two - dimensional polyacrylamide gel electrophoresis ( 2-de ) is the most widely used method for comparison of quantitative changes in the protein proles of cells , tissues or whole organisms ( herbert et al . ,
2-de is capable of resolving hundreds , and in some cases thousands , of proteins in a single separation procedure .
one of the major limitations of 2-de methods in proteomics is the fact that low - abundance proteins are rarely seen in traditional 2-d maps , whilst most proteins in vivo are present at low abundance .
these low - abundance proteins obviously perform very important roles and include transcription factors , signal transduction proteins and receptors
. this limitation of 2-de methods could be due either to the presence of overwhelming quantities of abundant soluble proteins which obscure the detection of low - abundance proteins or to the fact that these low - abundance proteins may not be completely solubilized prior to 2-de .
the most critical step in any proteomics study is protein extraction and sample preparation ( rose et al . , 2004 ) .
better solubilization increases the separation and resolution of gels allowing identification of a higher number of proteins and more accurate quantification of differences in gene expression .
plant tissues , when compared to animal tissues , often contain lower protein concentrations and extraction is often rendered difficult by the presence of interfering compounds , such as secondary metabolites , phenolic compounds , lipids , nucleic acids , the cell wall and storage polysaccharides
. some of these compounds may interact with proteins and give poor resolution and high background in 2-de gels .
phenol extraction methods have been developed to overcome some of these problems , but this method may reduce protein yield , is time consuming and requires the use of toxic phenol .
several publications detailing soybean proteomics are available , with the majority of these publications addressing changes in the proteome of seeds , roots , nodules and hypocotyls ( mooney et al . , 2004 ; hajduch et al . , 2005 ;
, 2007 ; arai et al . , 2008 ; oehrle et al . , 2008 ; brechenmacher et al . , 2009 ;
, 2010 ; komatsu et al . , 2010 , 2011 ; zhang et al . , 2011 ) .
some of these results are also contained in the soybean proteome database ( sakata et al . , 2009 ) .
proteomic analyses of soybean leaf , on the other hand , are scarce . in the soybean database , only one gel is available , which presents poor resolution and sequencing of only 17 peptides ( one peptide per spot ) via edman degradation .
krishnan and natarajan ( 2009 ) , using a phenol extraction method , have reported a fractionation technique using 10 mm ca and 10 mm phytate to precipitate rubisco from soybean leaf soluble protein extract , and identified only 52 proteins .
( 2010 ) , using a tca / acetone method , reported the identification of 54 proteins in leaves under heat stress , the majority of these proteins being chaperones .
no published work exists where different methods are compared or optimized for analysis of the leaf proteome in soybean , and despite the great number of proteins expressed in leaves , the results together do not allow identification of more than 100 proteins in this organ . here
we report an improved method for soybean leaf proteome analysis and the identification of 122 leaf proteins , 72 of which , based on published articles and the soybean proteomics web site , have not previously been detected in leaves .
the tca / acetone protein extraction method was chosen for optimization ( shen et al . , 2002 ) .
around 0.2 g of insoluble pvp ( pvpp ) was added to 1 g fresh weight of frozen leaves and this mixture was ground under liquid nitrogen . immediately before thawing , 2 ml of a modified extraction buffer was added ( tris - hcl 40 mm ( ph 7.5 ) 250 mm sucrose , 10 mm edta , 1% triton x-100 , 1 mm pmsf ; 1 mm dtt ; 2% ( v / v ) -mercaptoethanol ) and extraction was performed for 2 h at 4 c .
cellular debris was removed by centrifugation at 6,000  g for 15 min at 4 c , and a second centrifugation of the supernatant was performed to completely remove any insoluble components .
the precipitation of soluble proteins was performed by adding trichloroacetic acid ( tca ) to a final concentration of 10% ( w / v ) in cold acetone for 16 h at 20 c .
the protein pellet was harvested by centrifugation at 6,000  g for 15 min at 4 c .
the sediment was washed 4 times with cold acetone ( 10 ml each ) , and a final wash with ethanol 80% was carried out to remove the excess acetone and to improve the further steps of solubilization of the pellet .
the pellet was dried by vacuum centrifugation at room temperature , and re - suspended in a modified 2-de buffer ( 7 m urea , 2 m thiourea , 2% chaps , 100 mm dtt and 2% ipg buffer ( ph 47 , ampholytes ) ) .
complete solubilization was achieved by sonication with three pulses of 15 s at 1500 hz .
protein concentration was determined by the bradford method ( bradford , 1976 ) , using bsa to construct a calibration curve .
the first - dimension ief was performed using 24 cm linear ipg strips ( ph 47 ) in the ipgphor system ( ge healthcare ) .
all ipg strips were rehydrated for 16 h with 250 l of rehydration buffer ( 8 m urea , 2% chaps , 0.5% ampholytes , 0.002% bromophenol blue ) containing 1 mg of protein .
voltage settings for ief were 250 v for 1 h , 500 v for 1 h , followed by a gradient from 10007000 v until achievement of a total of 5.2 kvh , followed by running at 8000 v to achieve 40 kvh .
the focused strips were either electrophoresed runned immediately on a 2-d gel or stored at 80 c until further use .
for the 2-d gel electrophoresis , the gel strips were incubated with equilibration buffer 1 [ 50 mm tris - hcl ( ph 8.8 ) , 6 m urea , 30% glycerol , 2% sds , 0.002% bromophenol blue , 1% dtt ] and equilibration buffer 2 [ 50 mm tris - hcl ( ph 8.8 ) , 6 m urea , 30% glycerol , 2% sds , 0.002% bromophenol blue , 2.5% iodoacetamide ] for 15 min each and subsequently placed onto 12.5% polyacrylamide gel ( 26  32 cm ) with a tris - glycine buffer system as described by laemmli ( 1970 ) .
strips were overlaid with agarose sealing solution ( 0.25 m tris base , 1.92 m glycine , 1% sds , 0.5% agarose , 0.002% bromophenol blue ) using an ettan daltsix electrophoresis system ( ge healthcare ) .
the initial 2d eletrophoresis setting was 5 w ( constant and maximal 20 ma ) , followed by a separation run for 6 h using 12 w per gel ( constant and maximal 50 ma ) .
the 2d - page gels were visualized by staining with colloidal coomassie blue g-250 as described by newsholme et al .
the gels were fixed overnight in 50% ethanol and 3% phosphoric acid and then washed three times for 30 min with distilled water .
gels were pre - stained for 1 h in 34% methanol , 17% ammonium sulfate and 3% phosphoric acid and then stained for 2 days in the same solution containing coomassie blue g-250 ( 0.066% ) .
image acquisition was done using an imagescanner iii ( ge healthcare ) and images were analyzed using imagemaster 2d platinum v. 7 software ( ge healthcare ) .
protein spots were excised from the stained gels and washed first with distilled water to remove ammonium sulfate and then with 50% acetonitrile containing 25 mm ammonium bicarbonate to destain the gel plugs .
the gel plugs were dehydrated with 100% acetonitrile , dried under vacuum , and then re - swollen with 20 l of 10 g / ml trypsin ( modified porcine trypsin , sequencing grade , promega , madison , wi , usa ) in 25 mm ammonium bicarbonate .
the resulting tryptic fragments were extracted by sonication in 50% acetonitrile and 5% trifluoroacetic acid .
maldi - tof - ms analysis of tryptic peptides was performed using a bruker ultraflexiii spectrometer .
samples were co - crystallized with chca matrix and spectra acquired with 40 shots of a 337 nm nitrogen laser operating at 20 hz .
protein identification was done by searching the national center for biotechnology information ( ncbi ) non - redundant database using the mascot search engine and a probability - based scoring system ( perkins et al . , 1999 ) .
the following parameters were used for database searches with maldi - tof peptide mass fingerprinting data : monoisotopic mass , 25 ppm mass accuracy , trypsin as digesting enzyme with one missed cleavage allowed , carbamidomethylation of cysteine as a fixed modification , oxidation of methionine , n - terminal pyroglutamic acid from glutamic acid or glutamine according to variable modifications . for database searches with ms / ms spectra ,
the following parameters were used : average mass ; 1.5 da peptide and ms / ms mass tolerance ; peptide charge of + 1 , + 2 or + 3 ; trypsin as digesting enzyme with 1 missed cleavage allowed ; carbamidomethylation of cysteine as a fixed modification ; oxidation of methionine , n - terminal pyroglutamic acid from glutamic acid or glutamine according to variable modifications . for maldi - tof - ms data to qualify as a positive identification , a protein s score had to equal or exceed the minimum significant score ( perkins et al . , 1999 ) .
positive identification of proteins by ms / ms analysis required a minimum of two unique peptides , with at least one peptide having a significant ion score .
to evaluate the effect of the modifications to the tca / acetone method , protein extractions were carried out using leaves and roots of soybean plants collected five weeks after germination . the phenol - base method ( wang et al . , 2003 ) , the original tca / acetone method and the modified tca / acetone method described here were tested using three biological replicates ( three different extracts for each method from three different plants ) .
an example of the 2-de gels using 1 mg protein extract is shown in figure 1 .
as evidenced in figure 1 a and c , the phenol - based method resulted in a higher level of subunits of the enzyme ribulose 1,5 bisphosphate carboxylase oxygenase ( rubisco ) .
this result is important for attempts to help improve the detection of low - abundance proteins in 2-de gels of leaf protein . in leaves
, rubisco represents around 50% and 30% of total protein in c3 and c4 plants respectively ( feller et al . , 2008 ) .
this high abundance hinders proteomic assessment of low - abundance proteins ( xi et al . , 2006 ) and masks the ability of neighboring proteins to be detected ( corthalis et al . , 2000 ;
it also significantly limits the dynamic resolution of the gel ( herman et al . , 2003 ) .
krishnan and natarajan ( 2009 ) described a method using a tca / acetone extraction procedure together with a phytic acid treatment to deplete rubisco in soybean leaf 2-de gels .
however , the number of proteins detected by these authors was in fact lower than that obtained when using the unmodified tca / acetone method ( 511 spots versus 582 when using the phenol method ) .
these results suggest that additional changes to the protocol , besides rubisco depletion , are needed to increase the number of detectable proteins .
in contrast to the situation with leaf tissue , the unmodified tca / acetone method increases the number of spots detected in roots ( 634 spots using the phenol method versus 705 ) .
these differences support the inference that different sample preparation methods are required to produce optimal 2-de results from different plant organs .
the effect of the modifications to the tca / acetone method can be observed in figure 2 .
a comparison between figures 2a and 2b indicates that the modifications made to the tca / acetone method improved both the solubilization of proteins and gel resolution , as indicated by the increase in spot number by 57% and 5.4% in leaf and root respectively ( in leaves , 802 spots in modified method versus 511 , and 743 to 705 in roots ) .
no significant differences in the content of the large and small rubisco subunits were observed with the modification of this method .
these results ilustrate that significant increase in protein detection in 2d gels could be obtained by increase in solubilizatio besides no changes in rubisco content .
particularly interesting is the increase in low molecular weight proteins in leaves resulting from the introduced modifications .
these differences were reproducible and seen in all three repetitions for each method ( data not shown ) .
effects of the modified method on the root proteome were also observed ( figures 2c , d ) , although the improvement was not as clear as that observed for leaf tissue . in roots ,
the number of spots also increased with the modifications made to the method , but the problem of low resolution in the region of low pi / higher molecular weight still remained , despite the improvement in resolution for higher pi proteins and the fact that more proteins were detected overall .
the improvements in the resolution of 2-de gels for leaf tissue ( figure 3 ) were reflected by the successful identification of 122 proteins , based on the peptides matched in the ncbi protein bank , as described in table 1 .
figure 3 also shows that proteins coding for the large subunit of rubisco are separated into several spots around the 53 kda region of the gels .
although several additional proteins could potentially be identified , we used a mowse score of 40 as a minimum confidence threshold for identification in this study .
this score varied from 44 to 175 across identified proteins , with an average of 80 in this dataset ( a score greater than 67% means identification is significant at p < 0.05 ) , while the number of peptide matches / protein identified varied between 4 and 27 ( average of 11 ) , and the sequence coverage varied between 13% and 100% ( average of 35% ) .
almost 50% of the identified proteins corresponded to proteins with unknown function , these representing the largest group .
proteins involved in primary metabolism represented the second largest group , which includes genes involved in photosynthesis , respiration and biosynthetic metabolism , the latter mainly represented by enzymes involved in amino acid biosynthesis .
the fact that a transcription factor and some proteins involved in signaling were present in the small part of the leaf proteome analyzed indicates that some low - abundance proteins could be quantified using the 2-de method presented herein . for yeast ,
the most abundant proteins are present at around 2,000,000 copies per cell , which represents 4% of total protein , whereas the least abundant proteins , which likely includes transcription factors and signaling proteins ( futcher et al . , 1999 ) , are present at around 100 copies per cell ( a difference of four orders of magnitude ) .
low - abundance proteins are generally considered difficult or even impossible to detect using 2-d gel electrophoresis ( grg et al . , 2004 ; krishnan and natarajan , 2009 ) , while genes of unknown function typically encode low - abundance proteins .
taken together , these facts are indicative that the modifications made to the tca / acetone method allow increased detection of proteins expressed at low levels .
based on published results and those available in the soybean proteomic database that describe soybean leaf proteins ( krishnan and natarajan , 2009 ; sakata et al . , 2009 ; ahsan et al . ,
2010 ) , we were able to add 72 new proteins to the soybean 2-de profile , the majority of these being proteins of low abundance .
 the most critical step in any proteomic study is protein extraction and sample preparation . 
 better solubilization increases the separation and resolution of gels , allowing identification of a higher number of proteins and more accurate quantitation of differences in gene expression . despite the existence of published results for the optimization of proteomic analyses of soybean seeds , no comparable data are available for proteomic studies of soybean leaf tissue . in this work 
 we have tested the effects of modification of a tca - acetone method on the resolution of 2-de gels of leaves and roots of soybean . 
 better focusing was obtained when both mercaptoethanol and dithiothreitol were used in the extraction buffer simultaneously . increasing the number of washes of tca precipitated protein with acetone , using a final wash with 80% ethanol and using sonication to ressuspend 
 the pellet increased the number of detected proteins as well the resolution of the 2-de gels . using this approach 
 we have constructed a soybean protein map . 
 the major group of identified proteins corresponded to genes of unknown function . 
 the second and third most abundant groups of proteins were composed of photosynthesis and metabolism related genes . 
 the resulting protocol improved protein solubility and gel resolution allowing the identification of 122 soybean leaf proteins , 72 of which were not detected in other published soybean leaf 2-de gel datasets , including a transcription factor and several signaling proteins . 
Introduction
Material and Methods
Results and Discussion