pH 7.7) for bsf cells and resuspended in the same buffer. Equal volumes of 8% paraformaldehyde (PFA) in PBS or TDB, respectively, were added and cells were fixed at 4° C. Subsequently, suspensions of fixed cells were spotted on cover slips, allowed to air dry, permeablized with 0.1% TX100 in PBS for 10 min and blocked in PBS, 5% fish skin gelatin, 10% normal goat serum and 0.05% TX100. Affinity purified anti-TbMPGT, anti-TbUGP and anti-TbGNA mouse antibodies were used at 0.5–1.0 μg / ml diluted in PBS, 1% fish skin gelatin, 0.05% TX100 and incubated for 1 h at room temperature in a humid chamber. Anti-TbGALE, anti-TbPAGM, anti-TbPMI, anti-TbPMM and anti-TbGMER mouse sera were diluted at 1:500 or 1:1,000 in the same buffer. Rabbit anti-GAPDH and rabbit anti-TbEnolase sera (generous gifts of Dr Paul Michels) were used at 1:2,000 and 1:4,000 dilution, respectively. Coverslips were washed in PBS, 1% fish skin gelatin, 0.05% TX100 and incubated for 1 h at room temperature with 50 μL goat anti-mouse IgG Alexa 488 conjugated mixed goat anti-rabbit IgG Alexa 594 conjugated, both diluted at 1:500 in same buffer. Coverslips were washed and mounted on glass slides using Prolong Gold (Invitrogen). Microscopic images were obtained in a Zeiss LSM 710 META confocal microscope.

Colocalisations were quantified using Volocity software (Quorum Technologies). Zeiss.lsm images were imported into the Volocity library and processed as follows: The thresholds for the red and green channels were calculated from a region of interest outside the cell images. Individual cells were selected using the region of interest (ROI) tool and colocalization statistics were calculated and recorded for each cell. Mander’s colocalisation coefficients (the fraction of green signal colocalising with red compartments) are reported in (Table 3), according to the recommendations of [84].

Digitonin latency

Digitonin latency was performed as previously described in [42]. Briefly T.brucei bloodstream or procyclic form cells (2 x 10⁹ cells) were washed twice with 10 ml 250 mM sucrose, 25 mM Tris-HCl pH 7.4 and 1 mM EDTA (STE buffer) and resuspended in 1.5 ml STEN (STE containing 0.15 M NaCl). Aliquots (0.15 ml) were treated with equal volume of digitonin at various concentrations in the presence of 0.1 mM TLCK, 1 μg / ml leupeptin, 1 μg / ml aprotinin and 1 mM PMSF. Digitonin stock was prepared at 10 mg / ml in DMSO and diluted in STEN to the required concentrations. Complete extraction was obtained in parallel by treating an equivalent number of cells with 0.1% Triton X-100 in STEN. The lysates were incubated for 5 min at room temperature and the insoluble materials removed by centrifugation at 16,000 g for 2 min. The pellets were discarded and samples of the supernatants were run on a reducing 4–12% gradient NuPage gel (Invitrogen) and transferred to nitrocellulose for Western blotting using antisera produced in mouse and at the dilutions described in the immunofluorescence section. Different amounts of same samples were loaded onto separate gels for each antibody Western blot, in order to keep within the linear range of film exposure. For TbPMM, TbGALE and TbGMER 1x10⁷ cell equivalents per lane, for TbPAGM, TbMPGT, TbUGP, TbUAP, TbPMI, 5 x 10⁶ cell equivalents per lane, for TbGAPDH, TbAldolase (Aldo), for TbEnolase (Eno) 1x10⁵ cell equivalents per lane and for TbPEX13 5x10⁶ cell equivalents per lane.

Subcellular fractionation by differential centrifugation followed by density gradient centrifugation

The pcf cells used for differential and density gradient centrifugation came from a stable isotope in cell culture (SILAC) labelling experiment. However, the isotopic labelling is not relevant to the specific experiments and results presented here. The starting pcf cell preparation (a total of 5.6 x 10⁹ cells washed in STE buffer) was a 1: 1 mixture of (i) GFP-tagged PEX13.1 pcf mutants labelled to steady state over 8 cell divisions, and to a final cell density of 1.1.x 10⁷ cells / ml, in R6K6 SDM-79 medium containing heavy isotopes of L-Arginine (R6) and L-Lysine (K6), and (ii) double marker cells grown in normal SDM-79 medium (R0K0) and also harvested at 1.1 x 10⁷ cells / ml.

The mixed cells were lysed using a One-shot high-pressure cell disruptor (Constant Systems, UK) operated at 10,000 psi as described in [54]. The lysate was submitted to differential centrifugation as described in [70]: After spinning at 1,500 g to the remove nuclei and any remaining intact cells and cell ghosts, the supernatant was submitted to centrifugation at 5,000 g to yield a Large Granular fraction (LG) pellet, enriched in mitochondria. Subsequent centrifugation of the supernatant at 15,000 g pelleted the Small Granular fraction (SG), enriched in glycosomes, with concomitant generation of a supernatant (C) enriched in cytosol and ER, Golgi and plasma membrane microsomes.

The SG fraction was subsequently submitted to density gradient centrifugation using iodixanol (Optiprep, Axis Shield, UK) according to [53], with some modifications: A discontinuous step gradient of iodixanol was prepared by layering 20, 25, 30, 35, 40 and 45% (w/v) iodixanol in STE buffer inside an Optiseal polyallomer tube (Beckman) using a peristaltic pump to apply each layer to the bottom of the tube, starting with the least dense layer and displacing it with next dense layer and so on. The SG fraction resuspended in STE buffer was loaded carefully on top of the gradient and centrifuged at 45,000 rpm using VTi-50 vertical rotor in a Beckman ultracentrifuge for 1h at 4°C. After ultracentrifugation, the tube was punctured with needle and syringe to collect 2 distinct opalescent bands, fraction 1 (top band) and fraction 2 (lower band). The refractive index of each fraction was measured using a refractometer (Abbe refractometer model NAR-1T) and plotted against a standard curve of 20, 25, 30, 35, 40 and 45% (w/v) iodixanol in STE. The density was calculated according to published tables of iodixanol percentage versus density (g/mL).

Proteomic analyses

Samples of fraction 1 and fraction 2 were reduced with dithiothreitol and alkylated with iodoacetamide and subsequently digested with trypsin and processed using FASP II (filter-aided sample processing), as described previously in [54]. Samples were desalted using a custom made Porous R2 columns packed with 1.5 mg Porous R2 reverse phase beads (ABI). The columns were pre-conditioned by washing with 90% CH₃CN containing 0.1% formic acid followed by equilibration in 0.1% formic acid in water. Samples were applied in 0.1% formic acid and the columns washed with 0.1% formic acid then subsequently eluted three times with 90% CH₃CN containing 0.1% formic acid. The eluates were combined, dried in a Speedvac vacuum concentrator, resuspended in 35% CH₃CN containing 0.1% formic acid and fractionated using strong cation exchange (SCX) chromatography: The combined eluates were applied into a custom made SCX column made by adding Poros 50 HS SCX beads (ABI) to a SCX Ziptip (Millipore). Bound peptides were eluted sequentially with 20, 100, 150, and 200 mM NaCl in 35% CH₃CN containing 0.1% formic acid, followed by final elutions with 0.5% NH₄OH in 50% isopropanol and 50% isopropanol (IPA sample). Each of these five sub-fractions were dried and re-suspended in 50 μL 1% formic acid and 15 μL aliquots were subjected to liquid chromatography on Ultimate 3000 RSLCnano system (Thermo Scientific) fitted with an Acclaim PepMap 100 (C18, 100 μM x 2 cm) trap cartridge. Peptides were separated on an Easy-Spray PepMap RSLC C18 column (75 μM x 50 cm) (Thermo Scientific) using a linear gradient from 2 to 40% buffer B (80% acetonitrile, 0.1% formic acid) in buffer A (0.1% formic acid) over 70 min. The HPLC system was coupled to a Orbitrap Velos Mass Spectrometer (Thermo Scientific) with a source voltage of 1.2 kV.

Proteomic data analysis

LC-MS/MS data were analysed for protein identification using MaxQuant 1.6.14 [85,86] with the in-built Andromeda search engine [87]. The raw files were searched against the T. brucei brucei 927 annotated protein sequences from TriTrypDB release 46 [88] supplemented with the T. brucei brucei 427 VSG221 (Tb427.BES40.22) protein sequence. The mass tolerance was set to 4.5 ppm for precursor ions and trypsin was set as proteolytic enzyme with two missed cleavage allowed. Carbamidomethyl on cysteine was set as fixed modifications. SILAC labelling [5] of heavy arginine (Arg-6) and Lysine (Lys-6) were specified. Oxidation of methionine and Acetylation of Protein N-term were set as variable modifications. The false-discovery rate for protein and peptide level identifications was set at 1%, using a target-decoy based strategy. The minimum peptide length was set to seven amino acids and protein quantification was performed on unique plus razor peptides [89]. “Reverse Hits”, “Only identified by site” and “Potential contaminant” identifications were filtered out. Only protein groups with at least two unique peptide sequences and Andromeda protein score greater than 5 were selected for further analysis. The high confidence glycosomal resident proteins were extracted from Güther et al., 2014 [54]. Gene abbreviations were retrieved from the TriTrypDB database. The iBAQ values [89,90] were extracted from the MaxQuant output and visualised as bar graphs. The analysis pipeline was implemented in python using the SciPy packages (https://www.scipy.org/) and Jupyter notebook (https://jupyter.org/).

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [91] partner repository with the dataset identifier PXD023124. Reviewer account details: Username: reviewer_pxd023124@ebi.ac.uk. Password: qqm7vZg3. The analysis pipeline is available in GitHub (https://github.com/mtinti/nucleotide_sugar) and it is archived in Zenodo (https://doi.org/10.5281/zenodo.4289929). The analysis pipeline is reproducible using the mybinder app with the link reported in the GitHub repository.