Prefractionation by Digitonin Extraction Increases Representation of the Cytosolic and Intracellular Proteome of Leishmania infantum Aude L. Foucher, Barbara Papadopoulou, and Marc Ouellette* Centre de Recherche en Infectiologie, Centre Hospitalier de l’Universite´ Laval, Sainte Foy, Que´bec Received March 7, 2006
Proteome coverage is limited by the dynamic range of proteins present in a sample and often is confined to the analysis of abundant proteins. We have developed a protein prefractionation protocol, based on the differential solubilization of membranes using digitonin, that has allowed an increase in the resolution and depth of comparative proteomic studies. This prefractionation protocol can also be used to infer the subcellular localization of hypothetical proteins as tested experimentally using green fluorescent fusion proteins. The abundant tubulins and associated proteins of the cytoskeleton were removed from the sample using digitonin extraction, hence facilitating the visualization of lower abundance proteins. The digitonin prefractionation protocol was applied for a comparative proteomic analysis of the promastigote and amastigote life cycle stages of Leishmania infantum and has allowed the identification of novel proteins expressed in a stage-specific manner. Keywords: fractionation • proteome • localization • Leishmania infantum
Introduction The genomes of the human kinetoplastid parasites Leishmania major, Trypanosoma brucei, and Trypanosoma cruzi have recently been completed1 and the genome of several other kinetoplastid parasites, including Leishmania infantum, are in various phases of completion (www.genedb.org). These parasites are responsible for widespread, often fatal, parasitic diseases including leishmaniasis, sleeping sickness, and Chagas disease, respectively. In kinetoplastidae, genes are present as large polycistronic transcription units with limited regulation at the level of transcript initiation. Instead, the main mode of gene regulation involves post-transcriptional, translational, and post-translational mechanisms.2-5 These parasites have small genome size containing between 8300 genes (L. major) to possibly more than 10 000 open reading frames (T. cruzi). The protein complement is expected to be higher, not by alternative splicing as most protein coding genes of kinetoplastids, unlike other eukaryotes, are uninterrupted, but by post-translational modification (PTM) and processing. The small proteome size of kinetoplastid parasites, their particular mode of gene regulation and the apparent abundance of PTMs4,5 make proteomics a technique of choice to study the response of these parasites to a number of stimuli. These parasites have natural life cycles and one of the first application of proteomics has been in the elucidation of differentially expressed proteins in various Leishmania species4,6-9 and in T. cruzi.10-12 Good proteome maps of total soluble proteins also exist for L. major13 and T. brucei.5 * To whom correspondence should be addressed. Centre de Recherche en Infectiologie, Centre Hospitalier de l’Universite´ Laval, Sainte Foy, Que´bec, Canada, G1V 4G2, Tel: (418) 654-2705. Fax: (418) 654-2715. E-mail:
[email protected]. 10.1021/pr060081j CCC: $33.50
2006 American Chemical Society
These recent proteomic studies of kinetoplastid parasites have shown that only a small portion of the proteome can be analyzed using a whole cell extraction. A high-resolution proteome map of L. major comprising about 3700 spots was made but it required the analysis of gels spanning 5 narrow pH ranges.13 In one extensive proteome mapping of T. brucei, only 770 individual proteins were identified, which is far from the 8000 predicted open reading frames contained in its genome.5 Several studies demonstrated that multiple protein isoforms are common in kinetoplastids and that tubulins (R and β), due to their abundance and their extensive modification and processing, could hide the low abundance proteome.5,12,13 The variety and amount of tubulin fragments are dynamic and increase with the age of the parasite culture.13 A proteomic map of T. brucei identified R-tubulin in 125 spots and β-tubulin in 84 spots out of the 880 spots analyzed by MS/MS.5 These included whole protein, isoforms (PTM or products of different genes/alleles) and degradation products. Prefractionation is thus a requisite to increase proteome coverage. The main techniques used to prefractionate the proteome are based on electrophoresis, chromatography, and gradient centrifugation techniques.14 While these techniques do efficiently prefractionate the proteome, they tend to require massive amount of starting material. There is a need for a prefractionation protocol which would decrease the protein complexity per spot, simplify the proteome representation on two-dimensional gel electrophoresis (2DE) and eliminate the cytoskeleton. Recently, we have applied ammonium sulfate precipitation as a fractionation mean to study stage-specific expression of proteins in Leishmania and this technique was found useful in detecting novel lower abundance proteins.4 Other simple fractionation protocols would be useful. Digitonin is a detergent that binds sterols and precipitates them out, Journal of Proteome Research 2006, 5, 1741-1750
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research articles allowing the formation of pores in the membranes, that make the protein content to leak out. At low concentration, digitonin only affects the cytoplasmic membrane, allowing the extraction of the cytosolic content. On the other hand, higher concentrations of digitonin affect the organelle’s membranes, enabling the extraction of organellar’s content.15,16 In addition, rat cells treated with digitonin retained their cytoskeleton intact.17 We have developed a protocol based on the ability of digitonin to permeabilise cytoplasmic membranes at low concentration and organellar membranes at higher concentration to obtain sub cellular fractions of Leishmania cells for proteomic analysis. This approach has allowed the cellular localization of several hypothetical proteins and was also proved to be useful for identifying novel proteins expressed in a stage-specific manner.
Materials and Methods Sample Preparation. Cells (10 mL culture) of either L. infantum promastigotes or amastigotes grown as described previously,4,18 were harvested at optical density of about 0.500 (at 600 nm) by centrifugation (2000 × g, 5 min). The pellet was washed twice in HEPES-NaCl. Whole cell extracts were obtained by incubating the pellet in T8 buffer (7 M urea, 2 M Thiourea, 3% CHAPS, 20 mM DTT, 5 mM TCEP, 0.5% ampholytes 4-6.5, 0.25% ampholytes 3-10) supplemented by 20 µL of a cocktail of protease inhibitors (Sigma no. P8340), for 1 h at room temperature with vortexing every 15 min. Digitonin fractions were obtained by resuspending the cells in 1 mL of resuspension buffer (145 mM NaCl, 11 mM KCl, 75 mM TrisHCl pH 7.4) in the presence of a cocktail of inhibitors and 1 mL of 20 µM digitonin (Sigma) solution (in 10% methanol). Digitonin was left to act for 5 min at 37 °C after what 200 µL of 0.3 M sucrose was added to the tubes to prevent the cells from bursting open during the centrifugation. The soluble fraction (fraction 1) was recovered by centrifugation (13 000 rpm, 4 °C, 5 min) and the insoluble fraction was resuspended in 1 mL of resuspension buffer in the presence of a cocktail of inhibitors and 1 mL of 200 µM digitonin solution. After 5 min at 37 °C the soluble fraction (fraction 2) was recovered by centrifugation (10 000 × g, 4 °C, 5 min). The insoluble fraction was treated as above first with 1 mM digitonin and then with 10 mM digitonin to obtain fractions 3, 4, and 5 (fraction 5 being the pellet left over after treatment with 10 mM digitonin). The proteins were then precipitated with acetone and resolubilized in T8 buffer. The protein concentrations were determined using the 2D Quant Kit (Amersham). Fractions 1 and fraction 2 were found to contain an identical set of proteins and thus only fraction 2 was further characterized. Two-Dimensional Gel Electrophoresis, Staining and Image Analysis. Two-dimensional gel electrophoresis and staining were performed as described previously on 24 cm IPG strips pH 4.0-7.0 and 12% polyacrylamide gels.19 Spot detection, spot matching and semiquantitative statistical analysis were performed using the Progenesis Discovery software version 5.0 (PerkinElmer). Mass Spectrometry. Gel plugs were excised from the gel using the ProXcission robot (PerkinElmer) and send for mass spectrometry sequencing (Eastern Quebec Proteomics Centre, Centre Hospitalier de l’Universite´ Laval, Que´bec). Peptide tandem mass spectra were obtained by capillary liquid chromatography coupled to an LCQ DecaXP (ThermoFinnigan, San Jose, CA) quadrupole ion trap mass spectrometer with a nanospray interface. An aliquot of the digested protein sample was diluted to 5 µL with 0.1% formic acid and loaded onto a 1742
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reversed-phase column (PicoFrit 15-µm tip, BioBasic C18, 10 cm × 75 µm; New Objective, Woburn, MA). Peptides were eluted from the column with a linear gradient of water/ acetonitrile in 0.1% formic acid at a flow rate of ∼250 nL/min. Mass spectra were acquired using a data-dependent acquisition mode in which each full scan mass spectrum was followed by collision-induced dissociation of the three most intense ions. The dynamic exclusion function was enabled, and the relative collisional fragmentation energy was set to 35%. The resulting mass spectra were search against a L. major/L. infantum database using the MASCOT algorithm (http://www.matrixscience.com/). The protein identification cut off was set at a confidence level of 95% (MASCOT score >33) with at least 2 peptides matching to a protein. Western Blot Analysis. Polyacrylamide gels (12%), used to separate 30 µg of proteins from each fraction, were blotted onto nitrocellulose membranes as described.20 The blots were blocked overnight in 5% skimmed milk in Tris-buffered saline (TBS). A monoclonal anti- R -tubulin antibody directed against an amino-terminal peptide of bovine R-tubulin highly conserved in Leishmania (A-11126, Molecular Probes) was diluted 1:2000 in TBS containing 0.05% Tween 20 (TBS-Tween) and incubated for 1 h with the blot. The blot was washed 3 × 5 min in TBS-Tween and incubated with horseradish peroxidaseconjugated sheep-anti mouse IgG (Amersham Biosciences) diluted 1:10 000 in TBS-Tween. The blot was washed as above, incubated with ECL Plus chemiluminescent substrate (Amersham Biosciences), and exposed to X-ray film. Fusion GFP. Genes of interest were amplified by PCR fromL. infantum genomic DNA using the Expand High Fidelity polymerase (Roche Applied Science). Primers used were: for LinJ36.2220, INF8F, 5-TCT AGA ATG CGT CGT CGT GCC TGC-3 and INF8R, 5-GAA TTC TTA GCG ACG CAC AGC CCA CAG-3; for LinJ31.3010, INF9F, 5-TCT AGA ATG CGC CGC GCC ATA TCG TCC-3 and INF9R, 5-GGA TCC TTA CTT ACG AGA GCG GCT CTT-3; for LinJ11.0180, INF14F, 5-TCT AGA ACT TCG CGA TGA CGA A-3 and INF14R2, 5-GGA TCC CAC GCT GAT GTG TGA-3. The PCR fragments were cloned in pGEM T-easy vector (Invitrogen), digested with XbaI and either BamHI (for LinJ31.3010 and LinJ11.0180) or EcoRI (for LinJ36.2220) (underlined) and subcloned in-frame with the enhanced green fluorescent protein (eGFP) gene in pBluescript. The resulting constructs were inserted into either the Leishmania expression vector pSP72RNEOR, encoding paromomycin/G418 resistance or pSP72RHYGROR, encoding hygromycin resistance.21 Each construction was verified by restriction digests and by sequencing the junction between the gene and eGFP. The resulting plasmids were transfected into L. infantum by electroporation as previously described22 and maintained with either 0.02 mg/ mL of G418 (Sigma) or 0.6 mg/mL of hygromycin B. Fluorescence Microscopy. Live parasites were mounted under poly-L-lysine-coated coverslips. Coverslips were sealed with nail varnish and air-dried for 15 min. Brightfield and fluorescence images were taken using a Nikon eclipse TE300 inverted microscope with a Photometrics coolSNAPfx camera. Visualization of the GFP fluorophore was achieved using a460/ 500 nm excitation filter and 510/560 nm emission filter with a 100× objective. The images were processed using the ImagePro Plus software (version 5.0).
Results Digitonin Fractionation Increases Proteome Coverage. Total cell extracts from 4 × 108 L. infantum cells were prepared
Digitonin Fractionation for Proteomic Studies
research articles
Figure 1. Fractionation of L. infantum protein sample by serial extraction using digitonin increases proteome coverage. Two-dimensional gels pH 4-7 were run with either whole cell extract (A), fractions 2 extracted with 100 µM of digitonin (B), fraction 3 extracted with 500 µM of digitonin (C), fraction 4 extracted with 5 mM of digitonin (D) and fraction 5 the left over of fraction 4 (E) and stained with SyproRuby. The number of protein spots, as determined using the Progenesis Discovery software, are indicated below each gel.
as described13 and yielded 1.35 mg of proteins. The protein content (150 µg) was separated in the first dimension on pH 4 to 7 gels and 1921 spots could be visualized on the gel (Figure 1A). Serial digitonin extractions of the same quantity of L. infantum cells led to better solubilization and/or recovery compared to our standard extraction procedure since fractions 1, 2, 3, 4, and 5 yielded 0.75 mg, 0.75 mg, 0.36 mg, 0.16 mg and 0.84 mg respectively increasing by 2.1-fold the amount of proteins. The protein contents of the digitonin fractions were also separated on pH 4 to 7 gels and their analysis indicated that approximately 3186 spots could be resolved (983 spots in fraction 2, 833 spots in fraction 3, 286 spots in fraction 4, and 1084 spots in fraction 5). Fraction 1 and 2 led to similar protein patterns on 2D gels (data not shown), and thus only fraction 2 was analyzed. As expected, fractionating the sample clearly increases proteome coverage. Indeed, protein patterns of fractions 2, 3, 4, and 5 were distinct from each others (Figure 1B-E). Almost no overlap in spot pattern between the different fractions could be seen on the gels, demonstrating the efficiency of the fractionation protocol (Figure 1). Protein Composition of the Different Fractions. To determine whether the digitonin prefractionation corresponded to subcellular fractions, we analyzed selected proteins from each digitonin fraction by mass spectrometry (MS). The MS analysis of 24 protein spots from digitonin fraction 2, enabled the identification of 60 individual proteins containing 46 proteins with known predicted function and 14 proteins labeled hypothetical (Table 1). All the protein sequences were blasted against the Human Protein Reference Database (http://www.hprd.org/) and from these analyses a putative subcellular localization could be determined (Table 1). From the 60 proteins identified by MS, 26 proteins (43%) have a human homologue known to be found only in the cytosol, 22 proteins (37%) are known to
be present in both the cytosol and some organelles and only 5 proteins (8%) are known to reside only into organelles (Figure 2 and Table 1). The remaining 7 proteins (12%) are either proteins with no human homologue or proteins whose human homologue has no known localization (Table 1). We investigated, using the MitoProt II software (http://ihg.gsf.de/ihg/ mitoprot.html), whether any of the proteins identified could be predicted to have a mitochondrial localization. The analysis showed that only 2 known proteins and 2 hypothetical proteins had a 50% probability to be in the mitochondria (Table 1). In summary, this analysis showed that digitonin fraction 2 contained mostly cytosolic proteins. The analysis by MS of 30 spots excised from a gel separating the content of digitonin fraction 3, allowed the identification of 49 individual proteins containing 32 proteins of known function and 17 hypothetical proteins (Table 1). The protein sequence of each of these proteins was blasted against the Human Protein Reference Database and a putative subcellular localization was derived from this analysis. Out of the 49 proteins identified, 4 proteins (8%) have human homologues known to localize only in the cytosol, 7 proteins (14%) are known to have multiple localization and 19 proteins (39%) are known to reside only in organelles. The remaining 19 proteins (39%) are either proteins with no human homologue or proteins whose human homologue has no known localization (Figure 3 and Table 1). The analysis of the proteins identified in fraction 3 using the MitoProt II software revealed that 18 proteins (37%) had a 50% probability of being mitochondrial proteins (Table 1). This would suggest that digitonin fraction 3 contains mostly organellar proteins with few cytosolic proteins. A similar analysis was carried out for the 15 proteins identified in digitonin fraction 4 (Table 1). The analysis using Journal of Proteome Research • Vol. 5, No. 7, 2006 1743
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Table 1. Identification of Proteins of L. infantum Subcellular Fractions Obtained by Digitonin Serial Extraction protein name
accessiona
poly(a)-binding protein, putative hypothetical protein hypothetical protein 3-hydroxyisobutyryl-coenzyme a hydrolase-like protein chaperonin Hsp60, mitochondrial elongation factor 2 coproporphyrinogen iii oxidase acetylornithine deacetylase like ribosomal protein 17a beta tubulin oligopeptidase B/serine peptidase hypothetical protein hypothetical protein protein kinase A regulatory subunit chaperonin TCP20 hypothetical protein glutathione synthase like enolase UDP glucose pyrophosphorylase like serine-threonine protein phosphatase N-acyl-L-amino acid amidrohydrolase 40S ribosomal protein S6 proteasome regulatory ATPase subunit 1 mannose 1 phosphate guanyltransferase T-complex protein 1, gamma subunit transketolase 3 hydroxy 3 methylglutaryl CoA synthase like ribosomal protein s25 GTP binding protein hypothetical protein T complex protein 1 beta subunit hypothetical protein 40S ribosomal protein S14 LACK||activated protein kinase c receptor HSP70 RAB-GDP dissociation inhibitor metallo-Peptidase 4,methyl,5 beta hydroxy thiazole monophosohate synthesis protein hypothetical protein ATP dependent RNA helicase hypothetical protein ethanolamine phosphate cystidyl transferase hypothetical protein hypothetical protein nucleoside diphosphate kinase b HSP83 hypothetical protein hypothetical protein ubiquitin conjugating enzyme peptidase like cystathionine gamma synthase 60S ribosomal protein L23 T-complex protein 1, eta subunit hypothetical protein clathrin heavy chain PMM||phosphomannomutase, putative S Adenosyl homocystein hydrolase 40S ribosomal protein SA branch point binding protein elongation factor 1 R
LinJ25.0080 LinJ28.1900 LinJ30.2680 LinJ32.4170
108 89 197 174
FRACTION 2 4 8% 2 10% 4 6% 4 8%
no no no no
p ) 0.0010 p ) 0.0061 p ) 0.0183 p ) 0.2944
cytoplasm cytoplasm no hit unknown
LinJ36.4560 LmjF03.0980 LmjF06.1270 LmjF07.0270 LmjF07.0500 LmjF08.1230 LmjF09.0770 LmjF11.0140 LmjF11.0180 LmjF13.0160 LmjF13.1660 LmjF14.0190 LmjF14.0910 LmjF14.1160 LmjF18.0990 LmjF20.0660 LmjF20.1550 LmjF21.1780 LmjF22.0570 LmjF23.0110 LmjF23.1220 LmjF24.2060 LmjF24.2110
656 83 231 69 267 83 356 111 100 439 101 341 126 264 144 123 181 118 99 377 144 99 162
9 2 6 2 8 2 15 3 4 12 4 8 7 7 3 3 8 2 3 15 3 2 4
14% 3% 19% 3% 23% 5% 9% 14% 13% 17% 7% 34% 6% 21% 6% 6% 11% 10% 7% 19% 6% 4% 7%
no no no no no no no no no no no no no no no no no no no no no no no
p ) 0.3446 p ) 0.5039 p ) 0.3327 p ) 0.2223 p ) 0.1688 p ) 0.0776 p ) 0.0766 p ) 0.0277 p ) 0.2652 p ) 0.0094 p ) 0.1315 p ) 0.0087 p ) 0.1112 p ) 0.3193 p ) 0.0855 p ) 0.0525 p ) 0.1453 p ) 0.0035 p ) 0.0208 p ) 0.3033 p ) 0.0613 p ) 0.4567 P ) 0.5039
mtochondrion cytoplasm mitochondrion cytoplasm cytoplasm cytoplasm cytoplasm no hit cytoplasm cytoplasm cytoplasm no hit unknown cytoplasm unknown unknown cytoplasm ribosome cytoplasm cytoplasm cytoplasm cytoplasm cytoplasm
LmjF25.1190 LmjF25.1420 LmjF26.0500 LmjF27.1260 LmjF27.2480 LmjF28.0960 LmjF28.2740 LmjF28.2770 LmjF29.2160 LmjF29.2240 LmjF30.0750
98 99 134 46 144 97 132 109 451 95 64
2 2 3 3 3 2 3 2 18 2 2
17% 9% 8% 5% 6% 23% 9% 3% 23% 2% 12%
no no no no no no no no no no no
p ) 0.1722 p ) 0.0197 p ) 0.6113 p ) 0.0774 p ) 0.7212 p ) 0.0152 p ) 0.1126 p ) 0.0322 p ) 0.0498 p ) 0.0139 p ) 0.0154
ribosome nucleus cytoplasm cytoplasm nucleus ribosome cytoplasm cytoplasm cytoplasm cytoplasm no hit
LmjF32.0050 LmjF32.0400 LmjF32.0630 LmjF32.0890
102 360 144 136
3 7 3 3
10% 14% 16% 8%
no no no no
p ) 0.0768 p ) 0.0771 p ) 0.4036 p ) 0.0128
er nucleus no hit cytoplasm
LmjF32.0950 LmjF32.2180 LmjF32.2950 LmjF33.0312 LmjF34.2570 LmjF34.2700 LmjF35.1300 LmjF35.2350 LmjF35.3230 LmjF35.3790 LmjF35.3860 LmjF36.0590 LmjF36.1630 LmjF36.1960 LmjF36.3910 LmjF36.5010 LmjF36.5420 LmjF17.0080
690 246 157 118 374 241 71 140 97 103 215 226 72 83 153 50 135 93
p ) 0.2281 p ) 0.1314 p ) 0.0542 p ) 0.0441 p ) 0.0388 p ) 0.0210 p ) 0.2682 p ) 0.0275 p ) 0.0156 p ) 0.1198 p ) 0.1039 p ) 0.2189 p ) 0.0328 p ) 0.0150 p ) 0.1004 p ) 0.0570 p ) 0.1692 p ) 0.0027
unknown no hit nucleus cytoplasm no hit no hit unknown cytoplasm cytoplasm ribosome cytoplasm no hit cytoplasm cytoplasm cytoplasm ribosome nucleus cytoplasm
hypothetical protein enolase hypothetical protein hypothetical protein vesicular-fusion ATPase-like protein hypothetical protein Histidyl-tRNA synthetase hypothetical protein dihydrolipoamide acetyltransferase HSP60, mitochondrial precursor hypothetical protein histone H2B R tubulin hypothetical protein lectin, putative nucleolar RNA binding protein peroxidoxin
LinJ14.1020 LinJ14.1250 LinJ24.1500 LinJ25.1040 LinJ27.0660 LinJ28.0060 LinJ30.0960 LinJ30.4100 LinJ36.1010 LinJ36.4560 LmjF09.1010 LmjF09.1340 LmjF13.0280 LmjF13.0450 LmjF13.0670 LmjF15.1380 LmjF23.0040
299 85 284 184 106 105 115 248 169 145 357 149 68 133 86 112 423
p ) 0.1434 p ) 0.2090 p ) 0.5327 p ) 0.5969 p ) 0.0622 p ) 0.9947 p ) 0.0416 p ) 0.0433 p ) 0.9816 p ) 0.3446 p ) 0.1197 p ) 0.9860 p ) 0.0166 p ) 0.0686 p ) 0.7212 p ) 0.1327 p ) 0.9868
no hit cytoplasm no hit no hit no hit no hit cytoplasm no hit mitochondrion mitochondrion no hit nucleus cytoplasm no hit ER nucleus cytoplasm
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scoreb peptc covc
signal Pd
23 19% no 5 30% no 3 34% no 3 5% no 17 21% no 10 16% no 2 15% no 2 5% no 2 5% no 3 17% no 7 14% no 6 21% p ) 0.82 3 2% no 3 11% no 4 10% no 2 8% no 2 10% no 2 5% no FRACTION 3 8 9% no 3 12% no 8 17% no 6 12% no 3 2% p ) 0.716 2 3% p ) 0.978 4 12% no 6 17% no 4 14% p ) 0.610 3 2% No 13 15% p ) 0.963 3 27% no 3 7% no 3 28% no 2 5% no 2 4% no 12 40% no
MitoProt IIe
primaryf
alternativef
nucleus
ribosome nucleus mitochondrion
nucleus
nucleus cytoplasm, mitochondrion
cytoplasm, nucleolus nucleus, plasma membrane mitochondrion
cytoplasm ER
cytoplasm nucleus
plasma membrane, golgi nucleus
nucleus
nucleus
nucleolus golgi plasma membrane
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Digitonin Fractionation for Proteomic Studies Table 1. (Continued) protein name
hypothetical protein hypothetical protein aldehyde dehydrogenase ATPase β subunit protein disulfide isomerase glycosomal phosphoenolpyruvate carboxykinase hypothetical, WD repeat hypothetical protein LACK activated protein kinase receptor c hypothetical protein carnitine/choline acetyltransferase paraflagellar rod protein 1D HSP 20 heat shock 70-related protein 1, METK|MAT2|S-adenosylmethionine synthetase 3,2 trans enoyl CoA isomerase, mito precursor hypothetical protein hypothetical protein 2,4-dienoyl-coa reductase fadh1 cysteine conjugate beta lyase malate dehydrogenase hypothetical protein ruvb like 1 DNA helicase acyl CoA dehydrogenase hypothetical protein fructose 1-6 bisphosphate aldolase chaperonin Hsp60, mitochondrial precursor chaperonin Hsp60, mitochondrial precursor succinyl coa ligase beta subunit eukaryotic translation initiation factor S adenosyl homocysteine hydrolase hypothetical protein
accessiona
scoreb peptc covc
signal Pd
MitoProt IIe
p ) 0.814 no no p ) 0.949 p ) 0.998 no
p ) 0.5039 p ) 0.5956 p ) 0.4242 p ) 0.9717 p ) 0.1290 p ) 0.3672
primaryf
LmjF24.2110 LmjF25.1010 LmjF25.1120 LmjF25.1180 LmjF26.0660 LmjF27.1805
288 102 65 675 277 178
7 3 2 13 6 7
11% 6% 2% 27% 17% 12%
LmjF27.2480 LmjF28.0060 LmjF28.2740
240 105 236
10 2 8
11% no p ) 0.7212 nucleus 3% p ) 0.943 p ) 0.9957 no hit 29% no p ) 0.1126 cytoplasm
LmjF29.1100 LmjF29.1310 LmjF29.1750 LmjF29.2450 LmjF30.2470 LmjF30.3500
151 612 429 90 212 97
3 14 11 3 4 3
10% 20% 16% 25% 7% 9%
LmjF31.2250
260
6
18% p ) 0.001 p ) 0.7949 mitochondrion
LmjF31.2770 LmjF32.2180 LmjF33.0830 LmjF33.1330 LmjF34.0140 LmjF34.0240 LmjF34.3500 LmjF35.2730 LmjF36.0440 LmjF36.1260 LmjF36.2020
41 115 54 96 86 42 85 113 253 250 775
2 2 1 3 3 4 2 3 4 7 4
5% 12% 1% 6% 12% 3% 4% 8% 10%
no no no no p ) 0.969 no no no p ) 0.998 no 28% no
p ) 0.8131 p ) 0.1314 p ) 0.1296 p ) 0.1464 p ) 0.7280 p ) 0.1032 p ) 0.0289 p ) 0.2924 p ) 0.9290 p ) 0.3327 p ) 0.2759
LmjF36.2030 1246
25
37% no
p ) 0.3570 mitochondrion
LmjF36.2950 LmjF36.3880 LmjF36.3910 LmjF36.5650
653 279 392 120
15 33% no 6 p ) 0.947 10 20% no 4 8% p ) 0.952 FRACTION 4 2 4% p ) 0.978
mitochondrial processing peptidase R subunit serine carboxypeptidase CBP1 succinate dehydrogenase flavoprotein pyruvate dehydrogenase E1 beta subunit vacuolar ATP synthase subunit B ATP synthase hypothetical protein dihydrolipoamide dehydrogenase ubiquitin conjugating enzyme cytochrome c oxidase subunit iv R tubulin hypothetical protein hypothetical protein peroxidoxin hypothetical protein
LinJ13.0800
77
LinJ18.0450 LinJ24.1630 LinJ25.1800 LinJ28.2540 LinJ30.4090 LinJ30.4200 LinJ32.3870 LmjF07.0850 LmjF12.0670 LmjF13.0280 LmjF15.0280 LmjF17.0120 LmjF23.0040 LmjF23.1410
83 384 297 398 86 189 440 85 43 196 316 93 60 131
hypothetical protein microtubule associated protein hypothetical protein hypothetical protein flagellar radial spoke protein dynein heavy chain hypothetical protein hexokinase hypothetical protein ATP synthase F1 subunit gamma protein Hypothetical protein hypothetical protein hypothetical protein hypothetical protein 2 oxoglutarate dehydrogenase 3 hydroxy methylglutaryl CoA reductase hypothetical protein hypothetical protein hypothetical protein elongation factor 1 R ATP dependent DEAD box RNA helicase NADH dependent fumarate reductase hypothetical protein 40S ribosomal protein SA ATPase R subunit 60S ribosomal protein L7a β tubulin
LinJ02.0380 LinJ05.0380 LinJ08.0920 LinJ08.1020 Linj13.0300 LinJ13.1370 LinJ14.1540 LinJ21.0140 LinJ21.1270 LinJ21.1530 Linj25.0610 LinJ25.1270 LinJ25.1530 LinJ27.0680 LinJ28.2530 LinJ30.3600 LinJ31.1400 Linj32.3560 LinJ33.1550 LinJ34.0630 LinJ35.0470 LinJ35.0970 LinJ36.3570 LinJ36.5570 LmjF05.0500 LmjF07.0500 LmjF08.1230
249 298 140 168 107 66 221 749 217 206 120 100 172 119 81 183 95 255 91 125 134 545 77 101 450 210 869
no no no no p ) 0.982 no
2 3% p ) 0.998 6 15% no 6 17% p ) 0.998 9 20% no 3 16% no 4 15% p ) 1.00 9 20% no 2 13% no 2 4% p ) 0.927 4 8% no 8 49% p ) 0.976 2 7% no 2 11% no 3 10% no FRACTION 5 7 33% no 9 8% no 4 8% no 4 8% no 3 5% no 2 0% no 6 8% no 14 39% no 6 21% p ) 0.640 5 21% no 4 18% p ) 0.698 2 2% no 4 7% no 3 3% p ) 0.948 2 3% no 4 16% p ) 0.995 2 5% no 9 14% no 2 10% no 3 13% no 3 13% no 14 15% no 2 2% no 3 14% no 10 20% p ) 0.674 4 21% no 23 43% no
p ) 0.0091 p ) 0.0709 p ) 0.0337 p ) 0.2158 p ) 0.9710 p ) 0.0121
p ) 0.9517 p ) 0.2479 p ) 0.1004 p ) 0.0728
no hit no hit mitochondrion mitochondrion ER glycosomeg
no hit mitochondrion cytoplasm unknown mitochondrion cytoplasm
alternativef
plasma membrane
nucleus, plasma membrane
cytoplasm
no hit no hit mitochondrion no hit mitochondrion plasma membrane nucleus plasma membrane mitochondrion no hit unknown mitochondrion
mitochondrion ribosome cytoplasm no hit
cytoplasm
p ) 0.4123 mitochondrion p ) 0.4812 p ) 0.9289 p ) 0.9909 p ) 0.0686 p ) 0.6995 p ) 0.1860 p ) 0.0841 p ) 0.1095 p ) 0.7811 p ) 0.0166 p ) 0.8995 p ) 0.0119 p ) 0.9868 p ) 0.9772
lysosome mitochondrion mitochondrion ER, golgi mitochondrion no hit mitochondrion unknown no hit cytoplasm unknown no hit cytoplasm mitochondria
p ) 0.1482 p ) 0.2203 p ) 0.0051 p ) 0.0154 p ) 0.0753 too long p ) 0.0038 p ) 0.2634 p ) 0.5993 p ) 0.0430 p ) 0.5536 p ) 0.0180 p ) 0.3212 p ) 0.0486 p ) 0.9486 p ) 0.4645 p ) 0.0125 p ) 0.9027 p ) 0.2289 p ) 0.0027 p ) 0.0666 p ) 0.0087 too long p ) 0.0547 p ) 0.9842 p ) 0.1688 p ) 0.0776
no hit no hit no hit no hit no hit cytoplasm no hit cytoplasm no hit mitochondrion no hit no hit no hit no hit mitochondrion ER no hit no hit no hit cytoplasm cytoplasm ER no hit ribosome mitochondrion cytoplasm cytoplasm
endosomes, etc
nucleolus plasma membrane
plasma membrane peroxisome
nucleus nucleus cytoplasm
nucleolus nucleolus
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Table 1. (Continued) protein name
accessiona
scoreb
peptc
covc
R tubulin pyruvate dehydrogenase E1 component R subunit
LmjF13.0280 LmjF18.1380
966 68
17 3
42% 7%
signal Pd
no no
MitoProt IIe
primaryf
alternativef
p ) 0.0166 p ) 0.7889
cytoplasm mitochondrion
nucleolus
a Accession number as listed in at www.geneDB.org. b MASCOT score, the cut off for the protein identification was set at a MASCOT score > 33 (p < 0.005) and at least 2 peptides matching. c Pept, number of peptides/protein identified onto the protein, cov, coverage of the protein. d Signal P, presence of a signal peptide as determined at http://www.cbs.dtu.dk/services/SignalP/. e MitoProt II, probability of a localization in the mitochondria as determined at http:// ihg.gsf.de/ihg/mitoprot.html. f Putative primary and alternative cellular localizations as determined by blasting the Leishmania protein sequences in the Human Protein Reference Database (http://www.hprd.org/). g The subcellular localization was determined at www.geneDB.org.
Figure 2. Venn diagram showing the putative subcellular localization of proteins from fraction 2 identified by mass spectrometry. Proteins from digitonin fraction 2 were separated on pH 4-7 2D gels and stained with SyproRuby. Gel pieces were cut out from the gel, trypsin digested and sequenced by mass spectrometry. 60 proteins were identified using the MASCOT algorithm (p < 0.05). Their putative subcellular localization was determined by blasting the protein sequences in the Human Protein Reference Database (http:// www.hprd.org/query).
the Human Protein Reference Database revealed that fraction 4 contained no cytosolyc proteins but 8 proteins (54%) known to reside only in organelles, 2 proteins (13%) known to have multiple localization and the remaining 5 proteins (33%) are either proteins with no human homologue or proteins whose human homologue has no known localization (Table 1). According to the MitoProtII software, 7 proteins (47%) identified in this fraction have a 50% probability of being in the mitochondria. The mitochondrial proteins identified in digitonin fraction 4 were different from the mitochondrial proteins identified in digitonin fraction 3, demonstrating that there are no redundancy between these 2 fractions. Finally, we identified 29 proteins from digitonin fraction 5. The analysis performed against the Human Protein Reference Database indicated that fraction 5 is made of 11 proteins (37%) that are constituents of the cytoskeleton or correspond to proteins known to be associated with the cytoskeleton, of 5 proteins (18%) predicted to be either cytosolic or organellar and of 13 proteins (45%) of hypothetical proteins all of which had no homologue in the Human Protein Reference Database (Table 1). The MitoProt II analysis on these proteins showed that only 6 proteins (20%) had a 50% probability of being in the mitochondria. 1746
Journal of Proteome Research • Vol. 5, No. 7, 2006
While tubulin is an abundant protein overrepresented in several proteomic efforts done so far in Leishmania,7,8,13 we were intrigued that it was mainly found in digitonin fraction 5. To further investigate for the presence of tubulins in the various digitonin fractions, we reacted each individual fraction in Western blots with an anti-R tubulin antibody. This analysis confirmed that the vast majority of the R-tubulin (and fragments) was indeed found in fraction 5, with very little contamination in the other fractions (Figure 4). Presence of a Protein in a Fraction is Predictive of its Localization. The identification of proteins from the different fractions by MS suggested that cytosolic proteins were mainly found in fraction 2, while mitochondrial proteins were recovered in fractions 3 and 4, and the cytoskeleton and cytoskeleton-associated proteins were enriched in fraction 5. To determine whether hypothetical proteins found in either fraction 2 or fraction 3 were respectively either cytosolic or intraorganellar, we generated fusion proteins with one hypothetical protein of each fraction and the green fluorescent protein (GFP). These constructs were transfected in Leishmania and the localization of the resulting protein was determined by fluorescence microscopy. The hypothetical protein identified in fraction 2 (LmjF11.0180/LinJ11.0180) was found to localize
Digitonin Fractionation for Proteomic Studies
research articles
Figure 3. Venn diagram showing the putative subcellular localization of proteins from fraction 3 identified by mass spectrometry. Proteins from digitonin fraction 3 were separated on pH 4-7 2D gels and stained with SyproRuby. 50 proteins were identified by MS using the MASCOT algorithm (p < 0.05). Their putative subcellular localization was determined by blasting the protein sequences in the Human Protein Reference Database (http://www.hprd.org/query).
Figure 4. Presence of the R-tubulin in the digitonin fractions. 30 µg of protein from fractions 1, 2, 3, 4, and 5 were run on SDSPAGE gels, and analyzed by Western blotting. Antibodies against R-tubulin were used to determine in which fractions the Rtubulin was recovered.
in the cytosol of L. infantum cell (Figure 5A), while the hypothetical protein from fraction 3 (LmjF36.0440/LinJ36.2220) was found to localize in an organelle resembling the mitochondria (Figure 5B). This localization was further confirmed by the MitoProtII algorithm which predicted LinJ36.2220 to have a 92% probability to localize in the mitochondria. To further address the mitochondrial localization of LinJ36.2220, we fused 3,2-trans-enoyl-CoA isomerase (LmjF31.2250, found in digitonin fraction 3 corresponding to LinJ31.3010) with GFP and determined its localization by fluorescence microscopy. This protein is known to localize in the mitochondria (according to the Human Protein Reference Database)23 and was found to localize in a similar organelle than the hypothetical protein LinJ31.3010 found in fraction 3 (Figure 5C). In ongoing work, we have shown by Western blot analysis that the mitochondrial HSP60 protein is in fraction 3 (unpublished observation) and that the LmjF31.2250-GFP fusion (Figure 5C) is co localizing with the mitochondrial dye mitotracker (result not shown).
Figure 5. Localization of proteins found in fractions 2 and 3 using GFP fusions. Leishmania infantum cells were transfected with plasmids expressing fusion proteins containing a protein found in digitonin fraction 2 or fraction 3, in frame with eGFP and cells were analyzed by fluorescence imaging. A, parasite transfected with LinJ11.0180 coding for a hypothetical protein found in fraction 2; B, parasite transfected with LinJ36.2220 coding for a hypothetical protein found in fraction 3; C, parasite transfected with 3,2 trans enoyl CoA isomerase (LinJ31.3010) known to be found in human mitochondria.23
Digitonin Fractionation and Proteomic Analysis of Leishmania infantum Differentiation. To determine the potential Journal of Proteome Research • Vol. 5, No. 7, 2006 1747
research articles
Foucher et al.
Figure 6. Representative comparative 2D gels with digitonin fractions from promastigotes and amastigotes of L. infantum. Portions of 2D gels of fraction 2 of promastigotes (A) and amastigotes (B) and of fraction 3 of promastigotes (C) and amastigotes (D) of L. infantum are shown. Spots with significant differences in expression were excised from the gel, digested with trypsin, sequenced by mass spectrometry, and their identification was determined using the MASCOT algorithm (see Table 2).
of the digitonin prefractionation protocol for proteomic studies, we performed a preliminary comparative proteomic analysis of promastigote and amastigote forms of L. infantum. The proteomes of amastigote and promastigote L. infantum were fractionated according to the digitonin fractionation protocol. Fractions 2 and 3 were run on 2D gels (pH 4-7) and a semiquantitative analysis of differential protein expression between amastigote and promastigote was performed using the Progenesis Discovery software (Figure 6). Statistical analysis (t test) was performed on 3 independent gels from each fraction and showed that 1.6% of the protein spots in fraction 2 and 3.1% of the protein spots in fraction 3 were differentially regulated between the two life stages. In fraction 2, there were 14 spots up-regulated in promastigotes, 21 spots up-regulated in amastigotes and 3 unique spots for each life stage (Table 2). MS analysis was performed on 5 promastigote protein spots and 4 amastigote protein spots from fraction 2 (Table 2). Several spots contained more than one protein but others contained only one protein such as LmJF14.0190 (spot 1633, hypothetical protein) in the promastigote or LmjF20.0910 (spot 1115, hypothetical protein) in the amastigote stage. In fraction 3, we found 5 spots up-regulated in promastigotes, 19 up-regulated spots in amastigotes and 2 unique spots in each life stage. MS analysis was performed on 3 promastigote spots and 6 amastigote spots (Table 2). Several spots from fraction 3 contained only one protein.
Table 2. Identification of Proteins Differentially Expressed in Promastigote vs Amastigote L. infantum line
spot
diff
Pro
766
7
Pro
1350
9
Pro
4514
unique
Pro Pro
1633 2227
16 unique
ama
1173
8
ama ama ama
828 1115 1410
unique unique 44
Pro Pro Pro ama
1073 3239 3263 391
4 unique unique 2.5
ama
415
ama ama
1260 535
ama ama
319 590
3 7 2.7 unique unique
protein name
accessiona
FRACTION 2 potein kinase A regulatory subunit LmjF13.0160 T complex protein 1, eta subunit LmjF35.3860 T complex protein 1, gamma subunit LmjF23.1220 HSP83 LmjF33.0312 poly(a)-binding protein, putative LinJ25.0080 chaperonin TCP20 LmjF13.1660 coproporphyrinogen iii oxidase LmjF06.1270 chaperonin Hsp60, mitochondrial precursor LinJ36.4560 LACK||activated protein kinase c receptor LmjF28.2740 hypothetical protein LmjF11.0140 40S ribosomal protein SA LmjF36.5010 chaperonin Hsp60, mitochondrial LinJ36.4560 chaperonin Hsp60, mitochondrial LinJ36.4550 hypothetical protein LinJ28.1900 hypothetical protein LmjF14.0190 nucleoside diphosphate kinase b LmjF32.2950 chaperonin Hsp60, mitochondrial precursor LmjF36.2030 PMM||phosphomannomutase, putative LmjF36.1960 3-hydroxyisobutyryl-coenzyme a hydrolase-like LinJ32..4170 hypothetical protein LinJ30.2680 trypanothione reductase LmjF05.0350 hypothetical protein LinJ20.0990 hypothetical protein LmjF08.1270 spermidine synthase LinJ04.0580 hypothetical protein LmjF19.1130 FRACTION 3 peroxidoxin LmjF23.0040 HSP 70 related protein, mitochondrial LmjF30.2460 HSP 60, mitochondrial LinJ36.4550 chaperonin Hsp60, mitochondrial precursor LmjF36.2030 chaperonin Hsp60, mitochondrial precursor LmjF36.2020 heat shock 70-related protein 1, LmjF30.2470 chaperonin Hsp60, mitochondrial precursor LmjF36.2030 chaperonin Hsp60, mitochondrial precursor LmjF36.2020 heat shock 70-related protein 1 LmjF30.2470 hypothetical protein LmjF13.0450 chaperonin Hsp60, mitochondrial precursor LmjF36.2030 R tubulin LmjF13.0280 vesicular-fusion ATPase-like protein LinJ27.0660 HSP 60, mitochondrial LinJ36.1940 peroxidoxin LinJ23.0050
scoreb
peptc
covc
MW exp/predd(Kda)
pI exp/predd
439 215 144 118 174 101 231 319 132 111 50 656 224 89 341 157 124 83 174 197 65 109 207 56 51
12 7 3 3 5 4 6 6 3 3 2 9 4 2 8 3 2 3 4 4 1 3 6 1 1
17% 14% 6% 5% 8% 7% 19% 8% 9% 14% 8% 14% 9% 10% 34% 34% 5% 11% 8% 6% 3% 10% 23% 4% 0%
50/56.846 50/62.507 50/60.974 50/80.997 50/61.102 50/59.170 35/34.690 35/59.663 35/34.891 35/33.122 35/27.767 30/59.663 30/60.859 30/25.899 24/22.485 15/16.801 15/59.623 15/28.311 60/39.541 60/38.511 66/53.738 60/39.329 50/32.372 50/33.248 50/61.544
4.75/5.01 4.75/6.59 4.75/5.89 4.75/5.08 4.75/8.98 4.75/6.54 5.6/5.32 5.6/5.33 5.6/6.05 5.6/5.53 5.6/7.67 5.6/5.33 5.6/5.38 5.6/5.93 4.45/4.63 5.4/7.71 5.4/5.33 5.4/5.18 5.5/5.26 5.5/5.45 6.0/5.79 5.8/5.67 5.0/5.03 5.0/5.18 5.03/7.8
140 60 393 1246 775 212 913 567 173 133 705 68 106 1001 46
4 1 7 25 4 4 19 2 3 3 13 3 3 19 1
15% 1% 18% 37% 28% 7% 26% 20% 6% 28% 20% 7% 2% 41% 7%
27/25.497 30/69.532 30/60.859 66/59.623 66/60.484 66/72.346 60/59.623 60/60.484 60/72.346 10/13.379 47/59.623 47/50.526 47/10.8808 66/59.663 30/25.582
5.6/6.43 5.6/5.81 5.0/5.33 5.0/5.33 5.0/5.38 5.0/5.68 5.0/5.33 5.0/5.38 5.0/5.68 5.4/5.26 4.9/5.33 4.9/4.89 4.9/5.41 5.4/5.33 6.6/6.43
a Accession number as listed in at www.geneDB.org. b MASCOT score, the cut off for the protein identification was set at a MASCOT score > 33 (p < 0.005) and at least 2 peptides matching. c Pept, number of peptides/protein identified onto the protein, cov, coverage of the protein. d MW, molecular weight and pI, isoelectric point as predicted (pred) at www.geneDB.org and as determined from the experiments (exp).
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Digitonin Fractionation for Proteomic Studies
While tubulins are absent from these studies, we have observed several full size heat shock proteins (HSPs) as well as several fragments (Table 2). Increased expression of HSPs in amastigotes has been reported in several proteomic efforts4,7,8 and is expected as amastigote cells are exposed to a heat stress. Interestingly, however, most HSP fragments were found in the promastigote stage while in amastigotes the full length proteins were mostly detected.
Discussion In this study, we have developed a simple and reproducible fractionation protocol using digitonin extraction which enrich the proteome representation in Leishmania and provide some clues on protein subcellular localization. Digitonin is known to bind specifically and rapidly to sterols, forming an insoluble complex24 that results in permanent membrane holes. The large differences in sterol contents of the plasma membrane and the mitochondrial outer and inner membranes25 allow selective lysis of the sterol-rich plasma membrane using an appropriate concentration of digitonin while keeping more or less intact sterol-poor intracellular membranes such as endoplasmic reticulum, lysosomes,26 and mitochondria.27 We have determined that for Leishmania, a concentration of 100 µM digitonin affects the cytoplasmic membranes, liberating the cytosolic proteins with little contamination from the organelles or the cytoskeleton. Extraction of the remaining cellular extracts with 500 µM digitonin liberates proteins contained in organelles (mostly mitochondrial proteins) with little contamination from cytosolic proteins and the cytoskeleton. Treating the left over pellet with 5 mM digitonin allowed the recovery of intracellular proteins, including mitochondrial proteins not identified in the previous fraction but no cytosolic proteins and little tubulins. Extraction with increasing concentration of digitonin has been used for characterizing the various compartments of the mitochondria such as proteins of the outer membrane, of the intermediate space, of the inner membrane and of the matrix.28,29 The majority of non extracted proteins remaining after 5 mM digitonin contain mostly tubulins and proteins possibly associated with them. Indeed, the elongation factor 1-R, ATP dependent DEAD box RNA helicase, pyruvate dehydrogenase E1 β-subunit and 60S ribosomal protein L7a, which were identified in fraction 5 (Table 1) are known to bind to tubulin in Arabidopsis.30 As the extraction using digitonin neither denature nor alter the conformation of proteins, several of the proteins linked to the cytoskeleton would be expected to be recovered in fraction 5. This protocol is robust and can be applied to several different species. Indeed, in addition to L. infantum, we found that digitonin extraction was useful for several other Leishmania species and that in all species tested the mitochondrial HSP60 protein is found in fractions F3-F4 while R-tubulin is found in fraction F5 (unpublished observations). A few proteins, such as the enolase and SAHH, were identified in fractions in which they were not expected to be. Enolase was identified in both fractions 2 and 3 while this protein should localize only in the cytosol according to the Human Protein Reference Database. While we expect some differences in protein localization between human cells and Leishmania, such as with glycolytic enzymes, most of the orthologous proteins should have a similar localization between the two organisms. Enolase is an enzyme of the glycolysis pathway. While most of the enzymes of the glycolysis pathway are located in the glycosome in the kinetoplastidae, enolase
research articles has been shown, in T. brucei, to be located in the cytosol.31 The analysis of the mass spectra obtained from the spots of fractions 2 and 3 confirmed that they both corresponded to the same enolase open reading frame which is a single copy in the L. major genome (www.genedb.org). The experimentally determined molecular weight indicates that enolase is a full length protein in both fractions. Either the presence of enolase in fraction 3 is a contamination from the cytosolic fraction or it raises the possibility that, at least in Leishmania, it is also present in an organelle, possibly the glycosome. Further analysis such as GFP fusions as we have carried out for several other proteins (Figure 5) may help in further delineating the possible cellular localization(s) of enolase in Leishmania. SAHH, a protein usually located in the cytosol was also identified in fractions 2 and 3. As for the enolase, the human SAHH is predicted to localize solely in the cytoplasm according to the Human Protein Reference Database. However, SAHH is also thought to function as a protective enzyme for adenosine toxicity by preventing the accumulation of S-adenosine homocysteine in the nucleus.32 SAHH was indeed found to relocalize to the nucleus during the embryogenesis of Xenopus laevis.33 Thus, there is precedent for differential localization of SAHH, although the SAHH found in fraction 3 is shorter (by about 9 KDa) than the predicted full length protein, suggesting that this spot is either a degradation product or that SAHH is processed during relocalization. Digitonin extraction has allowed the identification of proteins expressed in a stage specific manner. Our identification of HSP83 in promastigote is consistent with the RNA expression of HSP83.34,35 As with other proteomic efforts in Leishmania, we found several protein spots containing HSP 60 in both life stages, and this is consistent with the known role of HSP60 in the differentiation of the parasite.8 Interestingly, we observed mostly proteolytic fragment of HSP60 in promastigotes, while we identified mostly the full length protein in amastigotes. It is tempting to speculate on a stage specific processing of this protein. Our observation of overexpression of an activated protein kinase C in promastigotes and R-tubulin in amastigotes is consistent with other proteomic efforts,8 validating further our protocol. Most interestingly, however, digitonin extraction has allowed the identification of several novel hypothetical proteins such as LmjF14.0190 which is up-regulated 16-fold in promastigote cells or LmjF13.0450 which is up-regulated 7-fold in amastigotes. The promastigote hypothetical protein (LmjF14.0190) was found in fraction 2 and is thus probably cytosolic while the amastigote hypothetical protein was identified in fraction 3 and is thus probably localized within an organelle. The concentration of tubulins in fraction 5 is a marked advantage in proteomic efforts for Leishmania as these proteins are greatly overrepresented. However, our fractionation protocol leads to several spots corresponding to HSPs and a technique to remove these abundant proteins would be most useful. Digitonin extraction is simple and reproducible and has shown here its usefulness in allowing the identification of lower expressed proteins. Indeed, more than 70% of the proteins identified here have not been identified in other proteomic work.4,6-9,13,19,36 Interestingly, digitonin fractionation can at the same time provide tentative subcellular localization, as we have experimentally tested using GFP fusions. Since the concentration of sterols may vary from one organism to the other or even between the same organism grown in different medium such as for T. cruzi,37 it is advisable to address experimentally the Journal of Proteome Research • Vol. 5, No. 7, 2006 1749
research articles concentration of digitonin required to render membranes leaky. In comparative proteomic efforts, it would be advisable to react each individual fraction with antibodies to verify that digitonin fractionation worked similarly between the two conditions tested. This digitonin fractionation technique is a complement to other fractionation protocols that should allow a better representation of the proteome, which can lead to a better understanding of the biology of the kinetoplastid parasites. Abbreviations. DTT, dithiothreitol; eGFP, enhanced green fluorescence protein; HSP, heat shock protein; MS, mass spectrometry; PTM, post-translational modification; SAHH, S-adenosyl homocysteine hydrolase; TCEP, Tris (2-carboxyethyl) phosphine.
Acknowledgment. We thank Dr. Danielle Le´gare´ and Julie-Christine Le´vesque for help with the mitotracker colocalization work, our colleagues in the Eastern Quebec Proteomics Centre for their help with mass spectrometry and Dr Jolyne Drummelsmith for initial training with the Progenesis software. This work was funded in part by a CIHR group grant GR 14500 to M.O. and B.P and operating grants to M.O. A.F. is a “Strategic Training Fellow of the Strategic Training Program in Microbial Resistance (IRSC STP-53924), a partnership of the CIHR Institute of Infection and Immunity and the Fonds de Recherche en Sante´ du Que´bec.” M.O and B.P. are Burroughs Wellcome Fund Scholar and New Investigator in Molecular Parasitology respectively, and M.O. holds a Tier 1 Canada Research Chair in Antimicrobial resistance. References (1) El-Sayed, N. M.; Myler, P. J.; Blandin, G.; Berriman, M.; Crabtree, J.; Aggarwal, G.; Caler, E.; Renauld, H.; Worthey, E. A.; HertzFowler, C.; Ghedin, E.; Peacock, C.; Bartholomeu, D. C.; Haas, B. J.; Tran, A. N.; Wortman, J. R.; Alsmark, U. C.; Angiuoli, S.; Anupama, A.; Badger, J.; Bringaud, F.; Cadag, E.; Carlton, J. M.; Cerqueira, G. C.; Creasy, T.; Delcher, A. L.; Djikeng, A.; Embley, T. M.; Hauser, C.; Ivens, A. C.; Kummerfeld, S. K.; Pereira-Leal, J. B.; Nilsson, D.; Peterson, J.; Salzberg, S. L.; Shallom, J.; Silva, J. C.; Sundaram, J.; Westenberger, S.; White, O.; Melville, S. E.; Donelson, J. E.; Andersson, B.; Stuart, K. D.; Hall, N. Science 2005, 309 (5733), 404-409. (2) Ivens, A. C.; Peacock, C. S.; Worthey, E. A.; Murphy, L.; Aggarwal, G.; Berriman, M.; Sisk, E.; Rajandream, M. A.; Adlem, E.; Aert, R.; Anupama, A.; Apostolou, Z.; Attipoe, P.; Bason, N.; Bauser, C.; Beck, A.; Beverley, S. M.; Bianchettin, G.; Borzym, K.; Bothe, G.; Bruschi, C. V.; Collins, M.; Cadag, E.; Ciarloni, L.; Clayton, C.; Coulson, R. M.; Cronin, A.; Cruz, A. K.; Davies, R. M.; De Gaudenzi, J.; Dobson, D. E.; Duesterhoeft, A.; Fazelina, G.; Fosker, N.; Frasch, A. C.; Fraser, A.; Fuchs, M.; Gabel, C.; Goble, A.; Goffeau, A.; Harris, D.; Hertz-Fowler, C.; Hilbert, H.; Horn, D.; Huang, Y.; Klages, S.; Knights, A.; Kube, M.; Larke, N.; Litvin, L.; Lord, A.; Louie, T.; Marra, M.; Masuy, D.; Matthews, K.; Michaeli, S.; Mottram, J. C.; Muller-Auer, S.; Munden, H.; Nelson, S.; Norbertczak, H.; Oliver, K.; O’Neil, S.; Pentony, M.; Pohl, T. M.; Price, C.; Purnelle, B.; Quail, M. A.; Rabbinowitsch, E.; Reinhardt, R.; Rieger, M.; Rinta, J.; Robben, J.; Robertson, L.; Ruiz, J. C.; Rutter, S.; Saunders, D.; Schafer, M.; Schein, J.; Schwartz, D. C.; Seeger, K.; Seyler, A.; Sharp, S.; Shin, H.; Sivam, D.; Squares, R.; Squares, S.; Tosato, V.; Vogt, C.; Volckaert, G.; Wambutt, R.; Warren, T.; Wedler, H.; Woodward, J.; Zhou, S.; Zimmermann, W.; Smith, D. F.; Blackwell, J. M.; Stuart, K. D.; Barrell, B.; Myler, P. J. Science 2005, 309 (5733), 436-442. (3) McNicoll, F.; Muller, M.; Cloutier, S.; Boilard, N.; Rochette, A.; Dube, M.; Papadopoulou, B. J. Biol. Chem. 2005, 280 (42), 3523835246. (4) McNicoll, F.; Drummelsmith, J.; Muller, M.; Madore, E.; Ouellette, M.; Papadopoulou, B. Proteomics 2006, in press. (5) Jones, A.; Faldas, A.; Foucher, A.; Hunt, E.; Tait, A.; Wastling, J. M.; Turner, C. M. Proteomics 2005.
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