Novel Prefractionation Method Can Be Used in Proteomic Analysis Haixin Bai,†,‡ Fan Yang,† and Xiurong Yang*,† State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Renmin Street 5625, Changchun, Jilin 130022, China, and Graduate School of The Chinese Academy of Sciences, Beijing 100039, China Received October 25, 2005
For the first time, a novel prefractionation method used in proteomic analysis was developed, which is performed by a novel aqueous two-phase system (NATPS) composed of n-butanol, (NH4)2SO4, and water. It can separate proteomic proteins into multigroups by one-step extraction. The phase-separation conditions of n-butanol solutions were studied in the presence of commonly used inorganic salts. The NATPS was subsequently developed. Using human serum albumin, zein, and γ-globulin as model proteins, the separation effectiveness of the NATPS for protein was studied under affection factors, i.e., pH, n-butanol volume, protein, or salt concentration. The model and actual protein samples were separated by the NATPS and then directly used for gel electrophoresis without separating the target proteins from phase-forming reagents. It revealed that the NATPS could separate proteomic proteins into multigroups by one-step extraction. The NATPS has the advantages of rapidity, simplicity, low cost, biocompability, and high efficiency. It need not separate target proteins from the phase-forming reagents. The NATPS has great significance in separation and extraction of proteomic proteins, as well as in methodology. Keywords: novel aqueous two-phase system • separation • proteomic proteins • prefractionation method
1. Introduction
used in proteome analysis, the time-consuming could not be neglected.
The aim of expression proteomics is to get comprehensive cellular protein expression profiles. However, whole cell proteomics is challenging.1 Prefractionation technologies in proteomic analysis, which could offer a strong step forward in “unseen proteome”, had been a focus of proteome researchers.2-4 Two-dimensional gel electrophoresis (2D-PAGE) has been proved over the years to be a reliable and efficient method for the separation of proteomic proteins based on charges and masses. Nevertheless, even the simplest proteome limits the resolving power of 2D-PAGE due to the complexity. This limitation can be alleviated by sample prefractionation using a variety of techniques.4-9 Prefractionation can be achieved by a number of techniques such as step extraction proteins, purification of cell organelles or protein complexes, preparative isoelectric focusing, or chromatographic techniques.5,6,10-12 Many of these procedures are time-consuming, difficult to reproduce and scale-up, result in sample loss, and often require expensive instrumentation or reagents. One-step13 and two-step14 methods for extraction biomolecules also have been reported. But they cannot be used for the whole proteomic proteins since they only can extract or separate a certain types of proteins. Although serial extraction method15 had been
Among protein-extraction techniques,16-18 aqueous twophase system (ATPS) is formed by combining either two watersoluble polymers differing in their chemical structure, or a polymer and a salt in water above a certain critical concentration.19-21 The attractiveness of the technique is its biocompability, simplicity, amenability to linear scale-up, potential for continuous operation, and wide range of hydrophobicity differences between the two-phase systems.22,23 ATPS has been widely used as protein purification and separation means24-27 since its introduction in late 1950s.28 However, its application is still not widespread in the biotechnology industry. One of the main reasons is the high cost of the phase-forming polymers. The choice of the polymer used in ATPS is mainly restricted to poly(ethyleneglycol) and dextran or hydrophobically modified starch, e.g., hydroxypropyl starch (Reppal PES 200). Difficulties in separating target proteins from the phaseforming polymers become another main bottleneck of ATPS’ application.
* To whom correspondence should be addressed. Fax: +86-431-5689711. E-mail:
[email protected]. † State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. ‡ Graduate School of The Chinese Academy of Sciences.
840
Journal of Proteome Research 2006, 5, 840-845
Published on Web 02/23/2006
Inorganic salts, water, and n-butanol were typically used in extraction of proteomic proteins. Low concentration salts can accelerate protein dissolving, while high concentration salts will cause protein deposition from solution. Water is the most primary solvent in extracting proteomic proteins since most proteins are water-soluble. As one of typically used solvents in extracting proteomic proteins, n-butanol can dissolve hydrophobic proteins and not denaturalize them. Furthermore, n-butanol-extraction method has a wide selection scope for 10.1021/pr050359s CCC: $33.50
2006 American Chemical Society
research articles
Novel Prefractionation Method
pH and temperature. It is also suitable for extracting the materials of animal, plant, or microorganism.29 Therefore, we wish to develop a prefractionation method used in proteomic analysis, which can separate proteomic proteins into multigroups by one-step extraction. It performed by a novel aqueous two-phase system (NATPS) composed of inorganic salt, water and n-butanol. In contrast to the traditional ATPS and the three-step methods,11,12 the NATPS has the advantages of simplicity, rapidity, low cost and easily separating the target protein from the forming-phase reagents. Proteins are broadly classified into several fractions according to their solubility characteristics, i.e., albumin (water soluble), globulin (soluble in dilute salt solution), prolamin (soluble in 70∼80% alcohol) and so on.29 Three types of model proteins i.e., HSA, zein, and γ-globulin, were used to study the separation effectiveness of the NATPS.
2. Materials and Methods 2.1. Materials. Human serum albumin (HSA) (Type A-1887), zein (Type Z3625), γ-globulin (Type G5009), and reagents used for gel electrophoresis were purchased from Sigma Chemical Co.. HSA solution was prepared by dissolving a given amount of HSA in water. Zein solution was obtained by dissolving it in 75% (v/v) ethanol aqueous solution. γ-globulin was dissolved in 0.01 g/mL (NH4)2SO4 solution and used as its work solution. The protein concentrations were varied from 0.1 to 3 mg/mL. Their accurate concentrations were determined by spectrophotometric method.30 The buffer solutions at pH 5.0∼5.8, pH 6.0∼7.0, pH 7.1∼8.9, and pH 9.3∼10.0 were prepared by HAcNaAc, NaH2PO4-NaOH, Tris-HCl, and Na2B4O7-NaOH buffer system, respectively. The experimentally used water was purified by Millipore Milli-Q system until its resistance reached to 18.2 MΩ. (NH4)2SO4, n-butanol and all the other reagents were analytical reagent grade. 2.2. Preparation of the NATPS. After a given amount of n-butanol being added to a tube with a 10 mL graduation, water was added until the total volume reached to 10 mL. The mixture was shaken gently and a homogeneous phase, n-butanol solution, was obtained. Then a certain amount of salt was added to the homogeneous phase and mixed by shaking. After settled for less than 1 min, the mixture separated into n-butanol rich phase (top phase) and aqueous salt phase (bottom phase) with a clear interface. In separation experiments, proteins or buffer solutions were added before the addition of water and (NH4)2SO4. The concentration of (NH4)2SO4 in each phase was determined using the formaldehyde method 2(NH4)2SO4 + 6HCHO f 2H2SO4 + (CH2)6N4 + 6H2O and the H2SO4 formed was titrated by NaOH solution with a given concentration.31 The n-butanol concentrations in two phases could be determined directly by colorimetric analysis of cerium complex36 or calculated based on the concentrations of (NH4)2SO4 and water, which concentration could be determined by sepectrophotometry.32 2.3. Determination of the Partition Coefficient (Kr). A known amount of HSA, zein or γ-globulin was added to one of the identical two-phase systems, while the other served as a blank for protein concentration determination. The samples were gently shaken in order to mix completely with the n-butanol solution. Then a given amount of (NH4)2SO4 was added. After mixing the mixture by invertion and leaving it to
settle for less than 1 min, the homogeneous phase separated into two phases. In the presence of protein precipitate, the NATPS was centrifugated at low speed for protein precipitate separating with each phase and depositing at phase interface. Protein concentration in each phase was determined spectrophotometrically with a Cary Model 50 UV-visible spectrophotometer from Varian (USA).30 Protein concentrations determined were used to calculate the partition coefficient (Kr), which was defined as the ration of protein concentrations in the top phase [P]top to that in bottom phase [P]bottom22 Kr )
[P]top [P]bottom
The percent protein recovery (R) in the bottom phase was calculated according to R(%) )
[P]bottomVbottom [P]oVo
where Vbottom, [P]o, and Vo are the bottom phase volume, the initial protein concentration and its solution volume, respectively. 2.4. Prefractionation of Protein Samples Using the NATPS. Model protein sample was prepared by mixing the model proteins. Human blood plasma and mixture of plasma with zein were selected as actual protein samples. All the protein samples were separated by the NATPS according to the methods in 2.2, respectively. After phase separated completely, a given amount of each top and bottom phase was taken out for preparing sample solutions of gel electrophoresis. The protein concentrations, in sample solutions of gel electrophoresis, were kept in the range of 50∼100 µg/mL. For the actual protein samples, 500 µL human blood plasma and a mixture of the 500 µL plasma with 500 µL zein solution (5.432 mg/mL) were separated by the NATPS. A 100-µL bottom phase solution, 200 µL top phase solution and the precipitate at phase interface were prepared to 1000 µL sample solutions for gel electrophoresis, respectively. 2.5. Gel Electrophoresis Analysis. The same gel electrophoresis method as reference 33 was used for the gel electrophoresis analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed using 1.5-mm-thick separating gel containing 10% acrylamide. The proteins were separated at 20 mA constant current until there was a 1∼1.5 cm distance from the bromophenol blue dye front to the gel bottom then postfixed for 30 min in 9.2% acetic acid/45.4% methanol. A Coomassie staining was performed with 0.05% Coomassie Brilliant Blue R-250 in the aforesaid fixing solution for 1 h. Destaining was performed in 7.5% acetic acid/5% methanol until clear electrophoretogram presented in the gel.
3. Results and Discussion 3.1. Development of the NATPS. At 20 °C, solubility of n-butanol in water is 7.7% (w/w) and that of water in n-butanol is 20.1% (w/w). Base on solubility and density data,34 it can be concluded that n-butanol can dissolve in water when its concentrations range out of 9.3∼83.1% (v/v). Considering the cost, waste disposal and separation of target proteins, the n-butanol solutions with concentrations lower than 9.3%(v/v) was selected for developing the NATPS. The results indicated that the commonly used inorganic salts could induce n-butanol solution to separate into n-butanol rich phase (top phase) and Journal of Proteome Research • Vol. 5, No. 4, 2006 841
research articles
Figure 1. Phase diagram corresponding to n-butanol solution(NH4)2SO4 system.
Figure 2. Amount of salts needed for phase separation of n-butanol with water 900 µL n-butanol was used with a 10 mL total volume before salt was added. A 1.00 g different salt was used.
aqueous salt phase (bottom phase) as salt concentrations reached to a critical value. The phase-separation reason was that the salting-out effect promoted hydrophobic interaction as well as self-association/aggregation of n-butanol,18 which caused n-butanol to separate from its solutions and hence formed a new phase. Phase diagram corresponding to nbutanol solution-(NH4)2SO4 system was shown in Figure 1 as a representative. Since n-butanol and water are partially miscible, which differ from the polymer and water in traditional ATPS, there were two segments of binodal curves for the NATPS. The tie-lines in Figure 1 indicated NATPS had similar phase compositions to the polymer + sulfate + water system.31 Increasing n-butanol volume decreased the salt needed for phase separation. On the contrary, the less n-butanol used, the more salt needed for phase separation. Keeping n-butanol volume as constant, the phase separated more thoroughly as salt concentration increased. But as the n-butanol volume was too small, phase separation would not appear even with higher salt concentration. Since too small volume of n-butanol would cause difficulties in determining the protein concentration in top phase, 900 µL n-butanol was used in developing the NATPS. The results in Figure 2 indicated that different inorganic salts had different phase-separation abilities. The more the top phase volume approximate to the added n-butanol volume (0.900 mL), the more thoroughly the phase separated. Known from Figure 2, sodium carbonate had the strongest ability of phase separation, while (NH4)2SO4 had a moderate one. Compared with other salts, (NH4)2SO4 is the most typically selected in protein extraction since its advantages such as almost independence on temperature, high solubility, good salting-out effect and hardly cause protein denaturalization.29 Thus, (NH4)2SO4 was used in developing the NATPS. 3.2. Effect of pH on the Kr of Proteins. To study the effect of pH, 900 µL n-butanol, 1000 µL protein solution, and 3000 842
Journal of Proteome Research • Vol. 5, No. 4, 2006
Bai et al.
Figure 3. Effect of salt concentration on the recovery of HSA and γ-globulin in bottom phase 900 µL n-butanol, 1000 µL protein solution and 3000 µL buffer solution were used with a 10 mL total volume before salt was added. Concentrations of HSA and γ-globulin were 0.8192 and 1.033 mg/mL, respectively.
µL buffer solution were used with a 10 mL total volume. Amount of added (NH4)2SO4 was 1.00 g. Concentrations of HSA, zein, and γ-globulin were 0.8192, 0.4060, and 1.033 mg/mL, respectively. The Kr of HSA, zein, or γ-globulin kept constant in pH 5.0∼10.0. HSA or γ-globulin was not detected in the top phase of the NATPS that indicated both types of protein could not dissolve in the top phase. So the Kr values of HSA and γ-globulin were zero. The 100% of recovery in the bottom phase proved that all HSA dissolved in the bottom phase and no protein separated out. As extracted by the NATPS, γ-globulin had a constant recovery around 30% in pH 5.0∼10.0 and part of γ-globulin precipitated. The reason was that γ-globulin could only dissolve in low concentration salt solution but had small solubility in high concentration salt solution. The Kr of zein was a constant around 120, which proved that most zein dissolved in the top phase. Even under phase-separation conditions, there was small amount of n-butanol in the bottom phase, which could be proved by evidences in Figure 2 and references 35-37. Because of this, zein had very low concentration in bottom phase. That is the reason Kr of zein had a bigger value. No effect of pH on protein Kr indicated that changing pH could not alter protein solubility in the NATPS. Since pH had no affection on the Kr or recovery, pH7.4, which is close to the physiological pH value, was selected for the further studies. 3.3. Effect of Salt Concentration on the Kr of Proteins. HSA and γ-globulin did not dissolve in the top phase. Hence each protein’s Kr was zero. But their recoveries in the bottom phase varied as the salt concentration increased (Figure 3). The recoveries of HSA approximated to 100% before the amount of (NH4)2SO4 more than 1.00 g. As the amount of (NH4)2SO4 reached to 1.50 g, small amount of HSA precipitate appeared in the top phase. HSA recoveries decreased from 95% to zero as the amount of (NH4)2SO4 increased from 1.50 to 3.00 g. The reason was that salting-out effect dominated for high concentration salt and promoted HSA to separate out, even though low concentration salt had a salting-in effect for protein. The recovery of γ-globulin increased with the addition of (NH4)2SO4 at low concentration and reached maximum 50% as 0.40 g (NH4)2SO4 was added. The recovery decreased from 50% to zero when added (NH4)2SO4 increased from 0.40 to 2.50 g. The recovery decreased to around 30% again when added (NH4)2SO4 increased to 1.00 g, which showed that most of γ-globulin separated out as precipitate under this condition. That was because that salting-in effect increased protein solubility in
Novel Prefractionation Method
Figure 4. Effect of salt concentration on the Kr of zein 900 µL n-butanol, 1000 µL zein solution (0.4060 mg/mL), and 3000 µL buffer solution were used with a 10 mL total volume before salt was added.
lower concentration salt solution, while salting-out effect decreased protein solubility in higher concentration salt solution. As shown in Figure 4, the Kr of zein had an evident decrease before the added (NH4)2SO4 reached to 1.50 g and increased slightly as added salt ranged from 1.50 to 3.50 g. These changes derived from the salt effect on the phase-separation extent and zein solubility in the bottom phase. The addition of salt caused increase in the top phase volume, which decreased the zein concentration in top phase as well as Kr value. On the other hand, salt effect also caused zein to transfer from bottom to top phase, which increased the Kr value. Before added (NH4)2SO4 more than 1.5 g, the former dominated the change of Kr and hence the Kr decreased accompanying the increase of (NH4)2SO4. After the salt amount more than 1.5 g, the latter dominated the change, which resulted in slight increase in Kr. Low concentration (NH4)2SO4 could result in a higher Kr for zein and also a 100% recovery of HSA in bottom phase, but the small volume of top phase caused difficulties in operations, e.g., determination of protein concentration or phase separation. On the other hand, high concentration salt could cause HSA and γ-globulin to deposit together. The results revealed that 1.00 g (NH4)2SO4 could extract all HSA in bottom phase, main-body part of zein in top phase and induce most γ-globulin to separate out as precipitate. Therefore, 1.00 g (NH4)2SO4 was used for the further studies. 3.4. Effect of Protein Concentration on the Kr of Proteins. Just as the effect of salt concentration, protein concentration did not affect the Kr of HSA or γ-globulin. HSA recovery was 100% in the bottom phase before it was saturated. Along with the increasing of γ-globulin concentration, its recovery increased to a maximum value and then decreased slightly, which is shown in Figure 5. The reason is that the salt effect upon the ionization of γ-globulin could increase its solubility.38 With the increase of γ-globulin concentration, more ionization interaction happened between γ-globulin and (NH4)2SO4 that increased the solubility and recovery of γ-globulin in bottom phase. In this case, the amounts of bottom phase or γ-globulin were constants and then γ-globulin solubility in the bottom phase would be a given value. At higher concentration, effect of ionization interaction for increasing the solubility became unconspicuous and γ-globulin concentration approximated to saturation. Thus at higher concentration, γ-globulin recovery in the bottom phase stopped increasing and then decreased slightly as its concentration increased. Figure 6 illustrated the effect of zein concentration on the Kr. At low concentration, zein had a relatively higher concen-
research articles
Figure 5. Effect of γ-globulin concentration on its recovery in bottom phase 900 µL n-butanol, 1000 µL γ-globulin solution, and 3000 µL buffer solution were used with a 10 mL total volume before salt was added. The added (NH4)2SO4 was 1.00 g.
Figure 6. Effect of zein concentrations on the Kr of zein 900 µL n-butanol, 1000 µL zein solution, and 3000 µL buffer solution were used with a 10 mL total volume before salt was added. The added (NH4)2SO4 was 1.00 g.
tration in top phase and a lower one in bottom phase because it preferred to dissolve in n-butanol rich phase. Furthermore, the small volume (0.5 mL) of top phase was easily saturated. Zein concentration in bottom phase had a more evidently increase corresponding to that in top phase as the concentration of added zein solution increased. Therefore, Kr of zein decreased with the increase of zein concentration. Known from the results, low concentration can result in most zein to be separated into top phase and most globulin deposited at phase interface. Thus, the NATPS has the best separation effectiveness for proteomic proteins at low concentration. 3.5. Effect of n-Butanol Volume on the Kr. Changing the volume of added n-butanol could alter the volume ratio of top phase to bottom phase and protein concentration in each phase, which would affect proteins’ Kr. The Kr of HSA or γ-globulin, was not affected by n-butanol volume. The changes in n-butanol volume had similar effects on the recoveries of HSA and γ-globulin to that of pH. Both proteins had constant recovery of 100% and around 30%, respectively. The reasons were that both HSA and γ-globulin could not dissolve in the top phase, and the change in n-butanol volume could not affect their solubility in the NATPS. The effect of n-butanol volume on the Kr of zein was shown as Figure 7. The addition of n-butanol increased the top phase volume, which decreased its protein concentration and hence resulted in decrease of Kr. On the other hand, more zein was transferred from bottom phase to top phase with the increase in n-butanol volume, which caused Kr increase. Since the former dominated the change of Kr, the Kr decreased with the increase of n-butanol volume. According to this conclusion, one can change alcohol soluble protein distribution in the two phases by altering the volume of added n-butanol. Journal of Proteome Research • Vol. 5, No. 4, 2006 843
research articles
Bai et al.
Figure 7. Effect of n-butanol volume on the Kr of zein A certain volume of n-butanol, 1000 µL protein solution (0.4060 mg/mL), and 3000 µL buffer solution were used with a 10 mL total volume before salt was added. The added (NH4)2SO4 was 1.00 g.
3.6. Separation of Protein Samples Using the NATPS. After the separation effectiveness of each model protein in the NATPS was separately studied, the model protein sample prepared by mixing the model proteins was separated using the NATPS. One-dimensional gel electrophoresis was used to separate the proteins in the top and bottom phase. The protein sample in each phase was directly analyzed by gel electrophoresis without separating proteins from phase-forming reagents (Figure 8a). As a control, the model proteins were separately analyzed by gel electrophoresis without preprocess by the NATPS (Figure 8b). Results revealed that the top and bottom phase contained different proteins, which indicated that proteins could be separated into different groups by the NATPS. The results also indicated coexisted proteins had similar behavior in the NATPS as they existed separately. Human blood plasma and mixture of plasma with zein were separated by the NATPS and then analyzed by gel electrophoresis. The Electrophoretogram (lanes labeled with Tplasma) corresponding to plasma sample in top phase indicated that there were very low content of alcohol soluble proteins in plasma. Results in Figure 8c also indicated that proteins in plasma or mixture of plasma with zein were separated into three different protein groups, i.e., water-soluble proteins (in bottom phase), alcohol soluble proteins (in top phase) and proteins deposited at phase interface. Therefore, it was concluded that the NATPS could separate proteomic proteins into multigroups by one-step extraction, which could be used as a prefractionation method for proteomic analysis.
4. Conclusion For the first time, a NATPS, which consists of n-butanol, (NH4)2SO4 and water, has been developed for separating proteomic proteins into multigroups by one-step extraction. It can be used as a prefractionation method in proteomic analysis. The whole separation process just need several minutes because of the rapid phase separation and simple operation step. Compared with other prefractionation techniques,4-12,15 this method just needed two cheap reagents (n-butanol and (NH4)2SO4). Simplicity, rapidity, and low cost are its highlight attractiveness. The NATPS has the attractiveness of traditional ATPS such as biocompability, simplicity, amenability to linear scale-up, potential for continuous operation and wide range of hydrophobicity differences between the two-phase systems.22,23 Since the NATPS is a homogeneous phase before phase separation, it realizes the extraction in homogeneous phase and separation in heterogeneous phase, which results in a best effectiveness of extraction and separation. In sum, 844
Journal of Proteome Research • Vol. 5, No. 4, 2006
Figure 8. (a) Electrophoretogram of model protein sample in top and bottom phase (b) Electrophoretogram of HSA, zein, and γ-globulin. (c) Electrophoretogram of plasma sample in top and bottom phase. In parts a and b, except each lane of T10 and B10 with a 0.5 µg protein amount, protein amounts in all the other lane are 1 µg. Lanes labeled with Tx and Bx are corresponding to the protein samples in the top and bottom phase, respectively.
the NATPS has the advantages of simplicity, rapidity, low cost, high efficiency, and biocompability. It need not separate target proteins from the phase-forming reagents. It also has potential application values in purification and concentration of proteins. Therefore, it has great significance in extraction and separation of proteomic proteins, as well as methodology.
Acknowledgment. This work was financially supported by the National Key Basic Research Development Project “Research on Human Major Disease Proteomics” with the Grant No. 2001CB5102 and the National Natural Science Foundation of China with the Grant No. 20475052. References (1) McCarthy, F. M.; Burgess, S. C.; van den Berg, B. H.; Koter, M. D.; Pharr, G. T.; J. Proteome Res. 2005, 4, 316-324. (2) Tu, C. J.; Dai, J.; Li, S. J.; Sheng, Q. H.; Deng, W. J.; Xia, Q. C.; Zeng R. J. Proteome Res. 2005, 4, 1265-1273.
research articles
Novel Prefractionation Method (3) Pedersen, S. K.; Harry, J. L.; Sebastian, L.; Baker, J.; Traini, M. D.; McCarthy, J. T.; Manoharan, A.; Wilkins, M R.; Gooley, A. A.; Righetti, P. G.; Packer, N. P.; Williams, K. L.; Herbert, B. R. J. Proteome Res. 2003, 2, 303-311. (4) Righetti, P. G.; Castagna, A.; Herbert, B.; Reymond, F.; Rossier, J. S. Proteomics 2003, 3, 1397-1407. (5) Figeys, D. Anal. Chem. 2003, 75, 2891-2905. (6) Gorg, A.; Boguth, G.; Kopf, A.; Reil, G.; Parla, H.; Weiss, W. Proteomics 2002, 2, 1652-1659. (7) Cordwell, S. J.; Nouwens, A. S.; Walsh, B. J.; Proteomics 2001, 1, 461-472. (8) Tang, H.-Y.; Speicher, D. W. Expert Review of Proteomics 2005, 2, 295-306. (9) Maccarrone, G.; Birg, I.; Malisch, E.; Rosenhagen, M. C.; Ditzen, C.; Chakel, J. A.; Mandel, F.; Reimann, A.; Doertbudak, C.-C.; Haegler, K.; Holsboer, F.; Turck, C. W. Clinical Proteomics 2004, 1, 333-364. (10) Schmidt, M.; Jain, A.; Wolf, D. A.: Multidimensional proteomic analysis of proteolytic pathways involved in cell cycle control. Cell Cycle Checkpoint Control Protocols Volume 241; Lieberman, H. B., Ed.; N. J. Human Press: Totowa, 2003, 235-245. (11) Molloy, M. P.; Herbert, B. R.; Walsh, B. J.; Tyler, M. I.; Trainni, M.; Sanchez, J. C.; Hochstrasser, D. F.; Wolliams, K. L.; Gooley, A. A. Electrophoresis 1998, 19, 837. (12) Lan, Y.; Qian, X.; Wang, G.; Li, Y.; Luo, L.; Liu, Z.; He, F. Chin. J. Prog. Biochem. Biophy. 2001, 28, 415-417. (13) Ruiz-Lo´pez, N.; Martı´nez-Force, E.; Garce´s, R. Anal. Biochem. 2003, 317, 247-254. (14) Colle´n, A.; Persson, J.; Linder, M.; Nakari-Seta¨la¨, T.; Penttila¨, M.; Tjerneld, F.; Sivars, U. Biochim. Biophys. Acta 2002, 1569, 139150. (15) Baginsky, S.; Siddique, A.; Gruissem, W. J. Proteome Res. 2004, 3, 1128-1137. (16) Nissum, M.; Schneider, U.; Kuhfuss, S.; Obermaier, C.; Wildgruber, R.; Posch, A.; Echerskom, C. Anal. Chem. 2004, 76, 2040-2045. (17) Schilling, E. A.; Kamholz, A. E.; Yager, P. Anal. Chem. 2002, 74, 1798-1804. (18) Li, Y.; Beitle, R. R. Biotechnol. Prog. 2002, 18, 1054-1059. (19) Bolognese, B.; Nerli, B.; Pico´, G. J. Chromatogr. B. 2005, 814, 347353.
(20) Zaslavsky, B. Y. In Physical Chemistry and Bioanalytical Applications; Zaslavsky, B. Y., Ed.; Marcel-Dekker Inc.: New York, 1994. (21) Albertsson, P. A° . Partition of Cell Particles and Macromolecules, second ed., John Wiley and Sons: New York, 1971. (22) Waziri, S. M.; Abu-Sharkh, B. F.; Ali. S. A. Biotechnol. Prog. 2004, 20, 526-532. (23) Li, C.; Bai, J.; Li, W.; Cai, Z.; Ouyang, F. Biotechnol. Prog. 2001, 17, 366-368. (24) Linder, M.; Selber, K.; Nakari-Seta¨la¨, T.; Qiao, M.; Kula, M.-R.; Penttila¨, M. Biomacromolecules 2001, 2, 511-517. (25) Haraguchi, L. H.; Mohamed, R. S.; Loh, W.; Pessoˆa Filho, P. A. Fluid Phase Equilibr. 2004, 215, 1-15. (26) Gavasane, M. R.; Gaikar, V. G. Enzyme Microb. Technol. 2003, 32, 665-675. (27) Fexby, S.; Nilsson, A. Biotechnol. Prog. 2004, 20, 793-798. (28) Albertson, P.-A° . Partition of Cell Particles and Macromolecules; Wiley: New York, 1986. (29) Chi, Y. Practical Techniques for Protein preparation; Harbin Engineering University Press: Harbin, China, 1999, P.15 (30) Militello, V.; Vetri, V.; Leone, M. Biophys. Chem. 2003, 105, 133141. (31) Salabat, A.; Dashti, H. Fluid Phase Equilib. 2004, 216, 153-157. (32) Chen, J. S.; Chen, F. P.; Chen Y. F. Chinese J. Phys. Test. Chem. Anal. B: Chem. Anal. 2003, 39, 408-409. (33) Li, J., Xiao, N., Yu, R., Yuan, M., Chen, L., Chen, Y., Chen, L., Eds.; Experimental principles and Methods in Biochemistry; Peiking University Press: Peiking, China, 1994, P.216. (34) Xu, K. Handbook of Raw Material and Intermediate for Fine Organic Chemical Industry, Chemical Industry Press: Beijing, China. 2001. (35) Santis, R. D.; Marrelli, L.; Muscetta, P. N. Chem. Eng. J. 1976a, 11, 207-214. (36) Santis, R. D.; Marrelli, L.; Muscetta, P. N. J. Chem. Eng. Data 1976b, 21, 324-327. (37) Li, Z.; Tang, Y.; Liu, Y.; Li, Y. Fluid Phase Equilib. 1995, 103, 143153. (38) Cohn, E. J. Proc. Natl. Acad. Sci. U.S.A. 1920, 6, 256-263.
PR050359S
Journal of Proteome Research • Vol. 5, No. 4, 2006 845
