Smit Proteomic(2010)

Smit Proteomic(2010)
Journal of Proteome Research
Proteomic profiling of Plasmodium falciparum through
improved semi-quantitative, two-dimensional gel
electrophoresis
Journal:
Manuscript ID:
Manuscript Type:
Journal of Proteome Research
pr-2009-009244.R1
Article
Date Submitted by the
Author:
Complete List of Authors:
Smit, Salome; University of Pretoria, Biochemistry
Stoychev, Stoyan; CSIR, Biosciences
Louw, Abraham; University of Pretoria, Biochemistry
Birkholtz, Lyn-Marie; University of Pretoria, Biochemistry
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Journal of Proteome Research
Proteomic profiling of Plasmodium falciparum through
improved, semi-quantitative two-dimensional gel
electrophoresis
Salome Smit1, Stoyan Stoychev2, Abraham I Louw1, Lyn-Marie Birkholtz1*
1
Department of Biochemistry, Faculty of Natural and Agricultural Sciences, University of Pretoria,
Pretoria, 0002, South Africa,
Telephone +27 12 420 2479, Fax +27 12 362 5302
2
CSIR Biosciences, Pretoria, 0001, South Africa
RECEIVED DATE:
TITLE RUNNING HEAD: Proteomic profiling of Plasmodial proteins
* Corresponding author: Prof Lyn-Marie Birkholtz, Department of Biochemistry, Faculty of Natural and
Agricultural Sciences, University of Pretoria, Pretoria, South Africa, 0002
Telephone +27 12 420 2479, Fax +27 12 362 5302, email: [email protected]
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ABSTRACT
Two-dimensional gel electrophoresis (2-DE) is one of the most commonly used technologies to obtain a
snapshot of the proteome at any specific time. However, its application to study the Plasmodial (malaria
parasite) proteome is still limited due to inefficient extraction and detection methods and the
extraordinarily large size of some proteins. Here, we report an optimized protein extraction method, the
most appropriate methods for Plasmodial protein quantification and 2-DE detection and finally protein
identification by mass spectrometry (MS). Linear detection of Plasmodial proteins in a optimized lysis
buffer was only possible with the 2-D Quant kit and of the four stains investigated, Flamingo Pink was
superior regarding sensitivity, linearity and had excellent MS-compatibility. 2-DE analyses of the
Plasmodial proteome using this methodology, resulted in the reliable detection of 349 spots and a 95%
success rate in MS/MS identification. Subsequent application to the analyses of the Plasmodial ring and
trophozoite proteomes ultimately resulted in the identification of 125 protein spots, which constituted 57
and 49 proteins from the Plasmodial ring and trophozoite stages, respectively. This study additionally
highlights the presence of various isoforms within the Plasmodial proteome, which is of significant
biological importance within the Plasmodial parasite during development in the intra-erythrocytic
developmental cycle.
KEYWORDS: 2-D gel electrophoresis, malaria, fluorescent dyes, proteome, isoforms
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INTRODUCTION
Global efforts to eradicate malaria in Third World countries are hampered by various factors including
global climate changes, increasing migration behavior, failing health care systems, absence of a licensed
vaccine and most disturbingly, the rapid development and dispersal of the respective drug- and
insecticide-resistant forms of the malaria parasite and the mosquito vector. About 40% of the world’s
population in 107 countries live under the constant risk of malaria infection and more than 80% of
malaria-associated deaths in the world occur in Africa south of the Sahara. Currently, only one drug,
Artemisinin, is still effective against the malaria parasite but the first signs of drug resistance has now
emerged at the Thai-Cambodian border1. This raises serious concerns and underscores the urgency for
innovative strategies to discover new and robust antimalarial drugs as well as new targets to combat the
disease. The latter can be expedited by the inclusion of techniques such as functional genomics in drugdiscovery pipelines2.
The Plasmodium parasite has complex life cycles in both the human host and the mosquito vector.
Pathogenesis is displayed during the 48 hour schizogony of the parasite in human erythrocytes (intraerythrocytic developmental cycle, IDC), where parasites mature from ring to trophozoite stages to the
ultimate production of daughter merozoites from schizonts. This asexual replication cycle is tightly
controlled in P. falciparum with a unique cascade of gene regulation resulting in the ‘just-in-time’
production of transcripts coordinated with expression of genes involved in related biological processes3.
Therefore, in the majority of cases, proteins are produced from their respective transcripts without
delay3, 4.
The resultant Plasmodial proteome is multifaceted and stage-specific, indicating a high degree of
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specialization at the molecular level to support the biological and metabolic changes associated with
each of the life cycle changes5, 6. Post-translational modifications are employed as a mechanism to
regulate protein activity during the parasite’s life cycle7 and certain proteins are predicted to act as
controlling nodes that are highly interconnected to other nodes and thus results in a highly specialized
interactome2. These enticing properties motivate studies focused on in-depth characterization of the
Plasmodial proteome including regulatory mechanisms and the ability to respond to external
perturbations. Analysis of the schizont stage proteome reinforced the notion that both posttranscriptional and post-translational mechanisms are involved in the regulation of protein expression in
P. falciparum8.
Due to the >80% AT-richness of the Plasmodial genome9, the resultant Plasmodial proteome contains
proteins in which long hydrophobic stretches and amino acid repeats (notably consisting of lysine and
asparagine) are found. Moreover, the proteins from this parasite are comparatively large, nonhomologous and highly charged with multiple isoforms within the parasite10. These properties have
confounded analyses of the Plasmodial proteome, including the recombinant expression of Plasmodial
proteins11, 12. Few studies attempted to describe the Plasmodial proteome, which is predicted to have
about 5300 proteins of which ~60% are hypothetical and un-annotated8, 13, 14.
The reported efficacy of two-dimensional gel electrophoresis (2-DE) to analyze the Plasmodial
proteome is relatively poor since only a low number of protein spots could be detected with various
protocols and stains13-17. The highest number of spots detected to date on Plasmodial 2-DE gels with
silver staining is only 23915 and recently, a total of 345 spots were detected for 4 time points in the
Plasmodial schizont stage using two-dimensional differential gel electrophoresis (2-D DIGE)8, of which
only 54 protein spots were identified. This clearly illustrates the need for an optimized protocol
including extraction, quantification and detection methods. This study details such an optimized 2-DE
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protocol, which was applied to the analysis of the Plasmodial proteome in the ring and trophozoite
stages. This study first optimized established methodology with regard to protein extraction,
quantification, detection and finally MS identification. Once the protocol was established, it was
subsequently applied to the analyses of the soluble Plasmodial proteome, resulting in the detection of
349 spots using the fluorescent stain, Flamingo Pink, with a 95% success rate achieved in the mass
spectrometry (MS) identification of a subset of these proteins, far exceeding previously reported
Plasmodial protein identification success rates of 50-79%13, 14. After the successful establishment of the
optimized 2-DE protocol, this methodology was applied to the Plasmodial ring and trophozoite
proteomes for which a total of 125 protein spots were identified. Several protein isoforms were also
identified in the two Plasmodial life-stages which has biological significance for the Plasmodial
parasite.
EXPERIMENTAL SECTION
Culturing of parasites
P. falciparum 3D7 (Pf3D7) parasites were maintained in vitro in human O+ erythrocytes in RPMI
1640 media (Sigma) with 0.5% (w/v) Albumax II (Gibco)18. Parasites were synchronized (~98%
morphological synchronicity) with sorbitol treatment for three consecutive generations. Thirty milliliters
of Pf3D7 parasite cultures at 8% parasitemia and 5% hematocrit were used per gel to establish the
proteomics methodology. Saponin was added to a final concentration of 0.01% (v/v) followed by
incubation on ice for 5 min to lyse the erythrocytes. Parasites were collected by centrifugation at 2500 g
for 15 min at room temperature, and washed in PBS at 16 000 g for 1 min at 4 C. This step was
repeated at least four times until the supernatant was clear instead of three times as previously reported7.
The parasite pellet was stored at –80 C. For analyses of proteomes of different developmental states of
the parasites, parasites were harvested from 60 ml cultures at 16 hours post invasion (HPI) (late rings)
and 20 HPI (early trophozoites).
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Protein preparation
Parasite pellets were suspended in 500 µl lysis buffer as described by Nirmalan et al. (8 M urea, 2 M
thiourea, 2% CHAPS, 0.5% (w/v) fresh DTT and 0.7% (v/v) ampholytes)7. Samples were pulsedsonicated on a Virsonic sonifier with microtip for 20 sec with alternating pulsing (1 sec pulse, 1 sec rest)
at 3 W output with 1 min cooling steps on ice (to prevent foaming and carbamylation) and repeated 6
more times. Sonication was followed by centrifugation at 16 000 g for 60 min at 4˚C, after which the
protein-containing supernatant was used in subsequent 2-DE.
Protein quantification
Four different protein quantification methods were tested on the samples obtained using two BSA
standard curves in each of the methods: firstly, BSA in 0.9% saline, and secondly, BSA in the
Plasmodial optimized lysis buffer, each containing the same amount of protein for analysis. The Quick
Start™ Bradford dye method (BioRad) was used for protein determination at an absorbance of 595
nm19. The Lowry method used a reaction mixture containing solution A (2% (w/v) NaCO3, 2% (w/v)
NaOH, 10% (w/v) Na2CO3), solution B (2% (w/v) CuSO4.5H2O), and solution C (0.5% (w/v) potassium
tartrate). Two hundred microlitres of the reaction mixture was added to each protein sample, mixed and
incubated for 15 min at room temperature. Six hundred microlitres of Folin Ciocalteau reagent (1:10,
FC reagent and H2O) were added and incubated at room temperature for 45 min in the dark. Absorbance
was measured at 660 nm20. Lastly, two commercial protein concentration determination kits were used
according to the manufacturer’s instructions and included the Micro BCA™ Protein assay kit (Pierce)
and the 2-D Quant Kit (GE Healthcare).
SDS-PAGE gels
Low molecular weight markers (GE Healthcare) were diluted in reducing buffer (0.06 M Tris-HCl,
2% (w/v) SDS, 0.1% (v/v) glycerol, 0.05% (v/v) β-mercaptoethanol and 0.025% (v/v) bromophenol
blue, pH 6.8), to provide a concentration range from 100 ng to 0.6 ng protein. Equal amounts of markers
were loaded onto four different 12.5% SDS-PAGE gels and the gels were subsequently stained with
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either Colloidal Coomassie, silver, SYPRO Ruby (Molecular Probes) or Flamingo Pink (Bio-Rad)
stains. The gels were scanned on a Versadoc 3000 and analyzed using the Quantity One Program (BioRad). The Rf values and the intensities of each band were compared, and used to determine the limit of
detection and linearity.
Two dimensional gel electrophoresis (2-DE)
For 2-DE, the protein concentration was determined with the 2-D Quant kit. Two hundred
micrograms of protein in rehydration buffer (8 M urea, 2 M thiourea, 2% (w/v) CHAPS). 0.5% (w/v)
DTT and 0.7% (v/v) IPG Buffer (pH 3-10 Linear) was applied to a 13 cm IPG, pH 3-10 L strip. First
dimensional isoelectric focusing was performed on an Ettan IPGphore Isoelectric Focusing Unit (GE
Healthcare), and commenced with a 10 hour active rehydration step. Isoelectric focusing time followed
an alternating gradient and step-and-hold protocol and was always allowed to proceed to a total of 18
500 Volt-hours, that completed within 15 hours. IPG strips were equilibrated for 10 min each in SDS
equilibration buffer (50 mM Tris-HCl, pH 6.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 0.002%
bromophenol blue) containing 2% DTT, and then incubated in 2.5% iodoacetamide. Finally, the strip
was placed in SDS electrophoresis running buffer (0.25 M Tris-HCl, pH 8.3, 0.1% SDS, 192 mM
glycine) for 10 min as a final equilibration step. Second dimensional separation was performed by
placing the IPG strips on top of the 10% SDS PAGE gel (Hoefer SE 600), covered with 1% agarose
dissolved in SDS electrophoresis running buffer. Separation was performed at 80 V at 20°C until the
bromophenol blue front reached the bottom of the gel. The gels were then fixed in the appropriate fixing
solution for each specific stain (see below). For the method optimization protocol, gel image analysis
was performed using PD Quest 7.1.1 (Bio-Rad). All 8 gels were filtered using the Filter Wizard. Spot
detection was performed on the gels by automated spot detection. The display of the gels stained with
SYPRO Ruby and Flamingo Pink was inverted for easier comparisons with the gels stained with CCB
and silver. Additional manual settings for spot detection were sensitivity (2.22), size scale (5) and min
peak (1244). For proteomic analyses of the different developmental stages of P. falciparum, 400 µg
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protein was applied to 18 cm IPG strips for separation and subsequently stained with Flamingo Pink and
scanned using the Versadoc 3000 as described below. PD Quest 7.1.1 was used to identify the number
of spots on each of the gels that were done for the ring and trophozoite 2-DE proteomes (8 gels for each
stage). First, all images were cropped to the same dimensions (1.59 Mb, 933 x 893 pixels, 303.7 x 290.7
mm) and filtered using the salt setting (light spots on dark background) of the Filter Wizard. The Spot
Detection Wizard was used to automatically detect spots on the selected master image by manual
identification of a small spot, faint spot and large spot. Additional settings for spot detection were
manually selected for sensitivity (5.31 for rings and 4.35 for trophozoites), size scale 5.0 (both), min
peak (808 for rings and 4712 for trophozoites). After automated matching of all the gels, every spot
were manually verified to determine correctness of matching. The master image contained 369 spots for
the ring stage proteome with a match rate of 98%, and the trophozoite master image contained 450 spots
with a match rate of 96%.
Flamingo Pink staining of 2-DE gels
Gels were fixed overnight in 40% (v/v) ethanol, 10% (v/v) acetic acid, and subsequently in 200 ml
Flamingo Pink working solution and incubated with gentle agitation in the dark for 24 hours, to increase
the sensitivity of the stain. The gels were washed in 0.1% (w/v) Tween-20 for 30 min to reduce
background. Finally the gels were rinsed in MilliQ water twice before scanning on the Versadoc 3000.
All gels were stored in Flamingo Pink at 4ºC until use for MS.
Silver staining of 2-DE gels
Gels were fixed in 45% (v/v) methanol, 5% (v/v) acetic acid overnight, followed by sensitizing for 2
min in 0.02% (w/v) sodium thiosulfate, and rinsing with MilliQ water twice. 200 ml ice cold 0.1% (w/v)
silver nitrate was added and incubated at 4°C for 30 min, rinsed twice with MilliQ water and developed
in fresh 2% (w/v) sodium carbonate with 0.04% (v/v) formaldehyde. Development was stopped by
adding 1% (v/v) acetic acid. All gels were stored in 1% (v/v) acetic acid at 4ºC in airtight containers
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until use for MS.
SYPRO Ruby staining of 2-DE gels
Gels were fixed in 10% (v/v) methanol, 7% (v/v) acetic acid overnight. The fixing solution was
replaced with 200 ml SYPRO Ruby stain and incubated with agitation for 24 hours in the dark, to
increase sensitivity. After staining, the gels were washed for 60 min with 10% (v/v) methanol, 7% (v/v)
acetic acid to reduce fluorescent background. Finally the gels were rinsed twice with MilliQ water
before scanning on the Versadoc 3000. Gels were stored in SYPRO Ruby at 4°C until use for MS.
Colloidal Coomasie Blue (CCB) staining of 2-DE gels
Colloidal Coomassie Brilliant Blue G250 stock solution (2% (v/v) phosphoric acid, 10% (w/v)
ammoniumsulfate, and 0.1% (v/v) Coomassie Brilliant Blue G250) was diluted (4:1) with methanol just
before use. The gels were immersed in the Colloidal Coomassie solution and left shaking overnight.
Gels were rinsed with 25% (v/v) methanol, 10% (v/v) acetic acid before destaining with 25% (v/v)
methanol, until the background was clear21. Gels were then scanned on the Versadoc 3000, and stored in
1% (v/v) acetic acid at 4°C until use for MS.
2-D spot identification by tandem mass spectrometry
For comparative purposes mostly the same 39 spots (154 in total) covering a wide range on the gels as
well as low molecular weight markers were cut from each of the 4 differently stained gels, dried and
stored at -20°C. The silver stained samples were first destained with potassium ferricyanide and sodium
thiosulfate to remove the silver before proceeding to wash steps22. All gel pieces was cut into smaller
cubes and washed twice with water followed by 50% (v/v) acetonitrile for 10 min each. The acetonitrile
was replaced with 50 mM ammonium bicarbonate and incubated for 10 min, repeated twice, except for
CCB samples, which had an additional wash step to ensure complete removal of the dye. All the gel
pieces were then incubated in 100% acetonitrile until they turned white. This was followed by another
ammonium bicarbonate, acetonitrile wash step as above, after which the gel pieces were dried in vacuo.
Gel pieces were digested with 10 ng/µl trypsin at 37°C overnight. Resulting peptides were extracted
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twice with 70% acetonitrile for 30 min, and then dried and stored at -20°C. Dried peptides were
dissolved in 10% (v/v) acetonitrile, 0.1% (v/v) formic acid and mixed with saturated alpha-cyano-4hydroxycinnamic acid before being spotted onto a MALDI sample plate. Experiments were performed
using Applied Biosystems QSTAR-ELITE, Q-TOF mass spectrometer with oMALDI source installed.
Laser pulses were generated using a Nitrogen laser with intensities between 15 and 25 µJ depending on
sample concentration and whether single MS or MS/MS experiments were performed. First, single MS
spectra were acquired for 15-30 sec. The 50 highest peaks from the MS spectra were automatically
selected for MS/MS acquisition. Tandem spectra acquisition lasted 4-8 min depending on sample
concentration. Argon was used as cooling gas in Q0 and collision gas in Q2. The collision energy was
first optimized using a 9 peptide mixture covering the scan range of 500–3500 Da and then
automatically set during MS/MS experiments using the Information Dependent Acquisition (IDA)
function of the Analyst QS 2.0 software. The instrument was calibrated externally, in TOF-MS mode,
via a two point calibration using the peptides Bradykinin 1-7 and Somatostatin 28 ([M+H]+ = 757.3992
Da and 3147.4710 Da, respectively). Data was submitted in MASCOT (www.matrixscience.com). For
the Plasmodial ring and trophozoite proteome analysis, spots of various intensities covering the whole
2-DE range (pI 4-9, and Mr 13-135 kDa) were selected, and subjected to MS/MS as described above.
For the ring stage 2-DE proteome analysis 77 spots were selected for MS identification and 63 spots
were selected for the trophozoite stage. The normalized intensities of these spots ranged from 58 to a
maximum of 9734 with 1963 as the average intensity per spot.
Results and Discussion
Optimization for 2-DE of Plasmodial proteins
The ability of 2-DE to provide a snapshot of the proteome at any particular time, is a distinct
advantage for a multistage organism such as Plasmodium. The 2-DE technique remains the most widely
used for proteomic investigation techniques23 due to several advantageous properties such as good
resolution of abundant proteins as well as information on protein size, quantity and isoforms with post-
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translational modifications or different pIs24. However, 2-DE gels are biased to the detection of
relatively high abundant proteins as well as soluble and mid-range molecular weight proteins25. Besides
the visual advantages of 2-DE in comparing protein levels, proteins are differentially stained due to their
specific chemical and physical properties, which necessitates careful selection of the staining method in
terms of its sensitivity, reproducibility, ease of use and cost-effectiveness. Most importantly, the stain
should be compatible with downstream applications such as MS. This study describes an improved
protocol for the detection and identification of Plasmodial proteins separated by 2-DE, which was then
also subsequently applied to identify the proteome of the Plasmodial ring and trophozoites stages.
The analysis of the Plasmodial proteome by 2-DE has been hampered by numerous technical
constraints. Plasmodial proteins are notoriously insoluble, comparatively large, non-homologous and
highly charged10 and therefore necessitates the use of optimized lysis buffers to ensure maximal
solubility of these proteins for 2-DE. The lysis buffer described by Nirmalan et al., is able to solubilize a
large proportion of Plasmodial proteins. In this study, the combination of 5-fold less saponin used,
increased washing steps and shorter sonication cycles (with prolonged cooling in between cycles),
contributed to the absence of hemoglobin on the 2-DE gels and the detection of proteins in the range of
pH 8-9 that was previously cumbersome in Plasmodial 2-DE. The use of this lysis buffer, however
precludes the use of traditional methods of protein concentration determination.
A two-pronged approach was used in this study to determine the most effective and reproducible
detection and staining method for Plasmodial proteins. Firstly, the effect of the extraction medium on
standard protein determination methods was established as well as the sensitivity of staining methods to
detect gel-separated molecular weight standards and secondly, for comparative purposes the sensitivity
and reproducibility of these staining methods to detect Plasmodial proteins on 2-DE gels. Four different
methodologies were evaluated to determine Plasmodial protein concentrations in the lysis buffer used
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for the protein extraction. The standard Bradford method as well as the Lowry and BCA methods was
found to be incompatible with the lysis buffer, (results not shown). The 2-D Quant kit conversely
provided reproducible and comparable data for both the saline (R2 = 0.9918) and lysis buffer (R2 =
0.9929) standard curves, most likely due to the quantitative protein precipitation step by which any
other interfering substances in the lysis buffer are also removed. Although various Plasmodial proteomic
studies have employed the Bradford method14,
15
, the present study confirms recent reports of the
reliability of the 2-D Quant method8, 26.
A second caveat in semi-quantitative proteomics is the sensitivity of the staining method used for the
detection of protein spots after 2-DE. The sensitivity, performance, and linear regression constants of
four different staining methods were compared in this study with quantitative 1-D analyses of standard
molecular weight markers. Four different SDS-PAGE gels were individually stained with Colloidal
Coomassie Blue (CCB), MS-compatible silver stain, SYPRO Ruby and Flamingo Pink, and compared
by using Quantity One 4.4.1 to determine the detection limits (Table 1, 1-D SDS PAGE MW analysis of
stains). SYPRO Ruby and Flamingo Pink achieved similar results, as both were able to detect up to 1 ng
of protein and were linear (R2 = 0.97). CCB was the least sensitive of the four stains with a detection
limit of 25 ng and relatively poor linearity (R2 = 0.89). The MS-compatible silver stain was able to
detect a minimum of 10 ng but has a very poor linear range (R2 = 0.83). The fluorescent stains, SYPRO
Ruby and Flamingo Pink, thus seem superior to CCB and silver in both sensitivity and dynamic linear
quantification range of standard protein molecular weight markers.
These same stains were subsequently tested on the proteome of Plasmodial proteins after 2-DE. The
total Plasmodial trophozoite proteome is predicted to contain 1029 proteins27, 28 (PlasmoDB 6.0), which
spans a wide molecular weight range and pI with different degrees of solubility. Filtering of this dataset
to represent the conditions used in this study for 2-DE resulted in 443 Plasmodial trophozoite proteins
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that should be detectable on a standard 2-DE gel in the molecular weight range of 10-110 kDa with a pI
range of 4-9. Duplicate 2-DE analysis were performed for all 4 stains used (n = 2 per stain and n = 8 in
total). The CCB stain performed poor in detection with an average of 126 spots detected, markedly less
than any of the other three stains tested (Table 1, 2-DE trophozoite analysis of stains). The MScompatible silver stain was superior in terms of sensitivity and 420 spots of the Plasmodial trophozoite
proteome could be detected (Fig. 1). This represents 95% (420/443) of the expected trophozoite
proteome that were detected here. However, the poor linearity and spurious artifacts associated with
silver staining of 2-DE could lead to unreliable results when groups of gels with differentially expressed
proteins are compared (Table 1)29.
Fluorescent stains have been developed with seemingly similar sensitivity to silver as well as being
MS-compatible, which include the earlier SYPRO Orange and SYPRO Red30,
31
, and the currently
commonly used SYPRO Ruby stain29. The latter is a fluorescent ruthenium-based stain that binds noncovalently to protein in gels, and is used to stain refractory proteins like glycoproteins and lipoproteins.
SYPRO Ruby has been reported to be a photostable end-point stain, with a good linear dynamic range29,
32
and to be MS compatible33. However, SYPRO Ruby was only able to detect 235 Plasmodial protein
spots after 2-DE with a MS identification rate of 85% (Table 1). These results are in sharp contrast to
those obtained with standard protein molecular weight markers and indicate that SYPRO Ruby is not an
appropriate stain to use with Plasmodial proteins. New generation fluorescent stains such as Flamingo
Pink are reported to be able to detect proteins across the full range of molecular weights and isoelectric
points separated on 2-DE with little gel-to-gel variability34, good linear dynamic range and MScompatibility. These properties seem to be supported by the results of this study since 79% (349/443) of
the Plasmodial trophozoite proteome were detected on 2-DE as predicted by our calculations.
In order to assess the overall MS-compatibility of the four staining methods, approximately 39 spots
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of four individual gels were selected consisting of 33 Plasmodial proteins (Fig. 1, 1-33) and 6 standard
molecular weight marker proteins (Fig. 1, marked Mr1 to Mr6), summarized in Table 1 (2-DE
trophozoite analysis of stains). Proteins were identified when a significant Mascot score was obtained
and further criteria of at least 5 peptides and sequence coverage of at least 10% was achieved
(Supplementary Tables 1 A-D). This was done to increase the MS/MS identification confidence. Silver
staining resulted in the least number of positive identifications (85%) with MS/MS (Table 1). This low
positive identification value was also observed for SYPRO Ruby staining. The most promising results
concerning protein identification were obtained with CCB and Flamingo Pink, which both had MS/MS
success rates in excess of 90% (CCB had positive identification for 35/37 proteins subjected to MS/MS
and Flamingo Pink had positive identification for 37/39 proteins subjected to MS/MS). The MScompatibility of CCB is well documented35,
36
, but literature evidence for the MS-compatibility of
Flamingo Pink is still lacking. However, for the Plasmodial proteins investigated here, Flamingo Pink
was superior to the other standard stains regarding its ability to provide excellent MS/MS identification
rates (95% success). Moreover, it provides an excellent linear dynamic range (R2 = 0.97) as detected on
1-D SDS PAGE gels with standard molecular weight markers and was able to detect 79% of the
predicted 2-DE trophozoite proteome under our experimental conditions. Thus, Flamingo Pink provided
high sensitivity to detect proteins on both 2-DE and 1-D gels, as well as good linear dynamic ranges
with the added advantage of excellent MS-compatibility. This suggests that Flamingo Pink may the
preferable stain as far as Plasmodial proteomics are concerned but this may also be generally true for
proteome analyses due to its superior detection and identification of proteins after 2-DE.
2-DE based analyses of the Plasmodial proteome is hampered by contaminating hemoglobin derived
products (HDP)37, possibly as a result of the thiourea/sonication steps during the extraction of
Plasmodial proteins, and the resultant destabilization of hemozoin. Typically, these HDPs are observed
as an intense smear focused around pI 7-10 with varying molecular weights. The less harsh sonication
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steps used in this study combined with extensive wash steps (to remove hemoglobin) and 5-fold less
saponin, resulted in discrete spots identified in the 2-DE based Plasmodial proteome described here.
Very little background and smearing were observed here compared to other Plasmodial proteome
studies7, 14, 15, 38 enabling the identification of several proteins in the pI 7.5-9 range (Fig. 1, e.g. LDH,
G3PDH, Adenylate kinase). Moreover, the protocol used here makes it unnecessary to use additional
fractionation steps to remove contaminating high pI fractions37 or two-step extraction procedures15.
Furthermore, the use of the 2-D Quant kit provided the only means of protein concentration
determination for Plasmodial proteins in the lysis buffer. Finally, Flamingo Pink proved to be superior
with regard to sensitivity as far as detection of spots on 2-DE is concerned and provided excellent
MS/MS compatibility for Plasmodial proteins.
Application of 2-DE optimized method on the Plasmodial ring and trophozoite stages
The successful establishment of an optimized 2-DE method allowed the comprehensive analyses of
the Plasmodial proteome during its IDC. The recent report of the schizont stage P. falciparum proteome
analyzed with 2-DE8 prompted the analyses of expressed Plasmodial proteins during the ring and
trophozoite stages of parasite development. Due to the just-in-time nature of transcript production per
life cycle stage in the parasite, and little delay between transcript and protein production, the majority of
this parasite’s proteins are relatively life cycle specific4. Proteins are therefore expressed 0.75 to 1.5
times of a life cycle3. Highly synchronized parasites were used where proteins were isolated from either
>98% pure ring stage or conversely trophozoite stage proteins. For the ring-stage parasite proteome, an
average of 328 spots were detected on 2-DE with Flamingo Pink staining, and of these spots, 73 protein
spots were identified by MS/MS. An average of 272 spots were detected on 2-DE with Flamingo Pink
staining for the trophozoite proteome, of which 52 protein spots were positively identified by MS/MS,
resulting in a total of 125 protein spots identified (out of 140 analyzed) in the late ring and trophozoite
proteomes (Fig. 2, Table 2 A and B). These results confirmed the high MS success rate (90%) that was
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achieved by applying the optimized methodology to the analyses of the Plasmodial proteome. The
identified proteins all had significant MASCOT scores, at least 5 peptides identified, and sequence
coverage of at least 10% each (Table 2 A and B). Of the 73 proteins spots identified in the ring stage
proteome, 57 proteins spots were from Plasmodial origin, and consisted of 41 unique Plasmodial protein
groups, where some groups contained multiple isoforms of the same protein. Therefore, protein
isoforms represented 28% (16 isoforms) of the total number of Plasmodial protein spots identified. The
trophozoite proteome consists of 52 protein spots identified by MS of which 49 protein spots were from
Plasmodial origin. Of these, 29% (14 protein spots) accounted for isoforms from the 35 unique
Plasmodial protein groups. From this data, it is clear that protein isoforms are prominent within both the
ring and trophozoite stages and may play an important role in Plasmodial protein regulation. Similarly,
this has also been demonstrated on 2-DE proteome maps for other protozoan parasites that also
highlighted the importance of isoform detection and PTM’s that regulate protein function39-41. The
significance of isoforms is further exemplified in a 2-DE proteomic study of T. brucei where the
absence of a single protein isoform was associated with drug resistance42.
Comparison of the positively identified proteins groups from the ring (41 Plasmodial proteins) and
trophozoite (35 Plasmodial proteins) stage proteomes to those of the schizont stage proteome (24
Plasmodial proteins)8 revealed only 9 proteins (~9%) which were shared between all three stages. These
include proteins involved in a variety of biological processes such as glycolysis, protein folding,
oxidative stress and the cytoskeleton. Nineteen (19) proteins are shared between the ring and
trophozoite stage whilst only 11 proteins were shared between the trophozoite and schizont. However,
14 proteins are shared between the ring and schizont stage parasites suggesting differentiation of the
schizont stage proteins in preparation for the next round of invasion by the merozoites and the formation
of the subsequent ring stage parasites. The remaining 39% of the proteins (39 proteins, 31 proteins from
ring and trophozoite stage and 8 from schizont stage) were not shared between the different life stages
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of the parasite, consistent with stage-specific production of proteins (and their transcripts) due to tightly
controlled mechanisms within the parasite3.
Comparison of the protein levels from the ring and trophozoite proteomes to the IDC transcript profile
demonstrated distinct similarities between transcript production profiles (obtained from PlasmoDB 6.0
www.plasmodb.org)28 and protein levels (Table 2A-B). Proteins that were up-regulated from rings to
trophozoites mostly exhibited a corresponding increase in transcript level when compared to IDC data
(Fig. 3, Table 2). Enolase, S-adenosylmethionine synthase, ornithine aminotransferase, uridine
phosphorylase and disulfide isomerase all demonstrated an increase in abundance of both the transcript
and protein expression levels. Similarly, eIF4A-like helicase and ribosomal phosphoprotein P0 all
exhibited unchanged transcript and protein expression levels from ring to trophozoite stage parasites.
These results emphasize the general observation of correspondences between transcript and protein
levels in P. falciparum4. Actin-1 was one of the few exceptions in which transcript levels remained
constant from ring to trophozoite stage parasites whilst protein levels were increasing. Similarly, the
transcript levels of 2-Cys peroxiredoxin remained constant over the two time points whilst the protein
was down-regulated. This could indicate possible differential regulation of these proteins at a posttranscriptional/translational level.
Of the 19 identified Plasmodial proteins shared between the ring and trophozoite stages of the
parasite, several proteins appear as isoforms (Fig 4, isoforms are also marked in Fig 2 and Table 2 A-B).
Moreover, some of these protein isoforms display differential regulation from the ring to trophozoite
stages (Fig. 4). An increase in both transcript as well as protein expression levels were determined for
the four enolase and phosphoethanolamine methyltransferase isoforms and the three glyceraldehyde-3phosphate dehydrogenase isoforms. The transcript levels of pyruvate kinase (2 isoforms) increased over
the specified period, but the protein expression levels for both isoforms declined. The transcript levels
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for both triosephosphate isomerase (2 isoforms) and eIF4A (2 isoforms) remained constant during this
period but the corresponding proteins increased in abundance. For glutamate dehydrogenase (3
isoforms) the transcript level decreased but the protein level remained constant from the ring to the
trophozoite stages. Unchanged transcript and protein levels were detected for eIF4A-like helicase (2
isoforms). These examples demonstrate the complexity of post-transcriptional and post-translational
regulation in the P. falciparum proteome.
Post-translational modification of proteins in P. falciparum has also been observed in the schizont
stage proteome8 similar to what has been detected within this study. Post-translational modifications of
Plasmodial proteins include at least phosphorylation43,
44
, glycosylation45-48, acetylation49 and
sulfonation50. The lateral shift of the eIF4A-like helicase isoforms in this study suggests
phosphorylation or sulfonation as potential modifications (Fig. 3)51, 52. However, only 2 isoforms of this
protein were observed in the trophozoite stage compared to 5 in the schizont stage, indicating additional
regulatory mechanisms e.g. increased phosphorylation in later stages of the parasite44 consistent with the
proposed involvement of this protein in controlling developmentally regulated protein expression.
Enolase seems to undergo post-translational modifications to produce 5 isoforms in P. yoelii, 7 isoforms
in the P. falciparum schizont stages8,
43
and 4 isoforms as described here. However, enolase
phosphorylation was not reported in the P. falciparum phospho-proteome44. Some of these enolaseisoforms have also been detected in nuclei and membranes in P. yoelii and therefore suggests
moonlighting functions including host cell invasion, stage-specific gene expression (Toxoplasma), stress
responses and molecular chaperone functions43. The biological significance of these isoforms is not yet
fully understood, but it clearly emphasizes the need for further in-depth investigations of posttranscriptional and post-translational modifications to further our understanding of the biological
regulatory mechanisms within the Plasmodial parasite.
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Concluding remarks
This is the first Plasmodial proteome study in which the 2-DE proteomic process was optimized in
detail, from sample preparation through spot identification with MS/MS. This resulted in a more
detailed description of the Plasmodial proteome due to the removal of contaminating hemoglobin
without additional fractionation steps or extraction procedures. The fluorescent stain, Flamingo Pink,
proved superior to the other stains tested and resulted in the detection of 79% of the predicted
trophozoite proteome after 2-DE and achieved exceptional protein identification by MS. The
reproducibility of the methods described here makes it highly expedient for the analysis of differentially
expressed Plasmodial proteins. The application of the optimized 2-DE method allowed the
characterization of 2-DE proteomes of the ring and trophozoite stages of P. falciparum, which showed
that some proteins are differentially regulated between these life cycle stages and included the
identification of a significant number of protein isoforms. Further analysis of the remainder of the
detected spots is ongoing. These results emphasize the importance of post-translational modifications as
regulatory mechanisms within this parasite.
Acknowledgements
Funding was provided by the National Research Foundation (NRF Grant FA2004051300055,
Thuthuka TTK2006061500031 and Prestigious Bursary to SS), the South African Malaria Initiative
(www.sami.org.za) and the University of Pretoria. Any opinions, findings and conclusions expressed in
this paper are those of the authors.
SUPPORTING INFORMATION PARAGRAPH
Supplementary table 1A. List of proteins identified by MS/MS for Colloidal Coomassie Blue
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Supplementary table 1B. List of proteins identified by MS/MS for MS-compatible silver stain
Supplementary table 1C. List of proteins identified by MS/MS for SYPRO Ruby
Supplementary table 1D. List of proteins identified by MS/MS for Flamingo Pink
This information is available free of charge via the internet at http://pubs.acs.org
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FIGURE CAPTIONS
Figure 1. Comparison of Plasmodial proteins on 2-DE gels using four different stains.
Two-hundred micrograms of Pf3D7 proteins were loaded onto 13 cm IPG pH 3-10L strips for 2-DE
analysis. After electrophoresis, the gels were stained with (A) Colloidal Coomassie Blue, (B) MS
compatible silver stain, (C) SYPRO Ruby, (D) Flamingo Pink. The number of spots was determined
using PD Quest 7.1.1 with n = 2 for each individual stain. About 39 similar spots were cut from each of
the stained gels to determine the MS efficiency. The spots that were identified are marked on the gels.
Figure 2. 2-DE of the rings and trophozoites stage P. falciparum indicating identified proteins.
2-DE of Plasmodial ring-stage proteome (A) and its master image (C) compared to the 2-DE of early
trophozoites stage proteome (B) and its corresponding master image (D). Master images were created
by PD Quest as representative of all the 2-DE gels performed for each of the time points and contains
spot information of a total of eight 2-DE gels. Plasmodial proteins are marked in white, human proteins
are marked in yellow and bovine proteins are marked in red. Isoforms are encircled with dotted lines.
The representing master images are also marked with identified proteins and all positively identified
proteins are listed in Table 1 A and B.
Figure 3. Proteins that are differentially regulated in the P. falciparum ring and trophozoite stage
proteomes.
Increased abundance is indicative of an increase in the abundance of the protein from ring to trophozoite
stage, while unchanged is indicative of proteins that did not change in abundance and decreased
abundance is indicative of a decrease in protein expression levels from the ring to the trophozoite stages.
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MAT: S-adenosylmethionine synthase, OAT: ornithine aminotransferase
Figure 4. Isoforms of proteins that are differentially regulated in the P. falciparum ring and trophozoite
stage proteomes.
The numbers are indicative of the number of isoforms per protein that were detected. Enolase, PEMT,
and G3PDH, TIM and eIF4A all increase in protein abundance from the ring to the trophozoite stage.
Pyruvate kinase decreased in protein abundance from rings to trophozoites, while glutamate
dehydrogenase and eIF4A-like helicase remained unchanged over the specified time in protein
expression levels. PEMT: phosphoethanolamine methyltransferase, TIM: triosephosphate isomerase,
G3PDH: glyceraldehyde-3-phosphate dehydrogenase.
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TABLES
Table 1. Comparative stain analysis for Plasmodial proteins analysed with 1-D as well as 2-DE SDS
PAGE. Spot detection and MS identification rates are included for each of the four different stains,
analysed on duplicate gels each (n=2).
1-D SDS PAGE MW analysis of stains
2-DE trophozoite analysis of stains
Stain
LOD (ng)
R2
(PD Quest)
for MS
by MS
Identification
success rate
(%)
CCB
Silver
SYPRO
Flamingo
Total
a
=average
25-90
10-90
1-90
1-90
0.89
0.83
0.97
0.97
126
420
235
349
1130
37
39
39
39
154
35
33
33
37
138
95
85
85
95
90a
Spots detected
Nr cut
Nr identified
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Table 2 A. List of proteins identified by tandem mass spectrometry for late rings.
Mascot
Score
MS/MSc
Seq
Da
pI
(PlasmoDB)
30862
21964
30008
30008
42022
42895
30027
28778
28620
28802
59816
59816
55808
55808
71454
45624
52647
52647
39464
95301
48989
48989
48989
48989
17791
33619
56405
53140
53140
5.18
6.65
5.91
5.91
5.27
5.41
6.1
6.63
6.65
6.63
6.95
6.95
5.56
5.56
5.37
5.48
5.68
5.68
4.49
5.28
6.21
6.21
6.21
6.21
5.42
5.1
7.12
7.48
7.48
150
540
152
146
627
573
441
531
845
320
659
425
693
1005
579
580
589
251
1135
298
313
373
414
1000
159
379
212
283
212
9
59
11
14
33
38
46
50
58
30
29
22
35
41
20
36
26
13
59
14
18
18
27
40
27
26
12
17
15
Mr (obtained)
Spot
nra
Transcript
trendb
PlasmoDB ID
Name
60
59
46
72
35
40
29
53
54
55
16
28
15
20
6
24
11
12
37
4
22
23
25
26
71
43
44
30
31
Up
↔
↔
↔
↔
Up
―
―
―
―
―
―
Up
Up
―
―
↔
↔
Up
Up
Up
Up
Up
Up
↔
↔
↔
Down
Down
PF10_0111
PF14_0368
PF10_0264
PF10_0264
PFL2215w
PF10_0289
―
―
―
―
―
―
MAL8P1.17
MAL8P1.17
―
PF14_0655
PFB0445c
PFB0445c
PF11_0098
PFL1070c
PF10_0155
PF10_0155
PF10_0155
PF10_0155
PFL0210c
PF14_0678
PF11_0165
PF14_0164
PF14_0164
20S proteasome beta subunit, putative
2-Cys peroxiredoxin
40S ribosomal protein, putative (1)
40S ribosomal protein, putative (2)
Actin-I
Adenosine deaminase, putative
Bisphosphoglycerate mutase (Homo sapiens)
Carbonic anhydrase 1 (Homo sapiens)
Carbonic anhydrase 1 (Homo sapiens)
Carbonic anhydrase 2 (Homo sapiens)
Catalase (Homo sapiens)
Catalase (Homo sapiens)
Disulfide isomerase, putative (1)
Disulfide isomerase, putative (2)
dnaK-type molecular chaperone hsc70 (Bos
Taurus)
eIF4A
eIF4A-like helicase, putative (1)
eIF4A-like helicase, putative (2)
Endoplasmic reticulum-resident calcium binding
protein
Endoplasmin homolog, putative
Enolase (1)
Enolase (2)
Enolase (3)
Enolase (4)
Eukaryotic initiation factor 5a, putative
Exported protein 2
Falcipain 2
Glutamate dehydrogenase (NADP+) (1)
Glutamate dehydrogenase (NADP+) (2)
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Down
―
Up
Up
Up
―
Down
↔
↔
―
Up
↔
↔
↔
↔
Down
Up
―
―
Up
Up
Up
Up
↔
↔
―
Up
Up
―
↔
Up
―
―
―
―
PF14_0164
PF14_0187
PF14_0598
PF14_0598
PF14_0598
PF11_0183
PF14_0078
PF08_0054
PF07_0029
―
PF10_0153
PF14_0439
PF13_0141
MAL13P1.283
PFE0585c
PFL0185c
PFF0435w
―
―
MAL13P1.214
MAL13P1.214
MAL13P1.214
PFI1105w
PF14_0077
MAL8P1.142
PFF0940c
PFF1300w
PFF1300w
PFI1270w
PF11_0313
PFI1090w
―
―
―
―
Glutamate dehydrogenase (NADP+) (3)
Glutathione s-transferase
Glyceraldehyde-3-phosphate dehydrogenase (1)
Glyceraldehyde-3-phosphate dehydrogenase (2)
Glyceraldehyde-3-phosphate dehydrogenase (3)
GTP binding nuclear protein Ran
HAP protein
Heat shock 70 kDa protein
Heat shock protein 86
Hemoglobin subunit beta (Homo sapiens)
Heat shock protein 60 kDa
Leucine aminopeptidase, putative
Lactate dehydrogenase
MAL13P1.283 protein
Myo-inositol 1-phosphate synthase, putative
Nucleosome assembly protein 1, putative
Ornithine aminotransferase
Peroxiredoxin-2 (Homo sapiens)
Peroxiredoxin-2 (Homo sapiens)
Phosphoethanolamine N-methyltransferase,
putative
(1)
Phosphoethanolamine
N-methyltransferase,
putative
(2)
Phosphoethanolamine N-methyltransferase,
putative
(3)
Phosphoglycerate
kinase
Plasmepsin 2
Proteasome beta-subunit
Putative cell division cycle protein 48 homologue,
putative
Putative pyruvate kinase (1)
Putative pyruvate kinase (2)
Putative uncharacterized protein PFI1270w
Ribosomal phosphoprotein P0
S-adenosylmethionine synthetase
Selenium binding protein 1 (Homo sapiens)
Serum albumin (Bos Taurus)
Serum albumin (Bos Taurus)
Solute carrier family 4, anion exchanger, member 1
(Homo sapiens)
53140
24888
37068
37068
37068
24974
51889
74382
86468
16112
62911
68343
34314
58506
69639
42199
46938
21918
21918
31309
31309
31309
45569
51847
31080
90690
56480
56480
24911
35002
45272
52928
71274
71274
101987
ACS Paragon Plus Environment
7.48
5.97
7.59
7.59
7.59
7.72
8.05
5.51
4.94
6.75
6.71
8.78
7.12
6.09
7.11
4.19
6.47
5.67
5.67
5.43
5.43
5.43
7.63
5.35
6.00
4.95
7.50
7.50
5.49
6.28
6.28
5.93
5.82
5.82
5.13
497
47
302
131
810
485
645
1378
1153
294
870
172
611
261
454
293
589
515
664
871
935
252
214
72
212
303
633
732
327
430
863
140
620
510
189
30
11
25
11
47
55
34
34
25
43
37
14
43
10
25
16
27
41
43
50
50
22
15
6
22
10
28
37
26
36
40
12
24
16
7
13
2
7
3
14
12
13
23
24
6
19
7
12
6
14
7
11
10
11
14
14
5
5
3
7
7
15
16
6
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48
1
70
39
62
63
45
63
8
9
―
―
↔
↔
↔
Up
Up
―
―
―
―
PFI0645w
PF14_0378
PF14_0378
PFE0660c
PFE0660c
PF13_0065
PF13_0065
Spectrin alpha chain (Homo sapiens)
Superoxide dismutase (Homo sapiens)
Translation elongation factor 1 beta
Triosephosphate isomerase (1)
Triosephosphate isomerase (2)
Uridine phosphorylase, putative (1)
Uridine phosphorylase, putative (2)
V-type proton ATPase catalytic subunit A (1)
V-type proton ATPase catalytic subunit A (2)
282024
16154
32121
27971
27971
27525
27525
69160
69160
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4.98
5.70
4.94
6.02
6.02
6.07
6.07
5.51
5.51
889
219
208
490
430
315
572
291
184
24
37
24
43
38
31
35
19
13
9
4
7
10
9
8
10
10
7
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23
24
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Table 2 B. List of proteins identified by tandem mass spectrometry for early trophozoites.
Mascot
Score
MS/MSc
Seq
Da
pI
(PlasmoDB)
21964
15558
15558
30008
30008
35972
42272
42022
42022
28620
55808
45624
45624
52647
52646
94546
94546
48989
48989
13608
56481
55928
67610
53140
33220
74382
62911
32112
23889
6.65
4.67
4.67
5.91
5.91
6.3
5.17
5.27
5.27
6.65
5.56
5.28
5.48
5.68
5.68
6.36
6.78
6.21
6.21
6.96
7.9
7.49
6.78
7.48
5.62
5.33
6.71
4.91
5.49
504
85
217
27
267
63
81
455
225
70
883
353
326
320
62
91
657
408
949
51
47
56
61
336
180
861
128
55
53
72
14
36
11
24
5
36
36
14
20
38
30
23
23
42
4
26
32
36
38
23
24
28
28
27
33
38
13
9
Mr (obtained)
Spot
nra
Transcript
trendb
PlasmoDB ID
Name
50
45
47
28
29
40
51
16
38
48
15
18
19
13
14
5
6
20
21
30
33
34
11
24
39
7
52
35
36
↔
Down
Down
↔
↔
Up
↔
↔
↔
―
Up
Up
Up
↔
↔
↔
↔
↔
↔
―
↔
↔
↔
Down
↔
↔
Up
Up
Up
PF14_0368
PFC0295c
PFC0295c
PF10_0264
PF10_0264
PF14_0036
PFL2215w
PFL2215w
PFL2215w
―
MAL8P1.17
PF14_0655
PF14_0655
PFB0445c
PFB0445c
PF14_0486
PF14_0486
PF10_0155
PF10_0155
PFD0615c
PF11_0165
PF11_0165
PF14_0341
PF14_0164
PF10_0325
PF08_0054
PF10_0153
PF11_0069
PF14_0138
2-Cys peroxiredoxin
40S ribosomal protein S12, putative (1)
40S ribosomal protein S12, putative (2)
40S ribosomal protein, putative (1)
40S ribosomal protein, putative (2)
Acid phosphatase, putative
Actin-1 (1)
Actin-1 (2)
Actin-1 (3)
Carbonic anhydrase 1 (Homo sapiens)
Disulfide isomerase precursor, putative
eIF4A (1)
eIF4A (2)
eIF4A-like helicase, putative (1)
eIF4A-like helicase, putative (2)
Elongation factor 2 (1)
Elongation factor 2 (2)
Enolase (1)
Enolase (2)
Eryhrocyte membrane protein 1 (fragment)
Falcipain 2 (1)
Falcipain 2 (2)
Glucose-6-phosphate isomerase
Glutamate dehydrogenase (NADP+)
Haloacid dehalogenase-like hydrolase, putative
Heat shock 70 kDa protein
Heat shock protein 60 kDa
Hypothetical protein
Hypothetical protein
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45
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48
23
17
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2
3
22
37
38
41
42
49
26
43
46
12
31
22
8
9
27
44
Up
Down
↔
Up
↔
↔
↔
Up
Up
Up
Up
Up
↔
Down
↔
―
Up
↔
Up
―
―
↔
Up
MAL13P1.23
7MAL8P1.95
PF14_0324
PF13_0141
MAL13P1.56
MAL13P1.56
MAL13P1.56
PFF0435w
MAL13P1.21
4MAL13P1.21
4MAL13P1.21
4MAL13P1.21
4
PF11_0208
PF14_0076
PF14_0716
PFL0590c
PFF1300w
PF11_0313
PFI1090w
―
―
PFI0645w
PF14_0378
Hypothetical protein MAL13P1.237
Hypothetical protein MAL8P1.95
Hypothetical protein, conserved
Lactate dehydrogenase
M1 family aminopeptidase (1)
M1 family aminopeptidase (2)
M1 family aminopeptidase (3)
Ornithine aminotransferase
Phosphoethanolamine N-methyltransferase,
putative (1)
Phosphoethanolamine
N-methyltransferase,
putative
(2)
Phosphoethanolamine N-methyltransferase,
putative (3)
Phosphoethanolamine
N-methyltransferase,
putative
(4)
Phosphoglycerate mutase, putative
Plasmepsin-1
Proteosome subunit alpha type 1, putative
P-type ATPase, putative
Putative pyruvate kinase
Ribosomal phosphoprotein P0
S-adenosylmethionine synthetase
Serum albumin (Bos Taurus)
Serum albumin (Bos Taurus)
Translation elongation factor 1 beta
Triosephosphate isomerase
42475
37933
66415
34314
126552
126552
126552
46938
31043
31043
31309
31309
28866
51656
29218
135214
56480
35002
45272
71274
71274
32121
27971
Page 28 of 40
7.14
4.13
6.63
7.12
7.3
6.68
7.3
6.47
5.28
5.28
5.28
5.28
8.3
6.72
5.51
6.13
7.5
6.28
6.28
5.82
5.82
4.94
6.02
574
385
66
100
102
124
107
637
69
261
177
722
401
540
268
54
101
121
480
466
822
488
183
37
25
7
12
26
26
27
29
9
26
22
48
36
35
31
18
51
13
32
24
36
35
22
13
8
4
3
23
25
23
12
2
6
5
13
10
12
6
16
16
3
10
15
21
9
6
Proteins identified are sorted alphabetically according to name with isoforms grouped together and the number of isoforms per protein is marked
in brackets.
a
Spot number corresponds to marked spots on the master image of ring stage parasites.
Trend of transcripts regulation from 16-20 HPI as acquired from the IDC database (http://malaria.ucsf.edu/comparison/index.php) for each of
the identified proteins. (↔) indicates unchanged transcript levels and (―) is indicative that result is not applicable.
b
c
Mascot scores are based on MS/MS searches and is only taken when the score is significant (p<0.05).
d
e
Sequence coverage is given by Mascot for detected peptide sequences.
Matched is the number of peptides matched to the particular protein
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SYNOPSIS
An optimized 2-DE method was established that enabled the detection of a large number of the
trophozoite proteome and achieved a 95% identification success rate by MS. Subsequently, this
methodology was applied to the Plasmodial ring and trophozoite proteome that allowed the positive
identification of 125 protein spots. The existence of various isoforms within the Plasmodial proteome
were identified that may have significant biological importance within the Plasmodial parasite.
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Figure 1. Comparison of Plasmodial proteins on 2-DE gels using four different stains.
Two-hundred micrograms of Pf3D7 proteins were loaded onto 13 cm IPG pH 3-10L strips for 2-DE
analysis. After electrophoresis, the gels were stained with (A) Colloidal Coomassie Blue, (B) MS
compatible silver stain, (C) SYPRO Ruby, (D) Flamingo Pink. The number of spots was determined
using PD Quest 7.1.1. About 39 similar spots were cut from each of the stained gels to determine
the MS efficiency. The spots that were identified are marked on the gels.
178x148mm (600 x 600 DPI)
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Figure 2. 2-DE of the rings and trophozoites stage P. falciparum indicating identified proteins.
2-DE of Plasmodial ring-stage proteome (A) and its master image (C) compared to the 2-DE of early
trophozoites stage proteome (B) and its corresponding master image (D). Master images were
created by PD Quest as representative of all the 2-DE gels performed for each of the time points
and contains spot information of a total of eight 2-DE gels. Plasmodial proteins are marked in white,
human proteins are marked in yellow and bovine proteins are marked in red. Isoforms are encircled
with dotted lines. The representing master images are also marked with identified proteins and all
positively identified proteins are listed in Table 1 A and B.
178x171mm (600 x 600 DPI)
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Figure 3. Proteins that are differentially regulated in the P. falciparum ring and trophozoite stage
proteomes.
Increased abundance is indicative of an increase in the abundance of the protein from ring to
trophozoite stage, while unchanged is indicative of proteins that did not change in abundance and
decreased abundance is indicative of a decrease in protein expression levels from the ring to the
trophozoite stages. MAT: S-adenosylmethionine synthase, OAT: ornithine aminotransferase
85x51mm (600 x 600 DPI)
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Figure 4. Isoforms of proteins that are differentially regulated in the P. falciparum ring and
trophozoite stage proteomes.
The numbers are indicative of the number of isoforms per protein that were detected. Enolase,
PEMT, and G3PDH, TIM and eIF4A all increase in protein abundance from the ring to the trophozoite
stage. Pyruvate kinase decrease in protein abundance from rings to trophozoites, while glutamate
dehydrogenase and eIF4A-like helicase remained unchanged over the specified time in protein
expression levels. PEMT: phosphoethanolamine methyltransferase, TIM: triosephosphate isomerase,
G3PDH: glyceraldehyde-3-phosphate dehydrogenase.
85x47mm (600 x 600 DPI)
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