the development of analytical tools for rapid analysis and identification of expressed protein profiles in cell , tissue or organism is currently an important area in biological research [ 13 ] . although two - dimensional gel electrophoresis ( 2de ) is a classical technique that monitors and distinguishes multiple forms of proteins with differences in molecular mass or pi values , it can face difficulties with proteins of extreme mass ( e.g. , > 200  kda or < 10  kda ) or pi values [ 46 ] . in addition , 2de is not readily amenable to automation . liquid - phase separation methods such as size - exclusion chromatography , affinity chromatography , and ion - exchange chromatography exhibited practical difficulties due to the lack of the isoelectric ( pi ) information and limited labeling efficiency [ 79 ] . alternatively , the proteomelab pf2d platform ( beckman coulter , usa ) that can be used for the separation / fractionation , as well as quantitative comparisons of various biological and clinical samples , works in full automation combining chromatofocusing separation and hydrophobic fractionation . during the first - dimension chromatofocusing of pf2d , proteins are separated by their pi and separated proteins with a ph gradient are collected using a fraction collector [ 11 , 12 ] . subsequently , fractions collected from the first dimension are separated using reversed phase chromatography in the second dimension , which separates on the basis of hydrophobicity . separated fractions are monitored with uv detection to observe changes in the proteome [ 1315 ] . although pf2d system offers high loading capacity and improved detection limit with lower abundance proteins [ 16 , 17 ] , its protein recovery efficiency during the chromatofocusing step will be low when the standard protocol recommended by the manufacturer is used . reported that the inclusion of 20% isopropanol in the isoelectric focusing ( ief ) buffer increased the number of proteins they could identify in the serum . they demonstrated improved recovery of protein , but purified bsa was used instead of complete serum . this buffer 's ability to improve the recovery of all proteins thus remains unclear . the columns used with pf2d require the use of nonionic detergents such as triton x-100 for the separation of proteins . triton x-100 is a low - cost mixture of octylphenol ethoxylates , with an average of about 9 - 10 ethylene oxide units per molecule . in search of an alternative method that can increase the recovery of a wide range of proteins , buffers recommended by beckman coulter 's proteomelab pf2d protocol was replaced by triton x-100 during protein profiling of escherichia coli k12 , in which its recovery efficiency was determined at various triton x-100 concentrations . subsequently , the elution accuracy of triton x-100 at its optimized condition was confirmed by the control protein mixture of ribonuclease a , insulin , -lactalbumin , trypsin inhibitor , and cholecystokinin ( cck ) . the eligibility of triton x-100 as a replacement for buffers used for proteomelab protein liquid chromatography was determined with escherichia coli k12 substrain w3110 and control mixture of proteins ( ribonuclease a , insulin , -lactalbumin , trypsin inhibitor , and cholecystokinin ( cck ) ) . e. coli k12 was cultivated at 37c in 200  ml lb medium ( 1% bacto tryptone , 0.5% bacto yeast extract , 0.5% nacl , ph 7.2 ) , to an od600 of 1.5 . the cells were harvested by centrifugation at 6000  g for 10  min at 4c and washed in 10  mm tris - hcl buffer ( ph 8.0 ) . after being resuspended in 10  ml of ice - cold lysis buffer ( 7  m urea , 2  m thiourea , 4% 3-[(3-cholamidopropyl ) dimethylammonio]-1-propanesulfonate ( chaps ) , 65  mm dithiothreitol ( dtt ) , 1.25  mm protease inhibitor ) , cells were sonicated on ice 30 times for 10  s each . obtained cell lysates were centrifuged at 6000  g for 10  min at 4c , and the final protein concentration in the supernatant was determined using the bradford protein assay ( biorad , usa ) . proteomelab pf2d protein test mix , which included 5 proteins ( ribonuclease a , insulin , -lactalbumin , trypsin inhibitor , and cholecystokinin ( cck ) ) , was purchased from beckman coulter ( ca , usa ) . before the chromatofocusing , cell extracts were exchanged to various start buffers using a pd-10 column ( ge healthcare life sciences , usa ) , and the first 3.5  ml fraction was collected . while the start buffer included in the proteomelab kit was designated as the  start buffer a , start buffer b , c , and d  were prepared with the following : 0.1% , 0.15% , and 0.2% triton x-100 in distilled water ( emd chemicals , inc . , usa ) at ph 8.4 for  b ,   c ,  and  d ,  respectively ; 6  m urea ; 25  mm bis - tris ; 1  m ammonium hydroxide . protein concentration was estimated using quant - it protein assay kits ( invitrogen , usa ) . all samples were diluted with each start buffer to obtain a final protein concentration of 1.5  mg / ml , and 2  ml of e. coli protein was injected into the chromatofocusing column . all protein samples were filtered through 0.2  m pes membrane filters ( millipore , usa ) . the chromatofocusing was performed using the proteomelab pf2d ( beckman - coulter , usa ) with an hpcf-1d column ( 250  mm  2.1  mm , eprogen , usa ) that was loaded with each starting buffer ( ph 8.5  0.1 ) for 120  min . each starting buffer was then equilibrated to the initial ph 8.5 , and protein sample was loaded at a flow rate of 0.2  ml / min for 45  min . the protein sample elution was initiated with a linear gradient of various elution buffers ( ph 4.0  0.1 ) that took ~60  min to complete . eluent buffer included in the proteomelab kit was labeled as  eluent buffer a.   eluent buffer b , c , and d  were prepared with the following : 0.1% , 0.15% , and 0.2% triton x-100 in distilled water at ph 4.0 , respectively ; iminodiacetic acid ; 6  m urea ; 10% v / v polybuffer 74 ( ge healthcare , usa ) . proteins were eluted and collected by their isoelectric point ( pi ) in 4.08.5 range with 0.2  ph intervals into a 96-deep - plate well using the fc / i module . remaining protein samples were finally eluted by washing the column with 1  m nacl for 40  min . the column was then rinsed with 10 column volumes of distilled water before the next sample injection . the entire chromatofocusing step was operated at 20c with a flow rate of 0.2  ml / min , and elution profiles were monitored at 280  nm by beckman 166  uv detector ( beckman coulter , usa ) . and the second - dimension separation was performed using hprp column ( 33  mm  4.6  mm , 1.5  m nonporous ods - iiie c18 silica beads , eprogen , usa ) at 50c with a flow rate of 0.75  ml / min . a 200  l from the first chromatofocusing fraction was injected into the column and eluted with a 0100% linear gradient of solvent a ( 0.1% w / v tfa in distilled water ) and solvent b ( 0.08% w / v tfa in acetonitrile ) for 35  min . at the end of second - dimension run , proteins were detected by a beckman 166  uv detector ( beckman coulter , usa ) at 214  nm . protein profiles obtained using uv detection were analyzed by proteovue 2d ( beckman coulter , usa ) . to investigate the elution accuracy of 0.15% triton x-100 , the mixture of five proteins purchased from beckman coulter ( usa ) was injected into the hpcf-1d column that allows the elution of proteins in ph range of 4.08.5 . the first- and second - dimension protein separations were achieved following the standard procedure described in section 2.2 using start buffer c ( ph 8.4 ) and eluent buffer c ( ph 4.0 ) . protein profile data obtained using uv detection were collected and analyzed by proteovue 2d software ( beckman coulter , ca , usa ) . the eligibility and efficiency of triton x-100 as an alternative to the buffers suggested for proteomelab ( beckman coulter , california , usa ) pf2d were evaluated with e. coli , using the standard proteomelab buffer as well as solutions prepared with 0.1% , 0.15% , and 0.2% triton x-100 . the first - dimension chromatofocusing separates proteins to differences in pi values , and the absorbance profiles created by each solution are shown in figure 1 . proteins were eluted in the order of decreasing pi values , and all four solutions eluted its first peak during the first 20  min of sample loading period . as the hpcf-1d column used in this study provides limited elution efficiency with proteins whose pi values are in 8.54.0 range , the first peak corresponds to protein unbound to hpcf-1d column because of its pi value being greater than 8.5 . the details of protein separation and ph gradient formation varied among four solutions . at first , e. coli chromatofocusing was performed using the buffer suggested for beckman coulter 's proteomelab platform . as shown in figure 1(a ) , first unbound protein was eluted during the first ~20  min , and the ph gradient started forming at 50  min . the ph gradient which started forming at 50  min ( from ph 8.1 ) lasted until 105  min ( to ph 3.5 ) , with a slight downward angle created at 92  min . during the ph gradient acidic proteins which remained in hpcf-1d column were eluted after 130  min as a result of washing the column with 1  m nacl . under given conditions , beckman coulter 's standard triton x-100 were prepared and used in place of standard buffers to perform first - dimension protein profiling of e. coli . when the solution that included 0.1% triton x-100 was used the first protein peak created in ~20  min was inverted , giving a negative au280 reading ( figure 1(b ) ) . this peak was followed by another inverted peak that was created during 4858  min . when ph gradient formed during 50130  min , proteins of low pi values ( 5.0 > pi ) were eluted indistinctively . after 130  min , remnant protein in hpcf-1d column was eluted with column washing and created a large peak area . figure 1(c ) of 0.15% triton x-100 showed significantly improved protein profiling results : an unbound protein was eluted at < 20  min , small amounts of proteins were eluted during 2060  min ( ph 8.57.74 ) while the sample was being loaded , and a ph gradient was created during 60110  min ( ph 7.743.95 ) . unlike 0.1% triton x-100 , 0.15% triton x-100 created a linear ph gradient , an indication that the capacity is even throughout its ph range . chromatofocusing that took place over the ph gradient finally , 0.2% triton x-100 was used to determine whether increasing the concentration of triton x-100 would bring further improvements for chromatofocusing of e. coli . interestingly , after the first unbound protein was eluted at < 20  min , no significant protein elution was observed until the start of the ph gradient at 50  min ( figure 1(d ) ) . as the ph gradient for 0.2% triton x-100 was created over the shortest time frame ( during 5080  min , ph 7.113.89 ) , a high volume of proteins were eluted abruptly during ph gradient thus creating unreliable profiling data ( figure 1(d ) ) . the performance of each buffer was judged upon its ability to achieve a well - defined ph gradient as well as an accurate pi - based protein separation at a given ph gradient range . indistinct chromatofocusing results and negative au280 readings made 0.1% triton x-100 inadequate for pf2d . while the performance of 0.15% triton x-100 was comparable to that of standard buffers suggested for proteomelab pf2d , 0.2% triton x-100 created a protein profile whose pi values were dubious as it happened in a shorter time frame . however , au280 readings of 0.15% and 0.2% triton x-100 were as much as 9 - 10 times higher than those of beckman coulter 's . such high au280 readings obtained using 0.15% triton x-100 were investigated further during the second - dimension separation that was performed on 16 first - dimension protein fractions generated using the standard and 0.15% triton x-100 solution . second - dimension of pf2d fractionates in the order of increasing hydrophobicity . as shown in figure 2(a ) , 16 protein fractions in ph 7.923.95 obtained using beckman coulter buffers were fractionized , and a thick protein band indicating a large quantity of proteins was seen around ph 5.014.70 . the second - dimension result of 0.15% triton x-100 that began from 16 first - dimension fractions ( ph 7.743.95 ) showed distinctive bands for wider pi ranges . this novel methodology that utilizes 0.15% triton x-100 enhances protein recovery efficiency by at least tenfold . first- and second - dimension chromatography results shown in this study confirmed protein recovery can be increased in the presence of 15% triton x-100 during pf2d analysis of e. coli , but its reliability with regard to accurate pi separation is yet to be judged without comparing the results with the standard proteins whose pi values are known . once again , the elution profiles of five - protein test mixture ( ribonuclease a , insulin , -lactalbumin , trypsin inhibitor , cholecystokinin ( cck ) ) were generated using 0.15% triton x-100 ( figure 3(a ) ) . multiple peaks were eluted in high resolution during its ph gradient . the accuracy of such protein elution was to be determined using the second - dimension protein chromatogram with a mixture of 5 proteins whose theoretical pi values are known ( figure 3(b ) ) . the theoretical pi value of ribonuclease a is pi > 8.5 but the actual elution took over pi 8.1 - 8.0 ( shown with the protein band e in figure 3(b ) ) , possibly due to a limited ph elution range set by the hpcf-1d column . the elution intervals of insulin , -lactalbumin , trypsin inhibitor , and cck ( shown by protein bands d , c , b , and a , resp . ) were similar to their theoretical ranges . the detailed comparison of the experimental elution intervals of five control proteins with regard to theoretical values is summarized in table 1 . triton x-100 is a common nonionic surfactant , and the experimental results of this study affirmed that 0.15% triton x-100 can be applied towards pf2d of a protein . not only can 0.15% triton x-100 greatly increase amount of protein recovery from chromatofocusing column , but it also enables pf2d analysis of protein with low pi . combining the beneficial qualities mentioned thus far , 0.15% triton x-100 for pf2d system can be exploited for further analyses of metaproteome originating from various sources .