Analysis of Membrane-Enriched and High Molecular Weight Proteins

Article pubs.acs.org/jpr

Analysis of Membrane-Enriched and High Molecular Weight Proteins in Leishmania infantum Promastigotes and Axenic Amastigotes Marie-Christine Brotherton, Gina Racine, Amin Ahmed Ouameur, Philippe Leprohon, Barbara Papadopoulou, and Marc Ouellette* Centre de Recherche en Infectiologie du Centre de Recherche du CHUL and Département de Microbiologie et Immunologie, Faculté de Médecine, Université Laval, Québec, Canada S Supporting Information *

ABSTRACT: Membrane and high molecular weight (HMW) proteins tend to be underrepresented in proteome analyses. Here, we optimized a protocol designed for the extraction and purification of membranes from the protozoan parasite Leishmania using a combination of serial centrifugation and free-flow zone electrophoresis (ZE-FFE). We also enriched for Leishmania HMW proteins from total extracts using the Gelfree 8100 fractionation system. This allowed the study of expression of both membrane-enriched and HMW proteins in Leishmania infantum promastigotes and amastigotes. We identified 194 proteins with at least one transmembrane domain (TMD) and 171 HMW proteins (≥100 kDa) in the invertebrate promastigote stage and 66 proteins with at least one TMD and 154 HMW proteins in the mammalian amastigote stage. Several of the proteins identified in one of the stages are part of pathways consistent with the known biology of the parasite, with many proteins involved in lipid synthesis, numerous dynein heavy chains, and some surface antigen proteins 2 detected in the promastigote stage. Notably, some proteins involved in transport and proteolysis were detected either in promastigote or amastigote. The present study is using improved proteomic methods for studying membrane-enriched and HMW proteins helping to achieve a better understanding of the parasite life cycle. KEYWORDS: Leishmania infantum, free-flow zone electrophoresis (ZE-FFE), gelfree 8100, membrane proteins, high molecular weight proteins, cytodifferentiation

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INTRODUCTION Protozoan parasites of the genus Leishmania cause a wide range of diseases affecting 12 million people worldwide with 1.5−2 million new cases each year.1 During their development, Leishmania alternate between the midgut of the sandfly vector as extracellular flagellated promastigotes and the phagolysosome of mammalian macrophages as round nonmotile amastigotes. The differentiation from promastigotes to amastigotes is triggered by elevated temperature and drop in pH2 within the phagolysosome, and these conditions can be reproduced in vitro for culturing axenic amastigotes.3−5 The control of gene expression in Leishmania occurs almost exclusively at the post-transcriptional and translational/posttranslational levels (reviewed in refs 6 and 7), and hence several proteomic studies were carried out to reveal the molecular mechanisms implicated in the cytodifferentiation of this parasite8 (reviewed in refs 9 and 10). Nonetheless, some classes of proteins such as low-abundant, membranous, extreme pI and extreme molecular weight (MW) proteins are difficult to analyze and are often underrepresented in proteomic screens.11,12 Although the analysis of the L. infantum predicted proteome using TMHMM server v. 2.0 (www.cbs.dtu.dk/ services/TMHMM/) indicated that 18.0% of the proteins are © 2012 American Chemical Society

predicted to contain at least one TMD, these remain underrepresented in proteomic screens from Leishmania whole cell extracts (reviewed in ref 10) because of their high hydrophobicity and poor solubility. Few studies have dealt with proteomics of membrane proteins in Leishmania. Surface biotinylation-streptavidin affinity separation and octyl glucoside detergent extraction was used recently to compare virulent and avirulent metacyclic forms of L. chagasi.13 Similarly, although in silico analysis revealed that 18.1% of the L. infantum predicted proteins have a molecular mass over 100 kDa (www.tritrypdb. org), these high molecular weight (HMW) proteins have a propensity to form aggregates in solution and are often missed during proteomic screens of Leishmania whole protein extracts (reviewed in ref 10). Furthermore, proteolytic protein products especially for HMW proteins were frequently reported in Leishmania, and evidence was provided that some of these processing events are stage-specific.14−20 Hence, technological improvements are needed to increase the coverage for these “hard-to-work-with” proteins. For example, free-flow electrophoresis (IEF-FFE) followed by hydroxyethyl disulfide-based Received: December 21, 2011 Published: June 20, 2012 3974

dx.doi.org/10.1021/pr201248h | J. Proteome Res. 2012, 11, 3974−3985

Journal of Proteome Research

Article

bromophenol blue front reached the bottom of the gel. The entire gel lanes were cut into a dozen bands. Second, membrane-enriched fractions were separated as above, but 5 spots corresponding to proteins with MW higher than 100 kDa were cut. Third, membrane-enriched fractions were separated on a 6% acrylamide gel until the 75-kDa band of the Precision Plus Protein standards (BioRad #161-0373) reached the bottom of the gel. These entire gel lanes were also cut into a dozen bands. These combined electrophoretic methods allowed a better separation of a broader range of transmembrane proteins. Bands on the gels were visualized using Coomassie blue staining.

2D gel separation was useful to study basic protein in Leishmania.20 In the present study, we describe a protocol combining sonication, serial centrifugations and free-flow zone electrophoresis (ZE-FFE), a continuous liquid-based method for the separation of organelles and membranes according to their net charge density (reviewed in ref 21), to enrich for Leishmania membrane proteins prior to their analysis by 1D gel electrophoresis. We also describe a protocol for the enrichment of HMW proteins from total protein extracts of Leishmania using the Gelfree 8100 fractionation system.22,23 Finally, these techniques allowed us to separate two classes of proteins usually underrepresented in proteomic studies11,12 and to compare both membrane-enriched and HMW proteins between L. infantum promastigotes and axenic amastigotes.

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Protein Extraction Prior to Gelfree 8100 Fractionation

L. infantum promastigotes (∼2 × 108 cells) and axenic amastigotes (∼6 × 108 cells) in their exponential phase of growth were pelleted by centrifugation (3000 rpm, 5 min). Pellets were washed twice in Hepes-NaCl (21 mM Hepes, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4·H2O, and 6 mM Dextrose, pH 7.05). Cells were resuspended in 500 μL of buffer B25 supplemented with 20 μL of protease inhibitors cocktail (Sigma #P2714) and then broken by sonication on ice at 50% amplitude (5 pulses of 10 s with 1 min interval). The supernatants were collected after centrifugation (3000 rpm, 10 min). Proteins were concentrated using Amicon Ultra 3K columns (Millipore), desalted with Zeba Spin Desalting columns (Thermo Scientific), and then quantified using the 2-D Quant Kit (GE Healthcare).

MATERIALS AND METHODS

Cell Culture

L. infantum (MHOM/MA/67/ITMAP-263) wild type promastigotes were grown in SDM-79 medium pH 7.0 at 25 °C, and axenic amastigotes were grown as described previously.24 Membrane Extraction

Five hundred milliliters of exponential phase culture (approximately 1010 cells) of each life stages were harvested and washed in buffers A and B as described previously.25 Cells were resuspended in 10 mL of buffer B supplemented with 50 μL of protease inhibitors cocktail (2 mM AEBSF, 14 μM E-64, 130 μM Bestatin, 0.9 μM Leupeptin, 0.3 μM Aprotinin, and 1 mM EDTA) (Sigma #P2714) and frozen overnight at −80 °C. Cells were thawed and broken by sonication on ice at 50% amplitude (5 pulses of 10 s with 1 min interval). Cell disruption was monitored by light microscopy. Centrifugations to pellet membranes were done as described previously.25 Membrane pellets were then washed and resuspended in 500 μL of buffer C supplemented with 2 mM MgCl2 and snap-frozen in liquid nitrogen until further use. Membrane proteins were quantified using the 2-D Quant Kit (GE Healthcare).

Gelfree 8100 Fractionation for HMW Protein Separation

Five hundred micrograms of proteins from two biological replicates for each life stage were loaded in the Gelfree 8100 Fractionation Station (Protein Discovery). The 5% TrisAcetate Cartridge (Protein Discovery) designed for the separation of HMW proteins were used according to the method recommended by the manufacturer (Protein Discovery Gelfree 8100 Fractionation System User Manual). Proteins were separated into 12 fractions according to their respective molecular weight. The last six fractions containing the lowabundant HMW proteins were concentrated with Amicon Ultra 3K columns (Millipore) prior to 1DE. Proteins were quantified using the 2-D Quant Kit (GE Healthcare).

Free-Flow Zone Electrophoresis (ZE-FFE)

Continuous ZE-FFE was performed using a BD FFE System (BD Diagnostics) following the protocol supplied by the manufacturer. For comprehensive reviews about this method, please see refs 26 and 27. ZE-FFE buffers required for Leishmania membrane purification were prepared as described (BD FFE application Manual), and electrophoretic conditions recommended by the manufacturer were used (10 °C, 750 V, media flow rate of 300 mL/h). Membranes were injected at a concentration of 2 mg/mL. Fractionated samples were collected into a 96-well plates, and the optical density at 280 nm (OD280) was measured to determine which wells contained Leishmania purified membranes that were usually recovered in 15−20 fractions that were pooled and pelleted by ultracentrifugation at 140000g for 1 h. Pellets were resuspended in ZE-FFE separation buffer and snap-frozen in liquid nitrogen until further use. Membrane-enriched proteins were quantified using the 2-D Quant Kit (GE Healthcare).

Sodium Dodecyl Sulfate (SDS)-PAGE (1DE) of HMW Proteins

The L. infantum promastigote and axenic amastigote Gelfree 8100 fractions and total sonicated proteins as a control were mixed with Laemmli sample buffer and separated on gradient 6−14% acrylamide gels until bromophenol blue front reached the bottom of the gel. Gels were then visualized using Coomassie blue staining, and bands corresponding to fractions 8−12 (above 150 kDa) were cut and sent for MS identification. In-Gel Trypsin Digest and Mass Spectrometry

Gel slices were excised manually and identified by tandem mass spectrometry (Proteomics Platform of the Eastern Quebec Genomics Center, Quebec, Canada). Gel slices were then deposited into a 96-well plate and washed with water. In-gel protein digestion was performed on a MassPrep liquid handling station (Waters, Milford, USA) according to the manufacturer’s specifications. Briefly, proteins were reduced with 10 mM DTT and alkylated with 55 mM iodoacetamide. Trypsin digestion was performed using 105 mM of modified porcine trypsin (Sequencing grade, Promega, Madison, WI) at 58 °C for 1 h. Digestion products were extracted using 1% formic acid, 2%

Sodium Dodecyl Sulfate (SDS)-PAGE (1DE) of Membrane-Enriched Fractions

Membrane-enriched fractions from L. infantum promastigotes and axenic amastigotes were mixed with Laemmli sample buffer. First, both life stages purified membrane-enriched fractions were separated on a 10% acrylamide gels until 3975



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described above. Total protein extracts were also obtained using 2D lysis buffer, as described previously.20 Five micrograms of total protein extract and membranes purified or not with ZE-FFE were used. It is important to note that samples were boiled (5 min at 95 °C) except when the blot was intended to be incubated with an anti-GFP antibody. SDSPAGE was run at 150 V on a 10% acrylamide gel. Separated proteins were then transferred to nitrocellulose at 100 V for 1 h. The membrane was blocked overnight in 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20. Then the membrane was incubated 90 min in the blocking solution containing a dilution of the primary antibody and washed 2 × 10 min with Tris-buffered saline containing 0.1% Tween 20. The primary antibody dilutions used were 1:1000 for the rabbit monoclonal GFP antibody (Invitrogen); 1:2000 for the mouse monoclonal EF-1α antibody (Millipore), and 1:5000 for the mouse monoclonal α-tubulin antibody (Sigma). The blots were then incubated for 1 h in the blocking solution with a dilution of the corresponding second antibody conjugated with horseradish peroxidase and washed three times as above. The secondary antibody dilutions used were 1:2000 for the ECL antirabbit IgG (GE Healthcare) and 1:10 000 for the goat-anti mouse IgG (Thermo Scientific). The blots were then incubated 1 min with Immobilon western chemiluminescent HRP substrate (Millipore) and exposed to X-ray film.

acetonitrile followed by 1% formic acid, 50% acetonitrile. Peptides were lyophilized in a speed vacuum and resuspended in 8 μL of 0.1% formic acid and 4 μL of this was used for mass spectrometry analysis. Peptide MS/MS spectra were obtained by capillary liquid chromatography coupled to an LTQ linear ion trap mass spectrometer equipped with a nanoelectrospray ion source (Thermo Electron, San Jose, CA, USA). Peptides were loaded onto a reversed-phase column (PicoFrit 15-μm tip, BioBasic C18, 10 cm ×75 μm; New Objective, Woburn, MA, USA) and eluted with a linear gradient from 2 to 50% acetonitrile in 0.1% formic acid at a flow rate of 200 nL/min. Mass spectra were acquired using a data-dependent acquisition mode (Xcalibur software, version 2.0) in which each full scan mass spectrum was followed by collision-induced dissociation of the seven most intense ions. The dynamic exclusion function was enabled (30 s exclusion), and the relative collisional fragmentation energy was set to 35%. Interpretation of Tandem Mass Spectra and Protein Identification

MS/MS spectra were analyzed using MASCOT (Matrix Science, London, UK; version 2.2.0) and searched against L. infantum in the GeneDB LeishPEP_2010 database (http:// tritrypdb.org/common/downloads/release-2.2/Linfantum/ LinfantumAnnotatedProteins_TriTrypDB-2.2.fasta) assuming a digestion with trypsin. A mass tolerance of 2.0 Da for peptides and 0.5 Da for fragments were tolerated, with 2 trypsin miss cleavages allowed. Carbamidomethylation of cysteine and partial oxidation of methionine were considered in the search. The Scaffold software (Proteome Software Inc., Portland, OR, USA; version 3.0.9.1) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they reached greater than 95% probability as specified by the Peptide Prophet algorithm. 28 Protein identifications were accepted if they reached greater than 80% probability and contained at least two unique peptides as specified by the Protein Prophet algorithm.29 Proteins that contained similar peptides and could not be differentiated according to MS/MS alone were grouped to satisfy the principle of parsimony. Transmembrane domains were predicted using TMHMM server v. 2.0 (www.cbs.dtu.dk/ services/TMHMM/), which is based on a hidden Markov model (HMM) and TMpred (http://www.ch.embnet.org/ software/TMPRED_form.html) (Supporting Information), a method using a combination of several weight-matrices for scoring. GPI-anchors were predicted using either FragAnchor (http://navet.ics.hawaii.edu/∼fraganchor/NNHMM/ NNHMM.html) or GPI-SOM (http://gpi.unibe.ch/).

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RESULTS

Enrichment in Membrane Proteins by ZE-FFE

To study membrane proteins of L. infantum, we developed a protocol combining sonication, ultracentrifugation and ZE-FFE purification. The enrichment of Leishmania membrane proteins was first confirmed using L. infantum promastigotes expressing a GFP-tagged version of the FBT protein LinJ.10.0390.30 This transporter contains 12 predicted TMDs, and we showed that this protein is localized to the plasma membrane using fluorescence microscopy (Figure 1A). Equal amounts of total proteins or membrane-enriched extracts (either purified by ZEFFE or not) derived from L. infantum pSPαNEOαLinJ.10.0390-GFP transfectants were separated by SDSPAGE, transferred to a nitrocellulose membrane, and reacted with anti-GFP antibody. A band of 103 kDa corresponding to

Western Immunoblotting

The gene LinJ10.0390 coding for a FBT (folate/biopterin transporter) protein, which is absent in the strain MHOM/ MA/67/ITMAP-263,30 was amplified from L. infantum JPCM5 using primers 5′-CGGGATCCGTGAAGTCGAACCATGTCC-3′ and 5′-CGTCTAGACTCCTTTTTATGCGTCTGCGC-3′, containing BamHI and XbaI sites, respectively (underlined). The PCR fragment was first ligated into the pGEM T-easy vector (Invitrogen), digested with the appropriate restriction enzymes, and cloned into the Leishmania expression vector pSPαNEOα-GFP.30 Cellular localization of the FBT-GFP fusion protein was determined by fluorescence microscopy as described previously.31 Membranes of L. infantum promastigotes containing pSPαNEOα-LinJ.10.0390-GFP were extracted and purified as

Figure 1. Enrichment in L. infantum membrane proteins after ZE-FFE. (A) Fluorescence microscopy using L. infantum transfected with pSPαNEOα-LinJ.10.0390-GFP indicating the localization of the fusion protein at the level of the parasite’s plasma membrane. (B) L. infantum pSPαNEOα-LinJ.10.0390-GFP total cell extract (lane 1), membraneenriched proteins before ZE-FFE purification (lane 2), and membraneenriched proteins after ZE-FFE purification (lane 3) were run on SDSPAGE and blotted. The blot was then incubated with anti-GFP (row 1), anti-EF1α (row 2), or anti-α-tubulin (row 3). 3976



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Table 1. MS/MS Identification of Membrane Proteins with at Least Two TMDs in L. infantum Promastigotes and/or Amastigotesa,b putative protein name Transport AAT8 amino acid permease ABC transporter-like protein ABCA10 ATP-binding cassette protein A10, putative ABCB1 ATP-binding cassette protein B1, putative ABCB3 ATP-binding cassette protein B3, putative ABCB4 (MDR1) p-glycoprotein ABCC1−2 ATP-binding cassette protein C1−2, putative ABCC3 (MRPA) ABC-thiol transporter ABCC7 (PRP1) pentamidine resistance protein 1 ABCG1−2−3 ATP-binding cassette protein G1−2−3, putative ABCG4 ATP-binding cassette protein G4, putative ABCG5 ATP-binding cassette protein G5, putative ANC1 ADP, ATP carrier protein 1, mitochondrial, putative calcium motive p-type ATPase, putative calcium-translocating P-type ATPase cation transporter, putative cation-transporting ATPase, putative COP-coated vesicle,ER–golgi transport protein erv25 precursor COP-coated vesicle, ER–golgi transport protein gp25L putative endomembrane protein, putative FT1 folate/biopterin transporter, putative FT5 folate/biopterin transporter, putative glucose transporter, lmgt2 H1A-1(2) P-type H+-ATPase, putative LPG2 lipophosphoglycan biosynthetic protein P-type ATPase, putative sre-2/carboxylate carrier-like protein transmembrane/endomembrane-like protein tricarboxylate carrier, putative vacuolar proton translocating ATPase subunit A, putative vacuolar-type Ca2+-ATPase, putative vacuolar-type Ca2+-ATPase, putative vacuolar-type proton translocating pyrophosphatase 1, putative zinc transporter-like protein Surface amastin-like surface protein-like protein extracellular receptor, putative MBAP membrane-bound acid phosphatase precursor surface antigen protein 2, putative surface antigen protein 2, putative surface antigen protein 2, putative surface protein amastin, putative Metabolism 3-oxo-5-alpha-steroid 4-dehydrogenase, putative C-14 sterol reductase, putative CDP-diacylglycerol-inositol 3-phosphatidyltransferase cytochrome p450-like protein dehydrogenase-like protein delta-12 fatty acid desaturase diacylglycerol acyltransferase, putative fatty acid desaturase, sphingolipid delta 4 desaturase, putative fatty acid elongase, putative, beta-ketoacyl-CoA synthase fatty acid elongase, putative, beta-ketoacyl-CoA synthase fatty acid elongase, putative, beta-ketoacyl-CoA synthase fatty acid elongase, putative, beta-ketoacyl-CoA synthase fatty-acid desaturase, putative GAT glycerol-3-phosphate acyl transferase glutathione-S-transferase/glutaredoxin,putative phosphatidylethanolaminen-methyltransferase-like protein

systematic IDsc

predicted Mr (kDa)

TMDsd

Proa

66 78 207 72 77 147 173 174 194 73 83 135 35 122 111 64 140 30 24 67 76 72 61 107 37 122 38 71 36 88 95 102 83 48

11 6 16 4 5 9 11 8 8 5 5 10 4 8 7 3 10 2 2 9 11 11 12 8 9 6 2 10 3 6 8 6 14 5

X X

X X X X X X X X X X X X X X X X X X X X X X X

LinJ.30.0930 LinJ.19.0640 LinJ.36.2700 LinJ.12.0663 LinJ.12.0665 LinJ.12.0666 LinJ.30.0920

24 100 57 71 49 44 21

4 2 2 2 2 2 4

X X X X X X X

LinJ.25.1850 LinJ.32.2470 LinJ.26.2500 LinJ.30.3610 LinJ.10.0070 LinJ.33.3420 LinJ.27.1460 LinJ.26.1670 (+1) LinJ.14.0670 LinJ.14.0680 LinJ.14.0700 LinJ.14.0760 LinJ.24.2340 LinJ.03.0070 LinJ.14.1580 LinJ.31.3250

34 49 25 58 45 45 176 43 32 32 33 37 49 71 35 67

2 6 4 2 2 4 30 5 7 6 5 5 4 3 2 8

X X X X X X X X X X X X X X X X

LinJ.31.1810 LinJ.32.2190 LinJ.29.0640 LinJ.25.0540 LinJ.32.3280 LinJ.34.1060 LinJ.23.0230 LinJ.23.0290 LinJ.31.1460 LinJ.06.0080 LinJ.15.0950 LinJ.23.0430 LinJ.19.0190 LinJ.35.2080 LinJ.04.0010 LinJ.19.1420 LinJ.07.1210 LinJ.36.2280 LinJ.35.1840 LinJ.13.1280 LinJ.10.0400 LinJ.10.0420 LinJ.36.6550 LinJ.18.1500 LinJ.34.4290 LinJ.17.0660 LinJ.05.0680 LinJ.34.3450 LinJ.01.0590 LinJ.23.1910 LinJ.07.0510 LinJ.07.0700 LinJ.31.1240 LinJ.31.2470

3977

(+3)

(+1)

(+2)

(+1)

(+1)

(+1)

Amaa

34

X X X X X X X X

refe

34 34

X 33,34 33

X X X X X X

33,34 13,33,34 33,34 33,34 34

X

33,34 33,34 34

34 34

X

33,34 34

X

34 34 34 34

X X X X

33 33,34 33,34 13,33,34

34

X 13 13

34

X

33,34

34 34 13,34

33,34 33,34 34

34

33,34



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Table 1. continued putative protein name

systematic IDsc

predicted Mr (kDa)

TMDsd

Proa

Amaa

refe

Metabolism 34 sterol C-24 reductase, putative LinJ.33.0730 58 7 X X Protein folding 33,34 QSOX quiescin sulfhydryl oxidase, putative LinJ.30.0440 63 2 X X Proteolysis 33,34 CAAX prenyl protease 1 metallo-peptidase Clan M- Family M48 LinJ.27.0040 49 3 X Transcription, Translation 13,33,34 60S ribosomal protein L7a, putative LinJ.07.0550 (+1) 39 2 X X 34 RNA-binding protein, putative, UPB1(2) LinJ.25.0500 (+1) 27 2 X X Others 34 glycosomal membrane like protein LinJ.24.0140 24 2 X 33 GPI16 gpi transamidase component, putative LinJ.34.2560 80 2 X mannosyltransferase-II, putative LinJ.18.0960 77 6 X 33,34 oligosaccharyl transferase subunit, putative LinJ.35.1170 92 11 X X 33 oligosaccharyl transferase-like protein LinJ.35.1150 87 9 X X 33,34 oligosaccharyl transferase-like protein LinJ.35.1140 96 11 X X palmitoyl acyltransferase 1, putative LinJ.04.0510 116 6 X 33,34 PAPLE22 reticulon domain, potentially aggravating protein LinJ.30.2570 22 3 X X 33,34 pretranslocation protein, alpha subunit, SEC61-like, putative LinJ.11.1050 54 9 X 13,33 protein tyrosine phosphatase, putative LinJ.05.0280 25 4 X X rer1 family like protein LinJ.22.0450 21 3 X X 34 Zn-finger domain protein, putative LinJ.29.1770 135 9 X Hypothetical (Includes 45 proteins with between 2 and 27 TMDs and between 19 and 153 kDa. See Tables S1 and S2 (Supporting Information) for the details.) a Mr, molecular weight; TMDs, transmembrane domains; Pro, promastigotes; Ama, amastigotes. bStatistical MS/MS data and identified proteins with no TMD are available in Tables S1 and S2, Supporting Information. cNumbers in brackets indicate the number of proteins with ambiguous identifications that is shown in Tables S1 and S2, Supporting Information. dTMDs were predicted using TMHMM v. 2.0. eAlso identified in other studies.

identifications, we processed the replicate extracts by varying migration conditions in 10 or 6% acrylamide gels (see Materials and Methods). For promastigotes, this enabled the identification of a total of 488 proteins (Table S1, Supporting Information), 194 (39.7%) of which contained at least one predicted TMD according to the TMHMM v2.0 (Tables 1 and S1, Supporting Information). Notably, we succeeded to identify a putative diacylglycerol acyltransferase (LinJ.27.1460) with 30 predicted TMDs, which is the highest number of predicted TMDs in the L. infantum proteome (www.tritrypdb.org) (Table 1). Approximately 21.3% of the proteins identified here in the promastigote membrane-enriched sample were also present in a previous study using L. chagasi.13 For amastigotes, 372 proteins were identified (Table S2, Supporting Information), with 66 (17.7%) containing at least one predicted TMD according to TMHMM v2.0 (Tables 1 and S2, Supporting Information). The protein with the highest number of predicted TMDs identified in this life stage was a hypothetical protein (LinJ.32.2940) containing 27 predicted TMDs (Table S2, Supporting Information). The TMHMM v. 2.0 algorithm seems to be stringent since when the TMpred (http://www.ch. embnet.org/software/TMPRED_form.html) software was used, 75% of proteins detected in both promastigotes and axenic amastigotes were predicted to have at least one TMD (Tables S1 and S2, Supporting Information). Moreover, 39.3 and 43.0% of the promastigote and amastigote membraneenriched identified proteins had already been identified in T. brucei plasma membrane extracts, respectively.33 Interestingly, 26.6% of the promastigote-enriched and 25.5% of the amastigote-enriched proteins were identified for the first time when compared with a previous large-scale L. donovani proteomic screen using MS-based techniques.34 Several of the proteins identified here did not contain TMDs (Tables S1 and

the LinJ.10.0390-GFP fusion was visible in the membraneenriched sample, but not in the total protein extract (Figure 1B, upper row, lane 1 vs 2). This band was further enriched when membrane extracts were purified by ZE-FFE (Figure 1B, upper row, lane 2 vs 3), hence validating the usefulness of our combined protocol for the enrichment of membrane proteins. This result also suggests that our membrane-enriched fractions contain plasma membrane proteins. We also reacted the blot with an antibody directed against the cytosolic elongation factor 1α (EF1α), and the intensity of the 49-kDa band corresponding to this protein was found to decrease substantially after the enrichment (Figure 1B, middle row, lane 1 vs 2) and purification (Figure 1B, middle row, lane 2 vs 3) of membranes. The band quantification corresponding to EF1α decreased by approximately 96-fold between the whole cell extract and the ZE-FFE-purified membranes (Figure 1B, middle row, lane 1 vs 3) as determined using ImageJ 1.38x (Wayne Rasband, NIH, USA, http://rsb.info.nih.gov/ij/). Our protocol did not completely exclude cytoskeleton proteins, however, as reacting the blot with an antibody directed against the abundant α-tubulin protein revealed only a partial decrease in the intensity of this protein (Figure 1B, bottom row). This was somewhat expected since the cytoskeleton is a structure tightly linked to the plasma membranes.32 Membrane-Enriched Proteome Analysis of L. infantum Promastigotes and Axenic Amastigotes

Confident with our workflow, we next analyzed the membraneenriched proteome of the two main Leishmania life stages. Triplicate membrane-enriched extracts enriched by ZE-FFE were prepared from L. infantum promastigotes and axenic amatigotes and were separately run on 1D SDS-PAGE prior to MS-based identification. To increase the coverage of protein 3978



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into 12 fractions of increasing MW (Figure 3), and the last five fractions corresponding to proteins having a MW higher than

S2, Supporting Information). One possibility is that some of them may be anchored to the membrane via GPI-anchors.35 By using two of the most common software for GPI-anchored protein prediction (GPI-SOM or FragAnchor-see Material and Methods), only 171 and 114 proteins of the entire L. infantum proteome were predicted to contain a GPI-anchor, respectively. We searched the proteins identified in the present study with those two algorithms and found only GP63 and three putative surface antigens protein 2 to have a high probability to be GPIanchored (Tables S1 and S2, Supporting Information). The other proteins without TMD identified here in our membraneenriched fraction analysis may thus correspond to proteins interacting with membrane proteins (see the Discussion), although we cannot exclude cytosolic contamination as we were still detecting low levels of EF1α in our membrane-enriched fraction after ZE-FFE purification (Figure 1B, middle row, lane 3). Proteins with predicted TMDs identified in promastigotes and/or amastigotes were sorted into functional groups according to BLAST analysis and Gene Ontology annotations (www.genedb.org). For both stages, the largest group consisted of hypothetical proteins, which represent more than 40% of the identified membrane proteins. Among the predicted membrane proteins with domains enabling functional assignment, 18 and 22% of the proteins detected in promastigotes and amastigotes, respectively, have predicted transport functions; 7 and 6% are possibly involved in host-parasite interactions; 7 and 12% are involved in metabolic processes, 2 and 6% are predicted to be involved in protein folding; and 1 and 3% are potentially involved in transcription or translation (Figure 2). Membrane

Figure 3. Fractionation of HMW proteins using the Gelfree 8100 fractionation system. Whole cell lysates of L. infantum axenic amastigotes were separated using the Gelfree 8100 fractionation system, and each fraction was run separately on SDS-PAGE and compared with sonicated total protein extract (lane T). Note that both biological replicates of promastigotes (not shown) and axenic amastigotes gave the same pattern for each fraction on SDS-PAGE.

150 kDa on SDS-PAGE were individually sent for MS/MS identification. This yielded a total of 178 and 162 protein identifications from the promastigote and amastigote extracts, respectively (Tables 2 and S3, Supporting Information), with more than 75% reproducibility between the two replicates in both life stages. Less than 20% of the proteins were found in more than one fraction, which indicates that we had only a minor overlap between adjacent fractions. Interestingly, 142 (79.8%) and 143 (88.3%) proteins identified in promastigotes and amastigotes, respectively, had a predicted MW between 100 and 299 kDa, with only 7 (3.9%) promastigote-identified and 8 (4.9%) amastigote-identified proteins having a MW under 100 kDa. Several very large proteins (MW > 300 kDa) like calpain-like cysteine peptidases, kinesins and dyneins were also detected (Table 2). Of interest, more than 50% of the proteins identified in promastigotes and/or amastigotes were identified for the first time when compared with a large-scale L. donovani proteomic screen previously done using MS-based techniques,34 which further indicates that the Gelfree 8100 Fractionation system is highly effective at enriching for HMW proteins that remain undetectable with conventional methods. Among the proteins identified using the Gelfree 8100 Fractionation system, 106 were common between promastigotes and amastigotes (Tables 2 and S3, Supporting Information). Thus, 72 and 56 proteins were identified either in the promastigote or the amastigote stages, respectively (Tables 2 and S3, Supporting Information). Interesting proteins detected in promastigotes include the ABC transporters ABCC3 (LinJ.23.0290) and ABCC7 (LinJ.31.1460), which were also detected in the promastigote stage using the protocol for membrane protein enrichment, five calpain-like cysteine peptidases (LinJ.20.1210, LinJ.21.0160, LinJ.21.0170, LinJ.27.2490 and LinJ.32.1020), and nine putative dynein heavy chains (LinJ.13.1390, LinJ.14.1130, LinJ.23.1570, LinJ.26.1000, LinJ.27.2460, LinJ.28.0650, LinJ.28.3110, LinJ.34.3690 and LinJ.36.1010) (Tables 2, S1 and S3, Supporting Information). On the other hand, interesting proteins detected in amastigotes include the ABC transporters ABCA4 (LinJ.11.1240) and ABCG5 (LinJ.23.0430), a putative myosin IB heavy chain (LinJ.34.1070), and a putative phospholipid transporting ATPase-like protein (LinJ.09.0940) (Tables 2 and S3, Supporting Information). Again, hypothetical proteins repre-

Figure 2. Functional assignment of proteins with at least one TMD in membrane-enriched fraction of L. infantum promastigotes and axenic amastigotes. Protein functional classification was based on GeneDB annotations and Gene Ontology.

proteins with predicted proteolytic functions have been detected in promastigotes (Figure 2). The remaining proteins implicated in other cellular processes were grouped together and represented 18 and 7% of the membrane proteins detected in promastigotes and amastigotes, respectively (Figure 2). HMW Proteome Analysis of L. infantum Promastigotes and Axenic Amastigotes Using the Gelfree 8100 Fractionation System

We also used the Gelfree 8100 Fractionation system with 5% Tris-Acetate Cartridge, which allows the enrichment of proteins having a MW higher than 60 kDa from whole protein extracts. We used two biological replicates of both L. infantum promastigotes and axenic amastigotes. The Gelfree 8100 Fractionation system enabled the recovery of HMW proteins 3979



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Table 2. MS/MS Identification of High Molecular Weight Proteins (Higher than 200 kDa) in L. infantum Promastigotes and/or Amastigotesa putative protein name calpain-like cysteine peptidase CA C2 putative calpain-like cysteine peptidase CA C2 putative ubiquitin-protein ligase-like, putative hypothetical protein, conserved hypothetical protein, conserved hypothetical protein, conserved hypothetical protein, conserved dynein heavy chain, putative dynein heavy chain, putative hypothetical protein, conserved hypothetical protein, conserved dynein heavy chain, putative dynein heavy chain, putative dynein heavy chain, putative dynein heavy chain (pseudogene), putative dynein heavy chain, putative dynein heavy chain, putative dynein heavy chain, putative dynein heavy chain, putative dynein heavy chain, putative dynein heavy chain, putative hypothetical protein, conserved ubiquitin-protein ligase, putative hypothetical protein, conserved kinesin K39, putative kinesin K39, putative hypothetical protein, conserved hypothetical protein, conserved kinesin, putative hypothetical protein, conserved P299 hypothetical protein, conserved TOR3, phosphatidylinositol 3-kinaselike hypothetical protein, conserved hypothetical protein, conserved hypothetical protein, conserved calcium channel protein ion transporter TOR1 target of rapamycin kinase 1, putative hypothetical protein, conserved phosphatidylinositol-kinase domain protein hypothetical protein, conserved hypothetical protein, conserved PRP8, U5 snRNA-associated splicing factor hypothetical protein, conserved hypothetical protein, conserved REH2 RNA editing associated helicase 2 hypothetical protein, conserved TOR2 phosphatidylinositol 3-kinase-like endosomal trafficking protein RME-8, putative phospholipid transporting ATPase-like protein hypothetical protein, conserved hypothetical protein, conserved

predicted Mr (kDa)

TMDsc

unique peptidesd (% seq. coverage)

assigned spectra

LinJ.27.0510

702

0

30 (7.9)

32

X

X

X

34

LinJ.27.0500

687

0

6 (1.1)

10

X

X

X

34

LinJ.07.0440 LinJ.16.0780 LinJ.30.1810 LinJ.26.2400 LinJ.31.1230 LinJ.23.1570 LinJ.25.1010 LinJ.20.0280 LinJ.13.1520 LinJ.13.1390 LinJ.34.3690 LinJ.27.1650 LinJ.27.2460

654 606 590 558 548 544 536 535 533 529 529 498 490

0 0 0 0 0 0 0 0 0 0 0 0 0

2 4 4 7 10 5 24 3 2 31 10 6 9

(0.4) (1.2) (0.9) (2.3) (2.9) (1.6) (7.0) (1.2) (0.7) (8.3) (2.8) (1.8) (2.7)

2 4 4 7 11 5 29 3 2 35 10 6 9

X X X X X X X X X X X X

X X

X

X

X

34

X

X X

34

LinJ.34.3990 LinJ.28.0650 LinJ.28.3110 LinJ.14.1130 LinJ.36.1010 LinJ.26.1000 LinJ.35.0680 LinJ.36.6600 LinJ.04.0410 LinJ.14.1180 LinJ.14.1190 (+1) LinJ.23.0130 LinJ.12.0260 LinJ.16.1550 LinJ.22.1470 LinJ.08.0630 LinJ.26.1790 LinJ.34.3750

478 477 474 473 473 458 457 455 433 361 326

0 0 0 0 0 0 0 0 2 0 0

8 11 7 3 7 5 3 2 8 9 8

(2.4) (3.2) (2.6) (0.9) (2.5) (1.9) (1.1) (0.8) (2.8) (3.3) (4.1)

10 11 7 3 7 5 3 2 8 18 17

X X X X X X X X X X X

313 308 305 303 299 297 296

0 0 0 0 0 0 0

8 (4.1) 2 (0.9) 5 (3.0) 7 (3.6) 3 (1.1) 49 (23.1) 14 (7.3)

9 2 25 8 3 113 14

X X X X X X X

X X

LinJ.36.2930 LinJ.32.3610 LinJ.32.3350 LinJ.34.0500 LinJ.36.6580

295 294 292 291 291

0 0 0 21 0

(4.6) (0.7) (0.9) (4.6) (9.0)

8 2 3 8 18

X X X X X

X X

LinJ.18.0820 LinJ.29.1550

290 290

0 0

61 (29.2) 44 (20.1)

84 77

X X

X X

LinJ.34.3940 LinJ.29.0110 LinJ.35.4000

282 278 278

0 0 0

13 (6.7) 9 (5.8) 52 (26.8)

13 11 59

X X X

X X X

LinJ.25.1390 LinJ.34.1470 LinJ.34.3010 LinJ.07.1330 LinJ.34.4160 LinJ.30.2220

278 277 277 276 275 273

0 0 0 0 0 0

2 (0.8) 37 (23.7) 20 (12.3) 25 (14.4) 15 (7.8) 10 (6.0)

2 48 21 29 17 10

X X X X X X

X X X X X

LinJ.09.0940

272

8

6 (3.4)

7

LinJ.09.1300 LinJ.08.0940

269 266

0 0

2 (1.1) 3 (2.4)

2 3

systematic IDsb

3980

8 2 2 8 18

Proa Amaa MEa

refe

34

34

X

X X

X

X

34

X

34

X X

X

34

34

X

34

34

X

34 34

34 34 34

X

34

X X

34

X

34

X X

X X X

X

34



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Table 2. continued putative protein name hypothetical protein, conserved hypothetical protein, conserved hypothetical protein, conserved hypothetical protein, conserved hypothetical protein, conserved hypothetical protein, conserved hypothetical protein, conserved hypothetical protein, conserved DNA polymerase epsilon catalytic subunit hypothetical protein, conserved hypothetical protein, conserved hypothetical protein, unknown function hypothetical protein, conserved RNA helicase, putative ubiquitin ligase, putative acetyl-CoA carboxylase, putative hypothetical protein, conserved hypothetical protein, conserved hypothetical protein, conserved hypothetical protein hypothetical protein, conserved hypothetical protein, unknown function cyclosome subunit-like protein hypothetical protein, conserved kinesin, putative ABCC8 ATP-binding cassette protein C8 hypothetical protein, conserved hypothetical protein, conserved hypothetical protein, conserved hypothetical protein, conserved hypothetical protein, conserved hypothetical protein, unknown function hypothetical protein, conserved hypothetical protein, conserved hypothetical protein, conserved hypothetical protein, conserved hypothetical protein, conserved hypothetical protein, conserved ABCA4 ATP-binding cassette protein A4 hypothetical protein, conserved ABCA10 ATP-binding cassette protein A10 carbamoyl-phosphate synthase, putative hypothetical protein, conserved hypothetical protein, conserved hypothetical protein, conserved hypothetical protein, unknown function hypothetical protein, conserved hypothetical protein, conserved hypothetical protein, conserved hypothetical protein, conserved ubiquitin hydrolase,cysteine peptidase CA C19 hypothetical protein, conserved DNA-directed rna polymerase I largest subunit hypothetical protein, conserved hypothetical protein, conserved

predicted Mr (kDa)

TMDsc

unique peptidesd (% seq. coverage)

assigned spectra

LinJ.35.0610 LinJ.07.1090 LinJ.34.2340 LinJ.36.6290 LinJ.29.1760 LinJ.26.1400 LinJ.08.0450 LinJ.35.2500 LinJ.35.4430

265 261 261 259 258 257 255 251 250

0 0 0 0 0 0 0 0 0

6 (3.9) 28 (18.3) 2 (1.5) 37 (22.2) 3 (1.5) 14 (9.3) 22 (12.7) 7 (4.4) 7 (3.8)

7 35 2 50 3 15 34 7 7

X X X X X X X

LinJ.21.0880 LinJ.03.0670 LinJ.27.1720 LinJ.35.3410 LinJ.13.0390 LinJ.09.0790 LinJ.31.3080 LinJ.36.4530 LinJ.35.0200 LinJ.07.0990 LinJ.03.0260 LinJ.07.0270 LinJ.13.0970 LinJ.30.1990 LinJ.03.0510 LinJ.21.1280 LinJ.34.0690

249 247 247 245 244 241 241 241 240 240 237 237 237 233 231 231 227

0 0 0 0 0 0 0 0 0 3 0 0 0 0 0 0 11

49 (30.4) 98 (50.8) 4 (2.8) 15 (11.9) 41 (26.6) 16 (11.7) 95 (51.0) 3 (1.7) 12 (7.2) 4 (2.5) 2 (1.4) 2 (2.0) 2 (1.6) 4 (3.1) 3 (2.6) 6 (3.7) 12 (9.8)

90 214 4 16 57 17 175 3 16 4 3 2 2 4 3 6 12

X X

LinJ.33.0570 LinJ.15.0840 LinJ.08.0850 LinJ.20.0940 LinJ.32.2790 LinJ.15.0830 LinJ.21.1780 LinJ.26.0700 LinJ.34.1830 LinJ.08.0430 LinJ.30.1760 LinJ.36.6150 LinJ.11.1240

226 225 223 223 222 222 221 213 213 212 211 209 209

0 0 0 0 0 0 0 0 0 0 0 0 13

36 (23.9) 3 (2.4) 5 (3.4) 9 (8.5) 15 (12.2) 2 (1.2) 2 (2.1) 2 (1.4) 2 (2.3) 2 (1.8) 4 (2.8) 21 (13.2) 5 (4.1)

48 4 5 9 29 2 2 2 2 2 4 28 5

X X X X X

LinJ.10.0300 LinJ.29.0640

209 207

0 16

3 (3.7) 13 (8.2)

3 13

X X

LinJ.16.0590 LinJ.15.0290 LinJ.19.0650 LinJ.02.0130 LinJ.25.1750 LinJ.36.6050 LinJ.29.1130 LinJ.04.0100 LinJ.29.2100 LinJ.16.0730

206 206 204 204 204 204 203 202 201 201

0 0 0 0 0 0 0 0 0 0

68 (47.2) 6 (4.0) 17 (15.0) 3 (2.8) 3 (3.2) 2 (1.7) 2 (1.3) 2 (1.8) 2 (1.6) 6 (6.3)

199 6 17 3 3 2 2 2 2 6

X X X

LinJ.16.1280 LinJ.16.1420

200 200

0 0

23 (18.7) 42 (34.0)

27 50

X X

X X

X X

LinJ.21.1220 LinJ.34.2220

200 200

0 0

38 (30.2) 5 (3.6)

48 5

X X

X X

X

systematic IDsb

3981

Proa Amaa MEa

refe

X X X X X

X

34

X X X X X X X X X

X

34

X

X X X X

X X X X X X X

34 34

X X

34

34

X X

34

X 34

X 34

X X X X X X X X

X X X

34

34

X

34 34

X X X

X

34

X

X

34

X

X

34

34

X X

34

X X X X X 34

X

34



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Table 2. continued putative protein name hypothetical protein, conserved

systematic IDsb LinJ.34.0580

predicted Mr (kDa)

TMDsc

200

0

unique peptidesd (% seq. coverage) 2 (1.8)

assigned spectra 2

Proa Amaa MEa

refe

X

a

Mr, molecular weight; TMDs, transmembrane domains; Pro, promastigotes; Ama, amastigotes; ME, membrane-enriched fraction. bNumbers in brackets indicate the number of proteins with ambiguous identifications that is shown in Tables S1 and S2, Supporting Information. cTMDs were predicted using TMHMM V. 2.0. dPeptide identifications were accepted if they reached greater than 95% probability as specified by the Peptide Prophet algorithm. eAlso identified in another study.

with ribosomal proteins (Tables S1 and S2, Supporting Information). This suggests that part of the endoplasmic reticulum (ER) and its associated ribosomes copurified with our membrane-enriched fractions. In Saccharomyces cerevisiae, approximately 1100 contact sites consisting of distances of less than 30 nm have been calculated between the plasma membrane and the ER membrane,41,42 which could allow proteins to interact with both membranes at the same time. In contrast, there are approximately only 80 contact sites between the ER and the mitochondria in yeast.43 Hence, this may explain the presence of proteins annotated as integral to the ER membrane in our membrane-enriched fraction (Table 1). Proteins primarily involved in metabolic functions are representing the second most important functional class identified from the membrane-enriched fractions (Figure 2). For instance, C-8 sterol isomerase-like protein (LinJ.29.2250) and CDP-diacylglycerol-inositol 3-phosphatidyltransferase (LinJ.26.2500) are respectively involved in ergosterol and phospholipid biosynthesis. Putative fatty acid desaturase (LinJ.24.2340) and fatty acid elongases (LinJ.14.0670, LinJ.14.0680, LinJ.14.0700 and LinJ.14.0760) are involved in fatty acid biosynthesis, while oligosaccharyl transferase-like proteins (LinJ.35.1140, LinJ.35.1150 and LinJ.35.1170) are part of a glycosylation complex (Table 1). It is worth noting that these proteins (except the oligosaccharyl transferase-like proteins that were detected in both stages) were detected in promastigotes, which is consistent with the reduced requirement of de novo lipid biosynthesis previously reported in amastigotes due to the scavenging of phospholipids and sphingolipids from their host macrophages.44−47 Finally, a putative GPI transamidase component GPI16 (LinJ.34.2560), a putative mannosyltransferase-II (LinJ.18.0960), and a hypothetical protein (LinJ.35.4420) expected to be involved in GPI anchor biosynthesis represent other ER membrane proteins recovered from our membrane-enriched extracts (Tables 1 and S1, Supporting Information). Several transporters with multiple TMDs have been recovered from our membrane-enriched and/or HMW fractions (Tables 1, 2 and S3, Supporting Information). The glucose transporter lmgt2 (LinJ.36.6550) was detected in promastigote membrane-enriched extract (Table 1), which is consistent with the increased need for glucose consumption in promastigotes compared to amastigotes.48 Among members of the ABC transporter superfamily, three ABCB proteins (ABCB1 LinJ.25.0540; ABCB3, LinJ.32.3280 and ABCB4, also known as MDR1, LinJ.34.1060); two ABCC proteins (ABCC3, also known as MRPA, LinJ.23.0290 and ABCC7, also known as PRP1, LinJ.31.1460), and one ABC protein that could not be assigned to one of the classic eukaryotic ABC subfamilies (LinJ.32.2190),49 were detected in the membraneenriched extracts of promastigotes (Table 1). Interestingly, ABCC3 and ABCC7 have also been found in promastigotes when using the protocol enriching for HMW proteins (Table S3, Supporting Information). These proteins have been

sented the majority of the HMW identified proteins in both stages (Tables 2 and S3, Supporting Information).

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DISCUSSION In trypanosomatid parasites, the control of gene expression occurs almost exclusively at the post-transcriptional level (reviewed in refs 6 and 7). Because of this peculiar mode of gene expression, several proteomic studies have been undertaken to look at stage-specific protein expression in these protozoan parasites (reviewed in refs 9 and 10). However, there is usually an underrepresentation of proteins with predicted membrane localization in the lists of identified proteins compared to what is expected from the genome (www.gendb. org),18,36 and only few proteomic studies have specifically focused on these more difficult proteins in Leishmania13,37 or Trypanosoma.33,38 A study using plasma membranes of L. chagasi promastigotes obtained either by surface biotinylationstreptavidin affinity separation or by octyl glucoside detergent extraction was recently published,13 and the number of proteins detected with at least one TMD was higher than in the present study. However, TMpred algorithm was used in the latter study instead of TMHMM v. 2.0 software used here. When we used TMpred, we found that close to 75% of proteins detected here had a least one TMD, a number similar to the study of Yao et al.13 We choose to use the TMHMM server v. 2.0 (www.cbs. dtu.dk/services/TMHMM/), because previous comparison studies proved that HMM-based prediction methods had better prediction accuracies than the other methods.39,40 In addition, the combination of sucrose centrifugation and carbonate extraction prior to gel-based separation methods and MS analysis allowed the recovery of Trypanosoma brucei membrane proteins with several TMDs, with 23% of the identified membrane proteins predicted to have more than 5 TMDs.33 This is in the same range as the 20 and 22% of membrane proteins with more than 5 TMDs identified here in L. infantum promastigotes and amastigotes, respectively. There are thus a number of proteins detected in our membrane-enriched fraction that appear to be without TMDs. One possibility is that these proteins may be associated to membranes via GPI-anchor. However, bioinformatic analysis has revealed that few proteins have a high probability of being GPI-anchored (Tables S1 and S2, Supporting Information). It is salient to point out that current algorithms predict less than 200 proteins that would be GPI-anchored in the whole Leishmania proteome, suggesting that either these algorithms are not optimized for Leishmania or they are not too many GPI-anchored proteins. Consistent with the latter, proteomic of plasma membrane proteins of L. chagasi only detected 5 GPIanchored proteins (all corresponding to GP63).13 In a study of the related parasite T. cruzi specifically focusing on GPIanchored proteins, only 23 predicted GPI-anchored proteins were identified, all corresponding to isoforms of 5 abundant GPI-anchored proteins. For both parasite life stages, our membrane-enriched fractions were substantially contaminated 3982



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implicated in resistance to heavy metals50,51 and pentamidine,52 respectively, and ABCC3 had also been previously shown to be significantly downregulated in mature amastigotes.34 However, ABCC3 and ABCC7 have previously been shown to be overexpressed in amastigotes in the context of resistance to antimony53 and pentamidine,54 respectively. Since these proteins have been shown to be implicated in resistance in the amastigote stage but are expressed at lower levels, it may explain why genes need to be amplified for achieving resistance. On the other hand, the ABC transporter ABCA4 (LinJ.11.1240) involved in phospholipid trafficking55,56 has been identified in amastigote HMW extract (Table 2). This is consistent with the identification of another phospholipid transporting ATPase-like protein (LinJ.09.0940) from the membrane-enriched extract of amastigotes (Table 1) and with the previously reported scavenging of host macrophage’s phospholipids by amastigotes.44−47 Three putative surface antigen proteins 2 (LinJ.12.0663, LinJ.12.0665 and LinJ.12.0666) were found in promastigotes (Table 1). This group of proteins had previously been thought to be promastigote-specific,57,58 but afterward some mRNA transcripts have also been found in the amastigote stage.59 Surprisingly, we did not find any member of the amastin superfamily in the list of amastigote membrane proteins (Tables 1 and S2, Supporting Information). These proteins are localized to the cell surface, and a subclass is known to be amastigote-specific.60,61 This is probably related to the low MW of amastins (below 30 kDa). Gel slices below 40 kDa almost exclusively yielded highly abundant ribosomal proteins and histones that could have masked lower-abundant amastin identifications. We also observed differences in proteins involved in proteolysis between the two life stages. Interestingly, we identified six different putative metallopeptidases (LinJ.18.0620, LinJ.19.0150, LinJ.19.1620, LinJ.23.1120, LinJ.27.0040 and LinJ.32.1570) (Tables 1 and S1, Supporting Information) and five putative calpain-like cysteine peptidases in promastigotes (LinJ.20.1210, LinJ.21.0160, LinJ.21.0170, LinJ.27.2490 and LinJ.32.1020) (Table 2), including two (LinJ.21.0170 and LinJ.27.2490) that were already known to be downregulated during the differentiation from promastigotes to amastigotes.34 Conversely, two putative ubiquitin hydrolases (LinJ.30.0250 and LinJ.35.1730) and a ubiquitin-like protein (LinJ.13.0620) were found in amastigotes (Table S2, Supporting Information). These results suggest that protein degradation processes might be carried out by different mechanisms between both life stages. Little is known actually about the regulation of protein turnover between the different stages of the parasite life cycle, and the proteins identified here suggest new avenues for further investigating stage regulated protein turnover. The motility of promastigote parasites is a major morphological difference between the two Leishmania life stages. Interestingly, several dynein heavy chains have been detected in the promastigote HMW-enriched extracts compared to the amastigotes (Table 2). These proteins are known to be essential for flagellar motility in T. brucei,62 and 9 out of the 12 dynein heavy chains identified were found in promastigotes (Table 2). Four hypothetical proteins (LinJ.08.0420, LinJ.10.1220, LinJ.25.0620 and LinJ.29.2740) and a putative MAP kinase kinase (MKK)-like protein (LinJ.35.4050) were detected in both membrane-enriched and HMW proteins derived from axenic amastigotes (Tables S2 and S3, Supporting Informa-

tion), and it would be interesting to further assess their role in the biology of amastigotes. In summary, the development of methods to investigate membrane-enriched and/or HMW proteins has allowed the identification of novel proteins, some of which have possible important roles in Leishmania differentiation.

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ASSOCIATED CONTENT

S Supporting Information *

Tables S1 and S2 show MS/MS identification of all the proteins found in membrane-enriched extracts of L. infantum promastigotes and axenic amastigotes, respectively. Table S3 shows MS/MS identification of proteins of MW lower than 200 kDa found in HMW extracts of L. infantum promastigotes and axenic amastigotes. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Telephone: 418-656-4141 ext 48184. Fax: 418-654-2715. Email: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We would like to thank Sylvie Bourassa, Michel Boutin and Isabelle Kelly for helpful proteomic discussions. B.P. and M.O. are members of a CIHR Group on Host−Pathogen Interactions and of the Centre for Host−Parasite Interactions “Programme Regroupements Statégiques” of the Fonds du Québec pour la Recherche sur la Nature et les Technologies. This work was funded by a CIHR grant to M.O. M.O. holds the Canada Research Chair in Antimicrobial Resistance.

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REFERENCES

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