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Research Article

Comparison of protein precipitationmethods for various rat brain structuresprior to proteomic analysis

Sample preparation is a fundamental step in proteomic methodologies. The quality of theresults from a proteomic experiment is dependent on the nature of the sample and the

properties of the proteins. In this study, various pre-treatment methods were compared by

proteomic analysis; we analysed various rat brain structures after chloroform/methanol,

acetone, TCA/acetone and TCA protein precipitation procedures. The protein content of the

supernatant was also examined by 2-DE. We found that for four of the rat brain structures,

precipitation with chloroform/methanol and acetone delivered the highest protein recoveryfor top-down proteomic analysis; however, TCA precipitation resulted in good protein

separation and the highest number of protein spots in 2-DE. Moreover, TCA precipitation

also gave high efficiency of protein recovery if prior sonication procedure was performed.

Keywords:

IEF / Protein precipitation / Proteomics / Rat brain / SDS-PAGEDOI 10.1002/elps.201000197

1 Introduction

The term ‘‘proteomics’’ refers to the analysis of all proteins in

a whole cell, including the description of co- and post-

translationally modified proteins and alternatively splicedprotein variants [1]. Proteomic methodologies have made a

great deal of progress in recent years with the development of

high-resolution 2-DE for protein separation and profiling,high-sensitivity MS for determination of the molecular weight

of peptides which result from enzymatic cleavage of proteins,

and sequencing of secondary ions, and bioinformaticapproaches to protein identification using software databases.

Sample preparation is crucial for conducting reliable

proteomic analysis [2, 3]. Samples should have a high-

protein concentration and be free of salt and other inter-fering components, such as detergents, nucleic acids, lipids,

etc [4, 5]. Precipitation is widely used for processing of

biological molecules such as proteins [6]. This procedure is

used to concentrate and fractionate the target molecule fromvarious contaminants. For example, in the biotechnology

industry, protein precipitation is used to eliminatecontaminants commonly contained in blood [7].

Sample preparation depends on the origin of the cells or

the tissue. The first step is usually homogenisation or

sonication followed by protein precipitation and solubilisa-

tion in a suitable buffer. Chloroform, methanol, acetone and

TCA are commonly used as protein precipitating reagents.

In this study, we investigated the efficiency of various

methods for protein precipitation using homogenatesderived from different rat brain structures. The rat brain is a

common model for studying the mechanism of action of

compounds that are used to treat human psychiatric and

neurological disorders [8]. Due to the complexity of braintissue, optimisation of protein precipitation methods is

crucial for the analysis of brain proteins. Proteomic studiescan assist in the identification of molecular biomarkers of

diseases and the elucidation of the mechanisms of drug

action. The results obtained in this study may facilitate the

choice of the most optimal methods for the study of

alterations in the rat brain proteome after various beha-vioural and/or pharmacological treatments. The efficiency

and specificity of contaminant removal were monitored by

the Bradford assay, 1-D SDS-PAGE [9] and 2-D SDS-PAGE

[10] before proteomic experiments were performed.We also analysed the supernatant (supernatants are

usually removed after protein precipitation) in order todetermine the types of proteins that were lost during sample

preparation; precipitation followed by re-solubilisation in

sample solution rarely gives a 100% yield.

2 Materials and methods

2.1 Tissues

The efficiency of various methods of protein precipitation

was investigated using four rat brain structures:

Ewelina Fic1

Sylwia Kedracka-Krok 1

Urszula Jankowska1

Artur Pirog1

Marta Dziedzicka-Wasylewska1,2

1

Department of PhysicalBiochemistry, Faculty of Biochemistry, Biophysics andBiotechnology, JagiellonianUniversity, Cracow, Poland

2Department of Pharmacology,Institute of Pharmacology PolishAcademy of Sciences, Cracow,Poland

Received April 6, 2010

Revised August 4, 2010

Accepted August 12, 2010

Correspondence: Dr. Ewelina Fic, Department of Physical

Biochemistry, Faculty of Biochemistry, Biophysics and Biotech-

nology, Jagiellonian University, Gronostajowa 7, 30-387 Cracow,

Poland

E-mail: [email protected]

Fax: 148-12-664-69-02

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

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protein were applied to an immobilised pH 3–10 nonlinear

gradient with 7-cm strips (GE Healthcare). Rehydration(14 h) was completed using a one-step procedure. IEF was

conducted on an Ettan IPGphor 3 IEF System (GE

Healthcare) at 201C with a current limit of 50 mA per strip.

An IEF program was applied that involved pre-focusing

(200 V for 5 h) followed by four steps: a gradient to 300 V for0.5 h, a gradient to 1000 V for 0.5 h, a gradient to 5000 V for1.5 h and 5000 V for 1 h. During pre-focusing, the electrode

wicks were used to improve disposal of excess water, salts

and proteins with pI values outside the pH range of the IPG

strips. Small electrode wicks immersed in miliQ-H2O(Millipore) were placed at the anode and cathode ends of

the IPG strips just beneath the electrodes. The wicks were

exchanged three times. In order to investigate the influence

of electrode wicks on the quality of protein separation, acomparative study was conducted.

Prior to conducting SDS-PAGE in the second dimen-sion, the IPG strips were equilibrated according to [10].

After the first dimension separation, the proteins wereseparated on Mini-Protean 3 using 12% polyacrylamide gels.

The gels were stained using silver nitrate according to [11]with minor modifications. The 2-DE analysis of protein

pellets and supernatants was performed in triplicate.

The gels were analysed using ImageMaster 2D

Platinum v6.0 (GE Healthcare) software.

2.6 Determination of protein concentration

The protein concentration of the re-solubilised samples was

determined in triplicate using the Bradford assay. The

efficiency of precipitation was determined as a ratio of the protein concentration before and after precipitation.

The results presented are an average of at least three

experiments.

The Bradford assay was also used to determine theprotein content after the supernatant analysis.

3 Results and discussion

Proper sample preparation is crucial in order to obtain reliable,reproducible and significant data, particularly in comparative

proteomic studies where minor differences between experi-

mental and control groups are often meaningful [12]. In thisstudy, we compared preparations of rat brain samples using avariety of applications for proteomic technology in

neuroscience. These methodologies are useful for identifying

changes in brain protein expression under different experi-

mental or disease conditions, profiling protein modifications

(e.g. phosphorylation) and mapping protein–protein interac-

tions in animal models of various diseases.The efficiency of the pre-treatment methods for protein

precipitation was studied using various rat brain structures.

Figure 1 shows a comparison of the 1-DE separation results.

Electrophoretic images representing the protein profiles of

each brain structure were very similar. The greatest protein

recoveries (Fig. 2 and Table 1) were achieved with thechloroform/methanol and acetone precipitation methods.

Precipitation of proteins from the amygdala, hippocampus

and striatum with chloroform/methanol gave a protein yield

greater than 70%. For the frontal cortex, the recovery was

surprisingly low; only 30% of the initial protein content wasrecovered. This is probably due to a larger concentration of hydrophobic proteins in the frontal cortex [13]. Hydrophobic

proteins are relatively difficult to dissolve in aqueous solu-

tions. The use of acetone resulted in slightly less than 70%

recovery of the proteins from the amygdala, hippocampusand striatum. However, for proteins from the frontal cortex,

the recovery was greater than 50%; therefore, precipitation

using acetone produced better results than those obtained

using the chloroform/methanol mixture (Fig. 2). Theadvantage of acetone precipitation is that it is a very feasible

procedure, but it requires a large volume of organic solvent.Precipitation with either TCA/acetone or TCA led to

approximately twofold lower recovery compared with acet-one precipitation. Especially, in case of TCA precipitation,

protein loss was probably due to incomplete solubilisation of the pellets and the acetone wash step. A portion of the pellet

remained insoluble despite the use of additional re-hydra-

tion buffer. It is important to emphasise that the recovery of

proteins was strongly dependent on pellet solubilisation.The solubilisation of protein pellets after precipitation with

methanol/chloroform took 30–60 min, and with acetone and

TCA, it took 90–120 min. With acetone/TCA, solubilisation

required approximately 180 min. Finally, we obtained a clear

solution of protein in the re-hydration buffer. A clear solu-

tion was observed, but it was not further analysed. There are

a few methods that have been shown to increase proteinrecovery, such as sample sonication after TCA precipitation

[14]; however, the insertion of the sonicator tip affects

protein recovery since proteins may coat the tip. The appli-

cation of the bioruptor UCD-200 TM (Diagenode, Liège,Belgium) (15 min, 320 W), which enabled to work in closed

tubes placed in ice bath, caused significant increase of

protein recovery after TCA precipitation (Fig. 2 and Table 1).

2-DE images obtained for hippocampal proteins that were

precipitated using each of the four various methods described

here are shown in Fig. 3. There was no significant differencein the final composition of proteins, and the number of

identified spots was very similar (approximately, 650 spots per

gel; Table 1). However, detailed analysis revealed that therewere few differences between the various precipitation meth-ods. The spots from the samples without precipitation

(Fig. 3A) were fuzzy and streaky. Precipitation with TCA/

acetone, as well as TCA precipitation, gave better IEF than the

other precipitation methods (Fig. 3B and C). This effect was

especially noticeable for basic proteins and small acidic

proteins. The presence of TCA improved the enrichment of alkaline proteins. Streaks in the alkaline range of the pH

gradient were common artefacts in the 2-D images. However,

a greater amount of alkaline proteins made the spots more

visible and detectable despite the presence of streaks.

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0

10

20

30

40

50

60

70

80

90

100

ch loroform/methanol acetone TCA/acetone TCA TCA (+ sonication)

P r e c i p i t a t i o n E f f i c i e n c y %

Amygdala

Frontal Cortex

Hippocampus

Striatum

Figure 2. Comparison of the precipi-

tation efficiency of different methods

for various brain structures. The

values of standard deviation errors

are presented as thin line bars at the

top of each column. Each experiment

was performed in triplicate.

Figure 1. 1-D SDS-PAGE electro-

phoresis. Analysis of proteins from

the homogenate of four different

brain structures after various preci-

pitation methods. Lane 1, homoge-

nate; lane 2, chloroform/methanol;

lane 3, acetone; lane 4, TCA/acetone

and lane 5, TCA. The quantity of the

proteins applied to each lane was

approximately 0.75mg. Gels werestained with silver nitrate, and each

experiment was performed in tripli-

cate.

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In addition, the presence of acetone in the TCA/acetone

mixture may cause more efficient delipidation of lipid-rich

biological material, such as brain tissue [10]. More complete

delipidation improves the accuracy of IEF. In the case of acetone, the small acidic proteins were rather weakly focused(Fig. 3E and G). The application of paper wicks significantly

improved the isoelectric separation and focusing of spots as

reflected in the 2-DE images (Fig. 3A–E). Image analysis

indicated that paper wicks give a sharper image and reducedstreaking. According to Fountoulakis [15], approximately 70%

of the brain proteins identified from the 2-D gels had theore-

tical pI values between 5 and 8, and 15% had pI values between

4 and 5. This is reflected in the 2-D gels shown in Fig. 3.Precipitation methods were recently compared by Jiang

et al. [16]; this study, which was performed using human

plasma, reported that TCA and acetone precipitation, as well

as ultrafiltration, yielded higher protein recoveries compared

with chloroform/methanol precipitation. TCA precipitation

was one of the best protocols. However, as we mentionedabove, the precipitation methods strongly depend on thestarting material.

The results of the supernatant analysis by 2-DE are

shown in Fig. 4. The protein concentration in the super-

natants was beyond the sensitivity of the Bradford assay.The quantity of loading on each 2-D gel was comparable and

estimated on the basis of 1-DE (computer densitometry

analysis). Figure 4 shows the type of proteins lost during

each precipitation procedure. The method that yielded thehighest protein recovery was acetone precipitation (as can

be concluded from the comparison of supernatants);

Table 1. Percentage of hippocampal protein recovery and number of protein spots after various precipitation procedures a)

Precipitation method Protein amount before

precipitation (mg)

Protein amount after

precipitation (mg)

Percentage of

recovery (%)

Number of spots

Chloroform/methanol 270.98732.51 242.77756.95 88.52711.62 667

Acetone 270.98732.51 186.15754.78 68.70717.81 665

10%TCA/acetone 270.98732.51 89.67714.83 33.0172.80 56310%TCA 270.98732.51 68.21736.72 24.08711.55 629

10%TCA (1sonication) 324.17741.22 249.60759.22 77.00719.69 699

a) Values are the mean7standard deviation of five independent experiments. The number of spots was determined using ImageMaster

2D Platinum v6.0 software.

Figure 3. 2-DE – IEF followed by SDS-PAGE. Analysis of proteins from rat hippocampus after various precipitation methods.

(A) Homogenate without precipitation, (B) TCA, (C) TCA/acetone, (D and F) chloroform/methanol, (E) and (G) acetone. Images

(A–D) represent samples prepared with paper wicks, whereas (F and G) represent samples prepared without paper wicks. During IEF,

small rectangular wicks were placed at the anode and cathode ends of the IPG strips just beneath the electrodes. The wicks absorb

excess water, salts and proteins with pI values that lie outside the pH range of the IPG strip.



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however, the worst results were obtained from TCA/acetone

precipitation (many spots are visible in the 2-D gels). As

shown in Fig. 4, precipitation of acidic proteins using themixture of chloroform and methanol gave a low efficiency,

whereas TCA was not efficient for precipitation of high-

molecular-weight proteins. The amount of proteins lost

during precipitation did not exceed 5%.

Unfortunately, despite the presence of denaturants anddetergents, the solubilisation of precipitated proteins wasoften incomplete. This may have yielded imprecise

results. The precipitate recovery often depends on

re-dissolving in a smaller volume followed by centrifugation

or filtration. Sonication significantly improved the solubility

of insoluble parts of supernatants. Better solubility may beachieved by intense and longer mixing or vortexing;

however, it is crucial to avoid excessive foaming

and temperature increases, which may cause protein

degradation.

4 Concluding remarks

The development of standardised, reproducible methods for

protein isolation from tissues offers great potential for thediscovery and validation of biomarkers.

The comparison of protein content in four supernatants

revealed that the TCA/acetone method produced the lowest

efficiency for protein precipitation. Reduced protein loss

(fewer proteins in supernatants) occurred with the metha-

nol/chloroform and TCA methods. The acetone methodyielded the lowest loss of proteins, as determined by analysis

of the supernatant. The acetone method yielded high

precipitation efficiency in comparison to precipitation with

methanol/chloroform, as determined by the protein

concentration. However, as has been shown by Simpsonand Beynon [17], acetone precipitation can, after proteolysis,

lead to selective modification of peptides, which makes MS

analysis more difficult.

Finally, the chloroform/methanol precipitation method

was regarded as the best precipitation method for top-downproteomics. The advantages of this method are: a lack of sample cooling, simplicity of performance and the short

duration of the procedure (about 45 min), which minimises

the risk of protease degradation. However, when time and

cost are important factors, one should use chloroform/methanol precipitation. It is advisable to keep the sample

preparation as simple as possible.

For 2-DE, TCA precipitation was optimal. This method

is laborious and requires low temperature; however, it

results in good IEF and clear spots. The proper sonication

procedure for complete TCA pellet solubilisation is stronglyrecommended. These results are presented for the hippo-

campus, but the conclusions can be extended to the other ratbrain tissues as well (data not shown).

In summary, it is important that the chosen

protein precipitation method is able to effectivelyconcentrate samples and eliminate contaminants, but such

methods may also have the disadvantages of causing

irreversible protein denaturation and protein insolubilisa-

tion. Therefore, special attention must be paid to the

complete solubilisation of protein pellets; however, precipi-

tation procedures rarely yield 100% recoveries. Carefulsample preparation is necessary for successful proteomic

analysis.

Figure 4. 2-DE – IEF followed by SDS-PAGE.Analysis of supernatants from rat hippo-

campus after protein precipitation using

various methods. (A) TCA, (B) TCA/acetone,

(C) chloroform/methanol and (D) acetone.

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This work was supported by grant 1/10/0-PBZ-MNiI-2/2/

1/2005 from the Ministry of Science and Higher Education. The research was carried out with the equipment purchased thanks to

the financial support of the European Regional Development

Fund in the framework of the Polish Innovation Economy

Operational Program (contract No. POIG.02.01.00-12-167/08,

project Ma"opolska Center of Biotechnology).

The authors have declared no conflict of interest.

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