趙新穎， 屈 鋒， 覃 浩， 羅愛芹

（1.北京理工大學(xué)生命學(xué)院，北京100081；2.北京市理化分析測試中心，北京100089；3.中國科學(xué)院遺傳與發(fā)育生物學(xué)研究所，北京100101）

Aqueous two－phase extraction（ATPE），a liquidliquid extraction method with advantages of simple operation，low cost，easy amplification and mild condition for keeping protein activity，has recently been applied in purification，separation and enrich－ment of proteins［1－7］as well as in proteomics research［8－13］.The separation of saliva proteins in 15% （mass ratio）polyethylene glycol（PEG）－4000／8% （mass ratio）NaH2PO4［12］and human serum proteins in15% （mass ratio）PEG－4000／10% （mass ratio）（NH4）2SO4［6］were reported previously by our group，in which HPLC was employed to analyze the protein distribution in PEG－rich top phase and inorganic salt－rich bottom phase.The ATPE employed in the above reports was based on onestep extraction，by which some proteins in complex samples could not be separated well.Therefore，the two－step ATPE combined with HPLC for saliva protein separation was studied in this work.With the optimization of HPLC gradient，more saliva protein information was obtained.For convenient discussion，protein peaks were divided into groups and sections based on the retention time on the HPLC column and the distribution in top and bottom phases，respectively.Moreover，saliva protein distributions between one－step and two－step ATPE were compared.The preliminary results show that two－step ATPE combined with HPLC presents a possibility for multi－dimensional selective separation of complex samples.

1 Experimental

1.1 Reagents and materials

PEG－4000，NaH2PO4（analytical grade）were purchased from Beijing Chemical Reagent Co.，Ltd.（Beijing，China）.Acetonitrile（ACN，analytical grade）and trifluoroacetic acid（TFA，chromatographic grade）were supplied by Fisher（USA）and MREDA Technology Co.，Ltd.（Beijing，China），respectively.Deionized water was purified by Millipore Milli－Q system.Saliva samples were collected from a volunteer in our laboratory.

1.2 HPLC experimental conditions

All experiments were performed on Shimadzu LC－20A HPLC with a Venusil XBP－C8（T）protein column（250 mm×4.6 mm，5μm，30 nm）supplied by Agela Technologies（Tianjin，China）.The eluent consists of solution A（ACN containing 0.1% （v／v）TFA）and solution B（deionized water containing 0.1% （v／v）TFA）.A linear gradient was applied by changing the ratio of solution A from10%to 60%in 50 min.UV detection：214 nm.

1.3 ATPE solution preparation and analysis

One－step ATPE solution was prepared by mixing 500μL saliva，0.375 g PEG－4000 and 0.125 g NaH2PO4，then complementing by the deionized water to 2.500 g.After the solution was centrifuged at 4 000 r／min for 10 min，the top phase（PEG－rich）and bottom phase （salt－rich） were formed.In addition，500μL saliva was diluted with water to 2.500 g as the control sample.

Two－step ATPE solution was prepared by adding 25% （mass ratio）NaH2PO4into the top phase from one－step ATPE with the volume ratio of 1∶5，and 0.200 g PEG－4000 into 1 mL bottom phase from one－step ATPE （Fig.1）.The two solutions were centrifuged at 4 000 r／min for 10 min，and then new top and bottom phases of the two－step ATPE were formed.

All the above phases were analyzed by HPLC.The protein group and amount were determined by the retention time and peak area respectively.

Fig.1 Scheme of two－step ATPE solution preparation

2 Results and discussion

2.1 Saliva protein separation

According to our previous results［12］，when solution A changed from30%to 60%in 30 min，saliva control sample showed nine component peaks in chromatogram.However，when solution A changed from10%to 60%in 50 min，ACN content increased slowly and more single peaks or split and cluster peaks appeared comparing with the control sample，which means the improvement of protein separation with solution polarity increasing.In addition，the saliva proteins with polarity difference could be separated with higher resolution（Fig.2）.

Since the silica particles in column are of 30 nm pore size，the protein larger than 30 nm is excluded without retention.Therefore the protein peak in chromatogram indicates that its size is less than 30 nm.The smaller its size is，the longer its retention time will be.

Therefore，saliva protein retention on the chromatographic column is depended on its hydrophobic property and molecular size.For convenient discussion，protein peaks in chromatograms are divided into groups and sections based on their retention times.

Fig.2 Chromatograms of saliva protein from one－step ATPEHPLC conditions：Venusil XBP－C8（T）protein column （250 mm×4.6 mm，5μm，30 nm）.Sections：a.9.01－19.00 min；b.19.01－23.00 min；c.23.01－30.00 min；d.30.01－37.00 min；e.37.01－39.00 min；f.39.01－50.00 min.

2.2 Saliva protein partition in one－step ATPE

Saliva protein from one－step ATPE was divided into six sections based on the retention time in two phases（Fig.2）.Section a distributed only in the bottom phase and Section c only in the top phase，while the other sections were in both phases except peak 8，which merely in the top phase.The split protein peak 4（Section b）was larger in the bottom phase than in the top.Reversely，the peaks 6，7 in Section d and peak 9 in Section e were obviously larger in the top phase than in the bottom.The peak 10（Section f）showed nearly equal peak areas in the top and bottom phases.

Protein partition in ATPS is the result of the surface hydrophobic and electrostatic interaction as well as the static repulsion.There are multiple interactions between protein and PEG－rich top phase as well as phosphate－rich bottom phase.The protein that is prone to be in the top phase might have stronger hydrophilic property and more surface negative charges.It is excluded by phosphate as well as with the compatibility in hydrophilic PEG－rich top phase.The protein that prefers to be in the bottom phase might have stronger hydrophobic property and more net surface positive charges.It is easier to be attracted by phosphate in the bottom phase and excluded by hydrophilic PEG in the top phase.Above all，the proteins（such as peaks 5 and8）with hydrophilic property and more surface negative charges are totally in the PEG－rich phase，while proteins in Section a with hydrophobic property and more surface positive charges transfer to phosphate－rich phase，which causes the results that proteins extracted in either top phase or bottom phase can be separated completely by the primary ATPE and other proteins distributed in both phases do not achieve separation.

2.3 Saliva protein partition in two－step ATPE

The top and bottom phases in the one－step ATPE were used as the new samples to perform further two－step ATPE separately.Some proteins exhibited redistribution in the second step ATPE，which in new top phase（Fig.3）and bottom phase（Fig.4）showed difference obviously.Comparing with the top phase in one－step ATPE，peaks existed in the new top phase did not change.Yet，only peaks 9 and 10 existed in new bottom phase.The area of peak 9 in the top was larger than in the bottom，and the areas of peak 10 were equal in the two phases.Comparing with the bottom phase in one－step ATPE，a new peak（peak 11）appeared before peak 10，which implied that more protein peaks might be obtained by two－step ATPE.Meanwhile，the split peak 4 became a single peak and transferred completely to the new top phase and peak 6 transferred to the bottom phase.However，peak 3 was always kept in the bottom phase.

Fig.3 Chromatograms of two－step ATPE for saliva protein in primary top phase

Fig.4 Chromatograms of two－step ATPE for saliva protein in primary bottom phase

Results of one－step and two－step ATPE show that some proteins can be separated completely in the first ATPE，for example peaks 1，2，3 and 5，which exist in either bottom or top phase.The remained components（e.g.peaks 4，6，7）can be separated completely in the following ATPE.Hence，some proteins existing in top or bottom phase can be separated completely by ATPE，but others distributing in two phases can not be separated.

3 Concluding remarks

Due to the differences of hydrophobic，hydrophilic，charge property as well as molecular size，proteins in saliva can be selectively separated by two steps ATPE combined with specific protein LC column.Elution optimization in HPLC achieves higher resolution of saliva proteins.Some proteins can be separated by one－step ATPE，and others have to be separated by two－step ATPE.The combination of two－step ATPE and HPLC provides a new way to achieve multiple－dimensional selective separation，which might be a useful method for protein sample separation，purification and proteomics research.

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