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.

All experiments were performed on Shimadzu LC-20A HPLC with a Venusil XBP-C₈(T) protein column (250 mm×4.6 mm,5 μ m,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 from 10% to 60% in 50 min. UV detection: 214 nm.

One-step ATPE solution was prepared by mixing 500 μ L 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 4000 r/min for 10 min,the top phase (PEG-rich) and bottom phase (salt-rich) were formed. In addition,500 μ L saliva was diluted with water to 2.500 g as the control sample.

Two-step ATPE solution was prepared by adding 25% (mass ratio) NaH₂PO₄ into 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 4000 r/min for 10 min,and then new top and bottom phases of the two-step ATPE were formed.

Fig. 1

Fig.1 Scheme of two-step ATPE solution preparation

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

According to our previous results ^{[ 12 ]},when solution A changed from 30% to 60% in 30 min,saliva control sample showed nine component peaks in chromatogram. However,when solution A changed from 10% 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).

Fig. 2

Fig.2 Chromatograms of saliva protein from one-step ATPE HPLC conditions: Venusil XBP-C8(T) protein column (250 mm×4.6 mm,5 μ m,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.

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.

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 and 8) 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.

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

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

Fig. 4

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.