Evaluation of Three Methods for Protein Extraction byTwo-Dimensional Electrophoresis
Sun Ningning, Wang Zijian, Gao Xiang,Cheng Xin, Liu Ning*
Central Laboratory, The Second Hospital of Jilin University, Changchun, China
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Abstract:
Protein sample preparation has long been a critical step in proteomics studies. A variety of methods for protein extraction has been developed for proteomics research, in which many reagents with different chemical properties have been used. Therefore, the ability of methods with different reagents to disrupt cell or tissue is very different. In the proteomics research, a method of choice should be carefully evaluated on the basis of both object and purpose of the study. In the present study, by using two-dimensional electrophoresis, the profiles of whole proteins in cultured 293T cellsextracted by three methods commonly used in proteomics field, such as Triton X-100 method、Urea method and Trizol method, were compared. The results indicated that both the numbersand intensities of proteins extracted by the three methods were different from each other.Thus, the present study provides a rapid, effective and comprehensive method for evaluation of protein extraction methods for proteomics study.
Keywords: Protein extraction, Urea, Triton X-100, Trizol, Two-dimensional electrophoresis
*Corresponding author
1 Introduction
In proteomics study, sample preparation is the primary issue. Different sample preparation methods may lead to different profiles of proteins extracted from specific cells or tissues [1, 2]. At present, a variety of sample preparation methods have been applied to proteomics research. A typical method for protein preparation uses high concentrations of urea, which arewidely used intwo-dimensional electrophoresis technique with Tris (pH8.5), Thiourea and CHAPS and so on[3, 4]. Another lysis buffer contains the surfactant Triton X-100, which is the main reagent in several commercially available kits for cell lysis, such as a product (# 9803) from Cell Signaling Technology. Besides these two chemicals, Trizol has been developed as reagent for protein extraction, which is commonly used to extract total RNA/DNA [5-8].
Until now, there’s no method toclaim that the whole proteome in cells or tissues can be extracted completely. Therefore, researchershave to choose one of the most suitable methods for protein extraction. However, it is not very clear to the difference between different methods for sample preparations, including the difference between different methods for the same sample and the difference of dissolving capacity between some proteins.Two-dimensional electrophoresis (2-DE)is the most common separation technologyin proteomics[4, 9], which has been widely used for separating proteins to demonstrate the difference of proteomes between two samples or among some samples [10-13].
In the present study, by using two-dimensional electrophoresis, the profiles of whole proteins in cultured 293T cells extracted by three methods commonly used in proteomics field, such as Triton X-100 method、Urea method and TRIzol method, were compared.
2 Experiment
2.1Chemicals and reagents
Trizol Reagent was purchased from Invitrogen (USA).Iodoacetamide, DTT, formic acid, ammonium bicarbonate (NH4HCO3), acetonitrile (HPLC grade) and Tris were purchased from Sigma Aldrich (USA);Urea,IPG strips ,IPG buffer and Bradford protein quantification reagent were purchased from Bio-Rad(USA); nucleasewas obtained from GE Healthcare (Sweden); Protease inhibitor was obtainedfrom Roche (Switzerland); Sequencing grade trypsin was purchased from Promega (USA); High glucose DMEM medium and Fetal bovine serum were obtained from Hyclone (USA); Cell lysis buffer # 9803 was obtained from Cell Signaling Technology(USA) ; 293T cells were stored in liquid nitrogen of our laboratory; Milli-Q water was used to prepare the protein extracts, 2-DE, and MALDI-TOF-MS/MS solutions.
2.2Methods
2.2.1 Cell culture
293T cellswere culturedin high glucose DMEM culture medium containing 10% fetal bovine serum in 10cm dishes andin a 37oC, 5% CO2 incubator. Until the density reached80% to 90%, 293T cells were collected and properly washed. The cell pellets were obtained by centrifugation (500g, 10 min, 4oC),followed bylysedby three different sample preparation methods as follows.
2.2.2 Protein extraction protocols
2.2.2.1 Triton X-100 method
The cell lysis buffer (Cell Signaling Technology, # 9803), of whichTriton X-100 isone of the main effective components, contains 20mM Tris-HCl (pH7.5), 150mM NaCl, 1mM Na2EDTA, 1mM EGTA, 1%Triton X-100, 2.5mM Sodium Pyrophosphate, 1mM β-glycerophosphate, 1mM Na3VO4, 1ug/ml Leupeptin.According to theinstruction,cell pellet was lysed in 100ul cell lysis buffer by pipettingrepeatedly.Spin down (12,000g, 10 min, 4 oC) and transfer the supernatant to a new EP tube.Place the supernatant in -80 oCto save quickly.
2.2.2.2 Urea method
The cell pellet was lysed in abuffer containing high concentrations of urea (100mM Tris-HCl, pH8.0, 8M Urea). 100ul of this buffer was used with addition of 1ul of nuclease and 10ul Protease inhibitor (10×). The supernatant was obtained by centrifugation (12,000g, 10 min, 4 oC) and transferredinto a new EP tube. Keep the supernatant in -80 oC.
2.2.2.3 TRIzol reagent method
According to theinstruction, add the Trizol reagent into cell pellet by pipettingrepeatedly to lyse cellscompletely. After adding chloroform, the solution is in three layers by centrifuging. Remove RNA in the upper aqueous phase. Remove DNA after adding ethanol in the intermediate phase.Finally, precipitate proteins after adding isopropanol in the lower phase.Rinse theprotein precipitation repeatedly; dissolve the precipitation in buffer(100mM Tris-HCl, pH8.0, 8M Urea) by vigorous vortexing. Spin down (12,000g, 10 min, 4 oC) and transfer the supernatant to a new EP tube. Keep the supernatant in -80 oC.
2.2.3 Two-dimensional electrophoresis (2-DE)
Equal amounts of protein samples by three extraction methods were diluted with rehydration solution (6 M urea, 2% w/v CHAPS, 65 mM DTT, 0.5% v/v pH 3-10 Bio-lyte, trace bromophenol blue). According to the operation manual of 2-DE (Bio-Rad), protein samples were passively absorbed into IPG gel strips (pH 3-10, 7 cm, Bio-Rad) for 16 h. Isoelectric focusing was performed at 20 oC on Protean IEF Cell (Bio-Rad). After equilibrated in equilibration solution, gel strips were applied on second-dimensional PAGE with 12% polyacrylamide. Separation was then carried out on a Protean II xi electrophoresis system (Bio-Rad) at a current setting of 15 mA/gel until the bromophenol blue reached the bottom of the gel. The gels were stained with silver and then scanned with a high-resolution scanner (GS800, Bio-Rad). The obtained gel images were analyzed using PDQuest software (Version 7.1.1; Bio-Rad) according to the instructions provided by the manufacturer.
3 Results and discussion
3.1 SDS-PAGE analysis of proteome from 293T cells using three different sample preparation methods
In our study, whole proteins from 293T cells were extracted by three different sample preparation methods respectively, which were subject to SDS-PAGE electrophoresis. As indicated in figure 1, the extracted proteins with molecular weight from 20kDa to 200kDa have been separated by 12% SDS-polyacrylamide gel electrophoresis, most of which distribute in the range of the molecular weight from 30kDa to 80kDa. The proteins obtained using three different sample preparation methods have a similar distribution profile, in which most proteins are distributed among the molecular weight from 30kDa to 46kDa.
Figure.1 SDS-PAGE of proteins extracted from 293T cells cultured in DMEM media by three different methods. Ten micrograms of each of three samples (lane 2: Urea method (UR); lane 3: TRIzol method (TR); lane 4: Triton X-100 method (TX)) were loaded per lane. Lane 1 was loaded with molecular weight marker (M).
3.22-DE analysis of proteome from 293T cells using three different sample preparation methods
Because 1D SDS-PAGE cannot find the subtle differences among the three different samples extracted by three methods, attempts have been made to apply other analytical approaches with high resolution and high performance. Therefore, we chose high resolution two-dimensional electrophoresis (2-DE) technique as a tool to compare the protein profiles of samples by these three extracted methods, as illustrated in figure 2. The 2-DE technology combines the isoelectric focusing electrophoresis (IEF) with polyacrylamide gel electrophoresis (PAGE) to separate a complicated mixture of proteins. Depending on the isoelectric point and molecular weight of proteins, 2-DE can present two-dimensional diagram. The horizontal direction represents the results which proteome are separated depending on the isoelectric point of proteins, and the vertical direction shows the results which proteome are separated relying on the molecular weight
of proteins.
Figure. 2. Comparison of 2-DE proteome maps of the protein samples extracted by Triton X-100 method (A), Urea method (B) and Trizol method (C), respectively.
Image analysis of the three 2-DE gels of different samples by three methods showed that the number of the identified proteins obtained by Triton X-100, urea and Trizol method was 661, 634 and 458, respectively. The number of the identified proteins obtained by Trizol method is nearly 30% less than those by other two methods (Triton X-100 and urea methods). In the last process of extracting protein by Trizol method, we found that part of the protein precipitation cannot completely be dissolved after adding the buffer (100 mM Tris, 8 M Urea). This might be the reason why the numbers of proteins extracted by Trizol method aremuch less than each of the other two methods.
Triton X-100 is a non-ionic surfactant, while only 1% concentration of Triton X-100 could disrupt cell membrane under non-denaturing conditions. Therefore, this kind of lysis buffer can maintain native conformation of protein [1, 6]. Our data showed that the numbers of proteins extracted by either Triton X-100 or urea were much more than that by Trizol. Although the Trizol reagent extracts less protein that other two methods, it is more suitable for the preparation of a biological sample containing virulent and contagious bacteria or viruses (e.g. avian influenza viruses), because of the violent lysing ability to bacteria and viruses. In conclusion, this study provides a tool for evaluating three universal sample preparation methods to obtain proteins of 293T cells, with the means of high-resolution 2-DE analysis.
Acknowledgements
This work was supported by Natural Science Foundation of China (21175055, 81472030), Jilin Province Science and Technology Department (20110739, 20150204001YY), Jilin University Bethune Project B (2012210).
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