the human placenta is unique among species . as a matter of fact , of all mammalian organs
the placenta shows the greatest variation in terms of anatomy . however , in all species it has the same fundamental function , to nourish the growing fetus , by establishing contact with the maternal blood circulation .
in addition , the placenta acts as a protective barrier ; as an example it prevents stress hormones to pass over to the fetus via active transport . in order to prevent rejection of the fetus
, the placenta expresses an intricate pattern of major histocompatibility complex molecules , immunizing the mother against the foreign fetal tissue . during pregnancy
the placenta also has several important endocrine functions , producing important hormones such as human chorionic gonadotropin and human placental lactogen [ 4 , 5 ] .
hence , due to its vast number of functions the placenta expresses more than 20 000 dna sequences and is perhaps the organ expressing the largest number of genes .
in fact , it has been used as a  fishing pond  for finding and sequencing new genes and has been used to identify and clone transporters and receptors , such as human growth hormone - variants , integrin alpha v ,   and subtypes of the angiotensin receptors [ 79 ] .  
it affects 3%7% of all pregnancies   and is a major cause of maternal and fetal mortality .
treatment for pe is symptomatic , and removal of the placenta is the only curative treatment , suggesting that the placenta is a key in the development of pe .
the etiology of pe is still largely unknown ; however , it is generally accepted that pe begins with inadequate placentation .
invasion of placental trophoblast cells into the maternal spiral arteries is too shallow , leading to inadequate placental perfusion and hypoxia [ 11 , 12 ] .   proteomics is a research field undergoing rapid development , especially in regards to the methods being used .
the most common method is two - dimensional polyacrylamide gel electhrophoresis ( 2d - page ) .
2d - page for proteome studies gives a map of proteins which reflect changes in protein expression levels , as well different isoforms and posttranslational modifications .
lately , three types of protein microarrays have become available , namely , analytical microarrays , functional microarrays , and reverse phase microarrays ( reviewed in ) .
highly sensitive and specific recombinant antibody microarrays allowing screening of crude protein extracts are also being developed . due to the large number of proteins ,
no protein array platform has yet been designed that covers the entire human placental proteome .
thus , when screening the placental proteome 2d - page is still among the most accepted methods giving the highest coverage although this may soon change .
however , there are several obstacles when working with placental tissue , since it contains large amounts of lipids and glycogen . in order for the first dimension , isoelectric focusing ( ief ) , to be successful
, proteins have to be efficiently extracted , and solubilized , and contaminants , such as lipids , polysaccharides , nucleic acids , and salt ions , must be removed .
lipids , polysaccharides and nucleic acids bind to proteins which increases the viscosity of the samples and often results in horizontal streaking on the 2d - page .
if the salt concentration is too high in the sample , the conductivity in the ipg strips increases and prolongs the ief , resulting in poor 2d - page resolution .
for example , precipitating samples with dichloromethane / methanol has been described to remove lipids whereas precipitation with ethanol will remove nucleic acids . for these reasons ,
the placenta proteome is not fully investigated although some progress has been made in the use of proteomics technology to study pe placenta [ 17 , 18 ] .  
the primary aim of this study was to establish a robust sample preparation protocol of human placental tissue , by comparing two different solubilization solutions in combination with six different precipitation methods .
sixty women admitted at the department of obstetrics and gynecology , lund university hospital , were included and assigned to two groups : pe ( n = 30 ) and control ( n = 30 ) ( table 1 ) .
g / l or rise in blood pressure above 20 mmhg from the first trimester of pregnancy .
a 10 mm cube of placenta tissue was collected immediately after removal of the placenta and was frozen on dry ice and stored at   80c .
this study was approved by the ethical committee review board for studies in human subjects at lund university .
human placental tissues were pulverized , and the frozen placental powder ( 25 mg frozen weight ) was homogenized with a lysis solution containing either 3 ml 8 m urea and 2% chaps or 3 ml ice cold 10 mm hepes , ph 7.5 , 24 mm kcl supplemented with a protease inhibitor cocktail ( complete mini plus edta , roche , basel , switzerland ) .
samples were stirred for 1 hour followed by centrifugation at 43 000 g for 2 hours .
protein concentrations were determined by the bicinchoninic acid ( bca ) method ( thermo scientific , rockford , usa ) with bovine serum albumin ( bsa ) as a standard . pooled samples were prepared by combining equal amounts of proteins from individual samples .
the pooled samples were either used directly ( fresh samples ) or stored at   80c prior to use ( frozen samples ) .
30 g were precipitated , and for small and large 2d - page , 100 g and 500 g were precipitated , respectively .
acetoneextracted samples were mixed with ice - cold acetone to final concentration of 80% acetone .
samples were incubated overnight at   20c followed by centrifugation at 9000 g for 2 minutes
. the supernatant was removed , and the protein pellets were allowed to air dry . extracted samples were mixed with ice - cold acetone to final concentration of 80% acetone .
samples were incubated overnight at   20c followed by centrifugation at 9000 g for 2 minutes .
the supernatant was removed , and the protein pellets were allowed to air dry . acidified acetonesamples were precipitated as above , but the acetone was acidified with acetic acid to a final concentration of 0.05% .
samples were precipitated as above , but the acetone was acidified with acetic acid to a final concentration of 0.05% .
samples were centrifuged as above , and the protein pellets were allowed to air - dry .
samples were centrifuged as above , and the protein pellets were allowed to air - dry .
briefly , one volume of sample was mixed with 4 volumes of methanol , 2 volumes of dichloromethane , and 3 volumes of distilled water .
the samples were centrifuged 9000 g for 2 minutes at room temperature ( rt ) .
the supernatant was discarded , and 3 volumes of methanol were added followed by mixing .
the samples were centrifuged at 9000 g for 2 minutes and the supernatant was again discarded after which pellets were air dried .
briefly , one volume of sample was mixed with 4 volumes of methanol , 2 volumes of dichloromethane , and 3 volumes of distilled water .
the samples were centrifuged 9000 g for 2 minutes at room temperature ( rt ) .
the supernatant was discarded , and 3 volumes of methanol were added followed by mixing .
the samples were centrifuged at 9000 g for 2 minutes and the supernatant was again discarded after which pellets were air dried .
trichloroacetic acid ( tca)one volume of 40% tca was added to one volume of samples and incubated for 1 hour on ice followed by centrifugation at 9000 g for 2 minutes .
the supernatant was removed , and the protein pellets were either directly dried or washed with ethanol and then allowed to air dry .
the dried pellets were either dissolved in 30 l sodium dodecyl sulphate ( sds ) sample buffer ph 8.5 ( 247 mm tris , 2% sds , 0.5 mm edetate ( edta ) , 16.5 mm dithiothreitol ( dtt ) , 10% glycerol , and 0.025% ( w / v ) bromophenol blue ) followed by 1d - page or dissolved in rehydration sample buffer containing 8 m urea , 2% chaps , 10 mm dtt , and 2% 310 nl ampholines ( ge healthcare , little chalfont , uk ) .
one volume of 40% tca was added to one volume of samples and incubated for 1 hour on ice followed by centrifugation at 9000 g for 2 minutes .
the supernatant was removed , and the protein pellets were either directly dried or washed with ethanol and then allowed to air dry .
the dried pellets were either dissolved in 30 l sodium dodecyl sulphate ( sds ) sample buffer ph 8.5 ( 247 mm tris , 2% sds , 0.5 mm edetate ( edta ) , 16.5 mm dithiothreitol ( dtt ) , 10% glycerol , and 0.025% ( w / v ) bromophenol blue ) followed by 1d - page or dissolved in rehydration sample buffer containing 8 m urea , 2% chaps , 10 mm dtt , and 2% 310 nl ampholines ( ge healthcare , little chalfont , uk ) .
samples ( 30 g ) were loaded onto 10-well 4%12% nupage bis - tris gels ( invitrogen , carlsbad , usa ) and run at 200 v for 45 minutes using 2-(n - morpholino ) ethanesulfonic acid ( mes ) as running buffer .
the first dimensional isoelectric focusing ( ief ) was performed on a horizontal multiphor ( amersham biosciences , usa ) .
immobilized ph gradient ( ipg ) blue native strips 310 nl ( 0.5  3  70 mm or 0.5  3  240 mm ) ( serva electrophoresis gmbh , heidelberg , germany ) were in - gel rehydrated in rehydration sample buffer over night . for 7 cm strips , 130 l ( containing 100 g of proteins ) and for 24 cm strips , 450 l ( containing 500 g of proteins ) rehydration sample buffer were used .
the focusing was performed at the following voltage gradients : 150 v for 30 minutes , 300 v for 30 minutes , 600 v for 30 minutes , 1500 v for 1.5 hours , 3000 v for 2 hours ( 7 cm strips ) and 25 hours ( 24 cm strips ) .
the strips were equilibrated for 10 minutes in a solution containing 65 mm dtt , 6 m urea , 30% ( w / v ) glycerol , 2% ( w / v ) sds and 50 mm tris - hcl ph 8.8 .
a second equilibration step was performed for 10 minutes in the same solution except for dtt , which was replaced by 259 mm iodoacetamide .
the strips were soaked in electrophoresis buffer ( 24 mm tris base , 0.2 m glycine and 0.1% sds ) just before the second dimensional gel electrophoresis .
the strips were applied on 12% homogeneous tris - glycine gels ( 0.1  8  8 cm or 0.1  20  24 cm ) and overlaid with a solution of 1% agarose in electrophoresis buffer ( kept at 60c ) .
electrophoresis was carried out in an xcell mini - cell apparatus ( invitrogen ) using electrophoresis buffer at 125 v for 1.5 hours or in an hoefer dalt gel apparatus ( ge healthcare ) at 20c and constant 80 v for 19 hours .
gel staininggels were either stained with silver   or stained with sypro ruby ( thermo scientific ) .
gels were either stained with silver   or stained with sypro ruby ( thermo scientific ) .
image analysissmall gels were scanned using a canoscan 995of ( canon , solna , sweden ) , and large gels were scanned using an amersham typhoon 9400 ( ge healthcare ) .
the images of the large gels stained with sypro ruby were submitted to ludesi 2d analysis ( ludesi , malmo , sweden , http://www.ludesi.com ) for spot detection , matching , and analysis .
small gels were scanned using a canoscan 995of ( canon , solna , sweden ) , and large gels were scanned using an amersham typhoon 9400 ( ge healthcare ) .
the images of the large gels stained with sypro ruby were submitted to ludesi 2d analysis ( ludesi , malmo , sweden , http://www.ludesi.com ) for spot detection , matching , and analysis .
the purified peptides were eluted directly onto the sample target ( anchorchip target , bruker daltonik gmbh , bremen , germany ) where 0.7 l of matrix , 2,5-dihydroxybenzoic acid ( 10 mg / ml in 30% acn ) had been added .
mass spectra of positively charged ions were recorded on a bruker reflex iii instrument ( bruker daltonik ) operated in the reflector mode .
the xmass 5.0 and ms biotools software packages provided by the manufactures were used for data processing .
the purified peptides were eluted directly onto the sample target ( anchorchip target , bruker daltonik gmbh , bremen , germany ) where 0.7 l of matrix , 2,5-dihydroxybenzoic acid ( 10 mg / ml in 30% acn ) had been added .
mass spectra of positively charged ions were recorded on a bruker reflex iii instrument ( bruker daltonik ) operated in the reflector mode .
the xmass 5.0 and ms biotools software packages provided by the manufactures were used for data processing .
database searchingfor protein identification , human protein sequences in the swissprot database were searched using the mascot software ( matrix science ltd .
parameters specified in the search were taxa , homo sapiens ; missed cleavages , 1 ; peptide mass tolerance + /0.1 da ; fixed modification , carbamidomethyl ( c ) ; variable modification , oxidized methionine . for protein identification , human protein sequences in the swissprot database were searched using the mascot software ( matrix science ltd . ,
parameters specified in the search were taxa , homo sapiens ; missed cleavages , 1 ; peptide mass tolerance + /0.1 da ; fixed modification , carbamidomethyl ( c ) ; variable modification , oxidized methionine .
proteins were separated by 1d - page using 10-well 12% nupage bis - tris gels ( invitrogen ) according to manufacturer 's instruction .
the gels were loaded with 2.5 g protein per well and run for 60 minutes at 200 v in mops running buffer under reduced conditions and blotted onto polyvinylidene fluoride ( pvdf ) membranes for 8 minutes at 23 v using an iblot dry blotting system ( invitrogen ) .
membranes were incubated with primary antibody for 1 hour ( mouse monoclonal anti - apoa1 , sc-69755 , 1 : 2000 dilution ) , followed by washes and then , incubated with the secondary antibody for 1 hour ( goat polyclonal antimouse igg , sc-2031 , 1 : 5000 dilution ) .
antibodies were obtained from santa cruz ( santa cruz biotechnology , santa cruz , usa ) .
subsequently , the membrane was exposed to enhanced chemilumeniscence ecl+ ( ge healthcare ) for 3 minutes .
autoradiographic film hyperfilm ecl ( ge healthcare ) was applied to the blot for various durations to obtain satisfactory exposure . when developed , films were scanned with ultra violet light using a gene flash scanner ( syngene , cambridge , uk ) and imported into syngene gene tools .
one reference sample was added to all gels to serve as an internal calibrator to normalize the quantification between gels .
a students t - test was used to determine statistical difference between the two groups .
immunohistochemistry was performed using envision+system - hrp ( dakocytomation , carpinteria , usa ) on 4 representative pe samples and 4 controls according to the manufacturer 's instructions .
briefly , fresh frozen sections , 12 m thick , of the placenta samples were fixed by immersion in 4% phosphate buffered saline ( pbs ) buffered formaldehyde for 15 minutes at rt .
sections were incubated in a blocking solution containing 3% h2o2 for 10 minutes at rt .
the apoa1 antibody was diluted ( 0.1 g / ml ) in pbs containing 0.25% triton x100 and 1.5% normal goat serum .
peroxidase labeled polymer was applied to cover the specimens and then incubated for 30 minutes .
after rinsing the sections , liquiddab substrate chromogen solution was applied and incubated for 8 minutes .
the sections were rinsed and stained with mayers hematoxylin after which sections were mounted with mountex ( histolab , gothenburg , sweden ) and cover slipped .
sections were viewed under an olympus bx-60 microscope and images captured using an olympus dp50cu digital camera ( olympus america inc . , center valley , usa ) .
two solubilization solutions were chosen : hepes buffer   and the commonly used urea / chaps .
the pellet resulted in smearing on 1d - page ( figure 1(a ) ) and without the centrifugation step ; no proteins entered the ief - strip resulting in a blank 2d - page ( data not shown ) .
there were no obvious differences in protein patterns between the supernatant and the starting material ( figure 1(a ) ) .
1d - page separation was used to determine protein expression from the two different solubilization solutions . a major difference in the protein pattern between hepes
minor differences at higher molecular mass range are also obtained when comparing the two different solubilization solutions ( figures 1(b ) and 1(c ) ) .
a number of the protein bands that visibly differed between the two solubilization solutions were identified using maldi - tof ms .
the obtained identities included annexin a1 , annexin a2 , endoplasmin , vimentin , and hemoglobin subunit alpha and gamma ( table 2 ) .
some of the bands from the 1d - page contained more than one identity due to the limited power of resolution using 1d - page .  
six different precipitation procedures were tested : acetone , acidified acetone , ethanol dichloromethane / methanol , tca , and tca followed by ethanol wash . to determine the effects of precipitation procedures on protein patterns , precipitated samples
were analyzed on 1d - page gels ( figures 1(b )   and 1(c ) ) .
samples precipitated with tca , with or without ethanol wash , showed a different protein expression profile compared to the other four precipitation methods .
no significant differences in the 1d - page expression profiles were detected between the other four precipitation procedures ( acetone , acidified acetone , ethanol and dichloromethane / methanol ) .  
samples solubilized with urea / chaps generally resulted in more protein spots and less horizontal streaking , regardless of precipitation procedure ( figure 2 ) .
the investigation of whether freezing of solubilized and centrifuged samples influenced the results , both fresh and frozen samples , was analyzed using 2d - page .
no major differences could be detected in the protein expression profiles for any of the different solubilization or precipitation methods ( data not shown ) .
an increased number of low molecular weight proteins were obtained with samples solubilized in urea / chaps precipitated with acidified acetone ( figure 2 , a5 ) .
these spots were not obtained with other combinations of solubilizations and precipitations ( figure 3 ) .
electroendosmotic flow ( eof ) was detected during the ief run with samples precipitated with acetone , acidified acetone , or ethanol .
this resulted in swelling of the ipg - strip in the alkaline ph gradient and might cause a poor protein separation at an isoelectric point above 8 .  
urea / chaps in combination with either acidified acetone or dichloromethane / methanol precipitation was judged to be the most efficient extraction methods for 2d - page .
however , we chose dicholoromethane / methanol since it is known to remove lipids of which there is abundance in the placenta . in a first attempt to investigate whether it is possible to detect differences between the pe and normal placenta in crude extracts , we pooled crude placenta extracts from 30 preeclamptic and 30 normotensive women respectively . in order to decrease the variation of the differently expressed proteins on the 2d - page , three replicates of each pooled group
the number of spots detected was 604 and 747 for preeclamptic and normotensive , respectively .
the number of spots was calculated based on the criteria that the spots were present in all triplicate gels in each group .
the analysis of the 2d - page showed that 23 proteins were increased and 28 were decreased in the pe placenta ( figure 4 ) .
two proteins have so far been identified with maldi - tof ms : apolipoprotein a1 ( apoa1 ) ( acc number : p02647 ) and tropomyosin-3 ( tpm3 ) ( acc number : p06753 ) .
apoa1 was 1.63 times increased in the pe placenta pool as compared to the control pool ( pe : 10871  1340 optical density ( od ) units and controls : 6663  1772 od units values presented as mean      sd ) .  
the increased accumulation of apoa1 in the pe placenta was validated by means of western blot ( figure 5(a ) ) .
after measuring the absorption of all the apoa1 bands , apoa1 was found to be 1.18 times increased in pe ( pe : 63120  14350 optical density ( od ) units and controls : 52810      13310 od units values presented as mean      sd ) .
the accumulation of apoa1 was significantly increased in pe ( p = 0.02 ) ( figure 5(b ) ) .
both normotensive and pe placenta sections showed a similar pattern with weaker staining in the control sections ( figures 5(c)5(e ) ) .
the tpm3 expression could not be validated by means of western blot since the recommended antibodies did not work in our hands ( sc-32516 and sc-18174 , santa cruz biotechnology , santa cruz , usa ) .
due to the vast number of functions and many different cell types besides the trophoblasts , including white and red blood cells and endothelial cells , the placenta is a complex organ to study .
in order to optimize the protein sample preparation for 2d - page , we compared two different solubilization solutions in combination with six different precipitation methods in order to obtain maximal separation of the proteins .
hepes buffer and urea / chaps showed different protein patterns in the low molecular mass range .
although no protease inhibitors were added to the urea / chaps solution , urea denatures and inactivates proteases and thus , it is unlikely that the differences are due proteolytic cleavage .
several steps in the sample preparations appear to be important to remove contaminating substances . during pregnancy
there is a gradual accumulation of glycogen as well as a large amount of lipids in the placenta .
glycogen and lipids are known to disrupt ief - separation by blocking the ipg - strip resulting in no or very few spots on 2d - page .
the centrifugation did not cause a selective loss of proteins , indicating that most of the placental proteins remained in the supernatant .
the freezing of samples directly after solubilization ( without the centrifugation step ) resulted in insoluble aggregates .
this observation may facilitate the work flow in proteomics where many samples are analyzed , and the samples can be stored in the freezer until further use without having a major influence on the results .  
our optimization procedures showed that solubilizing with urea and chaps and precipitating with dichloromethane / methanol or acidified acetone gave the highest number of spots on a 2d - page .
solubilization with hepes buffer in combination with dichloromethane / methanol also gave a high number of spots ( figure 2 , b1 ) , although there was considerably more horizontal streaking .
dichloromethane / methanol has the advantage or also removing lipids , and since the placenta is rich in lipids , this precipitation method may be the preferred choice .
hence , in a first study urea / chaps in combination with dichloromethane / methanol was chosen to examine the differences between the pe and normotensive placental proteome .  
the differentiating proteins have proven to be hard to identify due to the low concentration of the proteins in the differing spots .
the same problem was highlighted in a recent preeclamptic proteomic study , where , as a consequence of low amount of proteins , none of the differentially expressed proteins could be identified .
however , we have so far been able to identify two proteins differing between pe and controls : apoa1 ( increased in pe ) and tpm3 ( decreased in pe figure 4 ) .
the increase of apoa1 in pe was validated by western blot and immunohistochemistry ( figure 5 ) . due to the difficulties in identifying low abundance proteins in a crude extracted sample
, one might have to do purifications of specific cell types , for example , trophoblast to explore the proteome of the placenta .
another limitation with 2d - page is that membrane proteins do not easily enter ief , the first dimension since they often precipitate in the ipg - strip . in our first attempt to study differences between pe and normotensive placenta , we found apolipoprotein a1 , a lipid binding protein , to be accumulated in the pe placenta . of the lipoproteins ,
apoa1 is mainly synthesized in the liver and intestines and is the major fraction of the high - density lipoproteins .
the placenta has also been suggested as a site of synthesis for apoa1 although it is not clear whether this is an actual de novo synthesis or an accumulation from the blood stream . in a recent whole genome microarray analysis ,
it is unlikely that the accumulation of apoa1 in the pe placenta is due to high levels of apoa1 in the maternal plasma since plasma levels apoa1 are not increased in pe [ 26 , 27 ] . in a recent proteomics study of amniotic fluid , accumulation of pro - apoa1 was shown in the amniotic fluid in pe .
hence , amniotic fluid may be a possible source of the accumulated apoa1 seen in the pe placenta .
apoa1 may server either as nutrient or as a signaling molecule by affecting biological signaling pathways such as protein kinase c dependent pathway ( pkc and the mitogen - activated protein kinases ( mapk ) [ 29 , 30 ] .
in conclusion , two solubilization methods in combination with six different precipitation procedures were tested .
removal of glycogen by centrifugation is crucial since the glycogen clogged the ipg - strip .
both the solubilization solution and the precipitation procedure affected the placenta proteome maps . in this study ,
solubilization with urea / chaps in combination with dichloromethane / methanol or acidified acetone proved to be the best precipitation procedures , resulting in more spots and less streaking on 2d - page .
importantly , freezing samples following centrifugation of the solubilized samples did not affect the proteome map .
however , due to the limitation using crude extracts for 2d - page , low abundance proteins can not readily be detected , and thus subcellular fractioning may be required to fully investigate the placenta proteome .
 the human placenta is a difficult tissue to work with using proteomic technology since it contains large amounts of lipids and glycogen . 
 both lipids and glycogen are known to interfere with the first step in the two - dimensional polyacrylamide gel electrophoresis ( 2d - page ) , the isoelectric focusing . in order to gain the best possible protein separation on 2d - page , an optimized sample preparation protocol for placental proteins 
 was developed . 
 two different buffers , urea / chaps and hepes , were used for solubilization in combination with six different precipitation methods . 
 the removal of glycogen from the samples by centrifugation was crucial for the final proteome maps . 
 solubilization with urea / chaps in combination with dichloromethane / methanol or acidified acetone proved to be the best precipitation procedures . 
 when applied to clinical placenta samples apolipoprotein a1 was found to be accumulated in the preeclamptic placenta , where it may either have a nutritional effect or act as a modifier of signal transduction . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion