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298 Current Proteomics, 2013, 10, 298-311

A Simple Protein Extraction Method for Proteomic Analysis of Diverse Biological Specimens

Panga Jaipal Reddy1, Aishwarya Anand Rao

1, Darpan Malhotra

1, Samridhi Sharma

1,

Ravinder Kumar1, Rekha Jain

1, Kishore Gollapalli

1, Namita Pendharkar

2, Srikanth Rapole

2 and

Sanjeeva Srivastava1*

1Wadhwani Research Center for Biosciences and Bioengineering, Department of Biosciences and Bioengineering,

Indian Institute of Technology Bombay, Powai, Mumbai 400076, India; 2Proteomics Lab, National Centre for Cell

Science, Ganeshkhind, Pune 411007, Maharashtra, India

Abstract: The success of a proteomic experiment largely depends on the quality and quantity of the protein extract. Cur-

rently, various protocols are available for extraction of proteins from different types of samples; however, further optimi-

zation is required for every new sample type. Hence, a common protein extraction protocol is desirable. In the present

study, soluble proteins were extracted from six diverse samples using TRIzol without any additional clean-up step and

subjected to 2-DE and 2D-DIGE analysis for global protein expression profiling. Image analysis using IMP7 and DeCyder

showed good coverage, reproducibility and quality of the gel. MS analysis of 24 spots from all the six samples showed

good score and coverage for the identified proteins. Additionally, this method facilitated the concurrent isolation of RNA

from the same cell lysates with high integrity and quality, suitable for transcriptomic analysis. Thus, we demonstrate the

use of a common protein extraction protocol involving TRIzol reagent for 2-DE, 2D-DIGE and MS analysis using six

diverse samples and show its suitability for concomitant transcriptomic studies.

Keywords: 2-DE, 2D-DIGE, breast cancer, B. subtilis, glioblastoma cell line, mass spectrometry, protein extraction, S. coeli-color, TRIzol, yeast.

INTRODUCTION

Gel-based proteomics continues to be a powerful tool to

study high-throughput protein expression profile as a func-

tion of biological variation due to its simplicity, reproduci-

bility and cost-effectiveness [1]. More than 5000 publica-

tions have emerged using gel-based proteomics (2-DE and

2D-DIGE) on diverse samples in the last decade [2]. The

success of these methods largely depends on the choice of

the protein extraction protocol, which in turn determines the

purity of the protein preparation and also the reproducibility

[3]. Since each sample has its own complexity with respect

to the cell wall or membrane composition, metabolism and

functional regulation, extraction of protein from the diverse

sample is quite challenging. Moreover, polysaccharides, sec-

ondary metabolites, nucleic acids, lipids and pigments cause

improper separation in IEF resulting in streaking and arti-

facts across 2-DE and 2D-DIGE gels. In the past, protocols

involving TCA-acetone precipitation [4], acetone precipita-

tion [5], phenol/methanol-ammonium acetate precipitation

[6] and isopropanol precipitation [7] have been employed for

extracting the proteins from different samples. Though these

methods are widely employed, they pose a major hurdle for

protein solubilization and also necessitate the use of an addi-

*Address correspondence to this author at the Department of Biosciences

and Bioengineering, IIT Bombay, Mumbai 400 076, India;

Tel: +91-22-2576-7779; Fax: +91-22-2572-3480;

E-mail: sanjeeva@iitb.ac.in

tional clean-up step to remove the interfering agents [8, 9]. A

robust protein extraction protocol for proteomic analysis,

capable of removing all the impurities without any additional

clean-up steps and applicable to a variety of biological sam-

ples, is currently desirable.

TRIzol is a monophasic solution of guanidine isothiocy-

anate and phenol, which is routinely used for RNA isolation

and can also recover proteins from the sample [10]. Gua-

nidine isothiocyanate breaks the cell membrane and inhibits

RNase and proteases, while phenol/chloroform dissolves the

lipids and polysaccharides, and releases complex membrane

proteins [11]. After the removal of nucleic acids from the

whole cell lysate, acetone precipitation followed by gua-

nidine-HCl removes any remaining endogenous material

such as salts, phenol, and other metabolites (Table 1). At-

tempts have been made in the past to optimize the protein

extraction for 2-DE using TRIzol. Kirkland PA et al. (2006)

reported the advantages of TRIzol reagent for extraction of

proteins from H. volcanii DS70 cells for 2-DE [12]. Lee FW

et al. (2008) compared three different protocols for extrac-

tion of proteins from dinoflagellates and confirmed that TRI-

zol extraction gave best results [11]. Young C & Truman P

(2011) have also proved that proteins extracted from human

neuroblastoma cells using TRIzol reagent were predomi-

nantly unbiased, with no amino acid or mass modification

and exhibited compatibility for mass spectrometry analysis

[13]. Interestingly, TRIzol extraction has also been used for

preparing protein samples from kidney tissue [14], liver tissue

1875-6247/13 $58.00+.00 ©2013 Bentham Science Publishers

TRIzol Extraction Protocol for 2-DE, DIGE and Mass Spectrometry Current Proteomics, 2013, Vol. 10, No. 4 299

Table 1. Advantages of TRIzol protein extraction method as compared to other commonly used protein extraction methods.

Advantage TRIzol Extraction Method Other Published Methods

No nucleic acid contaminationTRIzol has ability to remove DNA and RNA in sequential

steps and doesn't require any additional enzymes [12].

Treatment with benzonase [23] or other nucleases [24] is

required for nucleic acid removal.

Salt removalAcetone precipitation followed by guanidium-HCl and

acetone washing removes excess salts [11].

Acetone or TCA-acetone is not sufficient to remove the

salt completely. Additional clean-up steps or MWCO need

to be performed prior to the 2-DE [25].

Dissolution of membrane

proteins

Phenol and chloroform dissolve the membrane lipids and

carbohydrates, and release the membrane bound proteins

[26].

Standard methods have problem with lipid and polysaccha-

ride contamination [27], and membrane protein extraction

is always challenging [28].

Protein solubility Protein pellet is easily soluble in rehydration buffer [29]. Protein pellet is sometimes insoluble in rehydration buffer

if DNA, lipid, polysaccharides are present [28, 30].

RNA isolation During sequential extraction, RNA can be isolated [29]. RNA extraction is not possible.

DNA extraction Genomic DNA can be extracted [29]. DNA extraction is not possible.

Table 2. TRIzol protein extraction reported in literature.

Technique

UsedSample

Quantification

Method

Quantity of

Protein Loaded

Length and pH

of the StripStaining No. of Spots Ref.

2-DE

Dermatophagoides

pteronyssinus (dust

mites)

BCA kit 0.80 g/L 7 cm (pH 3-10) CBB and silver

staining

LMW and HMW -

were identified[31]

2-DEHaloferax volcanii

DS70 cells_ 150 g 11 cm (pH 4-7)

SYPRO Ruby

fluorescent

protein stain

972 [12]

2-DE Dinoflagellate cells Bradford Assay 60 g 18 cm (pH 4-7) Silver-stained

1835, for Alexan-

drium spp. 1637 for

Scrippsiella spp.

[11]

2-DE Spinal cord Tissue

EZ-Q protein

quantitation

assay

100 g 7 cm (pH 3-10) SYPRO Ruby

staining _ [32]

2-DE SH-SY5Y cells (human

neuroblastoma cells)_ 200 g 11 cm (pH 4-7)

Biosafe

Coomassie stain _ [13]

2-DE

MCF-7, V. cholera, C.

irritans cyst and liver

abscess fat tissue (pus)

- 150 l 13 cm (pH 4-7) Silver staining

MCF7- 175 V. chol-

era- 595 C. irritans

cyst- 435 Pus- 306

[18]

2D-DIGENon-cancerous liver

tissues

Bradford

method 150 g 18 cm (pH 4-7) CyDyes 1229 [15]

2D-DIGE Kidney tissueBradford

method150 g

24 cm (pH 3-10

NL)CyDyes 989 [14]

2D-DIGE A. catenella DH01 - 200 g 24 cm (pH 4-7) CyDyes [17]

2D-DIGE MCF-7 cell line 2D-Quant kit 90 g24 cm (pH 3-10

NL)CyDyes - [16]

[15], MCF-7 cell line [16] and A. catenella DH01 [17] for

2D-DIGE. Though few studies exist in literature, most of the

attempts have been made on different systems in isolation

using the manufacture protocol. Recently, Tan et al. (2011),

have attempted to standardize the TRIzol extraction method

for 2-DE analysis for four samples using low-resolution

IPG strips [18] (Table 2).

In the present study we have focused on optimizing the

TRIzol protein extraction protocol for six diverse sample

types not reported in literature, and separating the proteins

300 Current Proteomics, 2013, Vol. 10, No. 4 Reddy et al.

by 2-DE and 2D-DIGE using high-resolution IPG strips. We

considered both the manufacturer’s protocol and the proto-

cols reported in literature and successfully tried some modi-

fications to make the method suitable for diverse sample.

The gels from all the samples showed good quality, cover-

age, reproducibility and resolution. Further, we explored the

compatibility of this method with mass spectrometry analy-

sis and the feasibility of simultaneous transcriptomic studies.

MATERIALS AND METHODS

Strains, Culture Conditions and Cell Lysis

Bacillus subtilis

B. subtilis AH75 (provided by Dr. Losick’s group) strain

was grown overnight in LB media having spectinomycin

antibiotic [19]. Culture was re-inoculated into fresh media

and grown till the culture reached mid-exponential phase. B. subtilis culture was centrifuged at 8000 x g for 10 min to

harvest the cells and resulting pellet was washed thrice with

PBS to remove any trace media. Pellet was resuspended in

PBS buffer containing 1 mg/mL lysozyme and kept at RT for

30 min. To lyse the cells completely, sonication was carried

out using a Vibra Cell sonicator for 4 cycles of 1 sec pulse

with 2 sec gap in between at 30% amplitude. Clear super-

natant was collected after centrifugation of the sample at

12,000 x g for 15 min at 4°C for protein isolation.

Streptomyces coelicolor A3

A wild-type strain M145 of Streptomyces coelicolor A3(2) was used in this study. Bacterial spores were pre-

germinated and then inoculated into liquid SMM media in

baffled Erlenmeyer flasks with SS springs sitting at the bot-

tom. To avoid frothing, 0.005 % (v/v) antifoam 204 was

added to the media. The cultures were shaken at a speed of

250 x g at 30°C. Exponentially growing culture was har-

vested and pellet was washed thoroughly with phosphate

buffer saline (pH 7.4) to remove all traces of media. Cell

lysis was performed for 1 hr at room temperature in TE

buffer containing 1 mg/mL lysozyme and protease inhibitor

cocktail. Cells were further broken by mild sonication, using

a Vibra Cell sonicator for 4 cycles of 1 sec pulse with 2 sec

gap in between at 30% amplitude. Cell debris was removed

by centrifugation at 12,000 x g, 15 min, 4°C.

Saccharomyces cerevisiae SK1 Background Strain

Diploid SK1 background yeast strain was grown on YPD

plate containing the following composition: 1% yeast ex-

tract, 2% peptone, 2% dextrose and 2% agar. 5 mL YPD

media was then inoculated with a single colony from YPD

plate and the tube was incubated at 30°C, 200 rpm overnight.

100 mL of YPD media was inoculated with the resulting

culture the following day such that initial OD600 was ~ 0.2

and the flask was further incubated at 30°C, 200 rpm till

OD600 reached 1.0. Cells were collected by centrifugation at

5,000 x g for 5 min at 4°C. The cell pellet was washed twice

with cold distilled water and centrifuged at 5,000 x g for 5

min at 4°C. Finally, the pellet was resuspended in 800 L of

homogenization buffer (50 mM Tris-chloride, 1 mM EDTA,

pH 7.4, 0.2% DTT). Cells were lysed using bead beater, op-

erated as 6 cycles of 1 min each, accompanied by intermit-

tent cooling of the tube on ice for 3 min. Cell lysate was col-

lected after centrifugation at 12,000 x g for 5 min at 4°C for

further processes.

U87 Glioblastoma Cell Line

Glioblastoma U87 cells were maintained in DMEM sup-

plemented with 10% FBS and 1% penicillin/streptomycin.

U87 cells were seeded at a density of 1x106 cells mL

-1 in 75

cm2 flasks and kept in a humidified incubator maintained at

37°C with 5% CO2. At 70-80% confluency, cells were

washed with PBS twice to remove debris, followed by addi-

tion of RIPA cell lysis buffer containing protease inhibitors

cocktail (Sigma, USA) and incubated on ice for 5 min. Intact

cells were scraped off the flask surface using a scrapper,

collected in an eppendorf tube and subjected to mild sonica-

tion using a Vibra Cell sonicator with 2 sec pulse and 2 sec

gap for 20 sec at 25% amplitude to break the intact cells.

Centrifugation was performed at 14,000 x g for 15 min and

clear lysate was collected for protein extraction using the

TRIzol protocol.

Breast Malignant Tissue

Breast malignant tissue samples were collected from

breast cancer patients after surgery. The use of human sam-

ples for research purpose was approved by the Institutional

Ethical Committee and informed consent was obtained from

all the patients and volunteers. Tissue samples (200 mg)

were washed thrice with PBS containing protease inhibitors.

Malignant tissue was ground using and pestle in

presence of liquid nitrogen. Ground tissue powder was trans-

ferred to a microcentrifuge tube. 1.5 mL of T-PER tissue

protein extraction buffer (Thermo scientific) was added

along with protease inhibitor cocktail. Tissue in T-PER

buffer was vortexed and sonicated 4 times with 10 sec pulse.

Tube was incubated on ice for 5 min after each pulse. Cell

lysate was collected after centrifugation at 12,000 x g for 10

min.

Human Brain Tissue

50 mg tissue (tumor or non-tumor brain tissue) was

weighed and washed with 0.5 mL of PBS (pH 7.4) contain-

ing protease inhibitor cocktail. Cell lysis was performed first

by four cycles of freeze-thaw method using liquid nitrogen

and then by 15 cycles of sonication on ice at 40% amplitude,

5 sec pulse with 5 sec gap. Centrifugation was performed at

10,000 x g for 10 min at 4°C and the cell lysate was used for

protein extraction using TRIzol method as discussed below.

TRIzol Protein Extraction Protocol

The TRIzol protocol was designed using

protocol with minor modifications. Complete protein extrac-

tion process from diverse sample types is described in (Table

3 & Fig. 1). In brief, to the cell lysate (obtained from any of

the above described samples), 1 mL of TRIzol reagent and

200 L of chloroform were added, mixed vigorously and

centrifugation was carried out at 14,000 x g for 15 min at

4°C. Upper clear phase was removed carefully and used for

RNA isolation. To the remaining phase 300 L of ethanol

was added and incubated at RT for 3 min followed by cen-

trifugation at 2,000 x g for 15 min at 4°C. To the super-

natant, 1:4 volume of ice-cold acetone (kept at -20°C) was

TRIzol Extraction Protocol for 2-DE, DIGE and Mass Spectrometry Current Proteomics, 2013, Vol. 10, No. 4 301

Fig. (1). Flow chart for TRIzol protein extraction protocol. TRIzol is a monophasic solution of guanidine isothiocyanate and phenol used to

separate major biomolecules in sequential steps. Chloroform addition leads to phase separation; aqueous phase contains RNA, while the

organic (phenolic) phase contains protein and DNA. Isopropanol precipitation of aqueous phase extracts RNA, which is further washed with

75% ethanol and dissolved in DEPC treated water. Ethanol precipitation of phenolic phase removes DNA followed by protein precipitation

with ice-cold acetone. Guanidium-HCl in 95% ethanol wash removes residual phenol from protein precipitant and further acetone wash

removes the salts. Finally, protein is dissolved in 2-DE compatible rehydration buffer.

Table 3. Summary of TRIzol protein extraction protocol.

Step 1: To the cell lysate (the cell number or tissue weight or volume of the sample mentioned above), 1 mL of TRIzol reagent (Invitrogen, USA) was

added and mixed well.

Step 2: To the above solution, 200 L of chloroform was added and vortexed for 20 sec. The tubes were kept at RT for 5 min to separate the phase and

tubes were centrifuged at 14,000 x g for 15 min at 4°C.

Step 3: The top colorless phase was collected carefully without disturbing the phase for RNA extraction using isopropanol and to the remaining phase,

300 L of absolute ethanol was added per mL of TRIzol and mixed the solution gently. The tubes were kept at RT for 5 min to precipitate the

DNA and centrifuged the tubes at 2000 x g for 15 min at 4°C.

Step 4: The supernatant was collected into a fresh tube and 1:4 volumes of ice-cold acetone (kept at -20°C) was added. Contents were mixed gently and

tubes were kept at 4°C at least for 30 min or overnight to precipitate the protein. Tubes were centrifuged at 14,000 x g for 5 min at 4°C.

Step 5: Protein precipitate was washed with 0.3 M guanidine-HCl in 95% ethanol. To the protein precipitant, 2 mL of 0.3 M guanidine-HCl in 95% etha-

nol was added and dispersed uniformly by sonicating the tubes at 25% amplitude for 1 min (2 sec pulse, 2 sec gap) on ice. Tubes were kept at 4°C

for 15 min and centrifuged the tubes at 14,000 x g for 5 min.

Step 6: Washing step was repeated thrice with 2 mL of 0.3 M guanidine-HCl in 95% ethanol without sonication in between to remove the residual phenol.

Step 7: Finally, 2 mL of chilled acetone was added to the protein pellet to remove the salt and kept the tubes at 4°C for 5 min. Tubes were centrifuged at

14,000 x g for 5 min. Washing step was repeated one more time.

Step 8: Protein pellet was air dried and redissolved in rehydration buffer (8 M urea, 2 M thiourea, 2-4% CHAPS (w/v), 40 mM DTT, 1% IPG buffer (v/v)

and bromophenol blue.

302 Current Proteomics, 2013, Vol. 10, No. 4 Reddy et al.

Table 4. Sample information used in present study for 2-DE and 2D-DIGE analysis.

Name of the Strain Buffer and Lysis MethodProtein Loaded for 2-DE/

IPG Strip

Protein Loaded for

2D-DIGE/ IPG Strip

No. of Spots in

2-DE

No. of Spots in

2D-DIGE

B. subtilis TE buffer, lysozyme and

sonication 600 g/ 24 cm (4-7 pH) 60 g/ 24 cm (4-7 pH) 1500±60 2000±50

S. coelicolor TE buffer, lysozyme and

sonication 800 g/ 24 cm (4-7 pH) - 1520±40 -

S. cerevisiae TE buffer having DTT,

bead beater 800 g/ 24 cm (4-7 pH) 60 g/ 24 cm (4-7 pH) 1150±30 1930±30

Human breast tissue

PBS buffer washing, ex-

traction using T-per tissue

buffer and sonication

1000 g/24 cm (3-10 pH) - 1400±100 -

Human brain tissue PBS buffer, freeze-thaw

and sonication - 60 g/ 18 cm (4-7 pH) - 1550±100

U87 glioblastoma cell

line RIPA buffer and sonication - 60 g/ 24 cm (4-7 pH) - 2070±140

added and kept for 30 min at -20°C for all the samples,

whereas in case of GBM tissue and GBM cell line overnight

precipitation was done at 4°C. Protein pellet was thoroughly

washed first with 0.3 M guanidium-HCl in 95% ethanol

three times and then two times with ice-cold acetone (kept at

-20°C). After air-drying, all protein pellets were dissolved in

rehydration buffer [7-8M urea, 2M thiourea, 2-4% CHAPS,

1% IPG buffer and BPB]. Protein quantification was per-

formed using either 2-D quant kit (GE healthcare) or Brad-

ford reagent (Bio Rad) with BSA standard.

Two-dimensional Gel Electrophoresis (2-DE) Analysis

B. subtilis, S. coelicolor and S. cerevisiae protein sam-

ples were rehydrated on 24 cm IPG strips of pH range 4-7

(GE healthcare), whereas breast malignant tissue protein was

loaded on 24 cm IPG strips of pH range 3-10 by adding 1%

DTT and 1% (v/v) respective IPG buffer. The amount of

protein load was different for each type of sample based on

literature reports (Table 4). First dimension was performed

using Ettan IPGphor 3 isoelectric focusing unit (GE

Healthcare) with following settings: 200V for 4 h (step and

hold), 500V for 1 h (step and hold), 1000V for 1 h (step and

hold), 8,000V for 13,500 Vh (gradient) and 8,000V for

76,000 Vh (step and hold), resulting in a total of ~ 93 .

Focused IPG strips were treated with equilibration buffer-I

and -II containing dithiothreitol (DTT) and iodoacetamide

(IAA) for 15 min each. The IPG strips were then placed onto

12.5% SDS-PAGE gels and the second dimension was car-

ried out. PhastGelTM

Blue R (GE healthcare) staining was

used for all the samples and gels were analyzed using IMP

7.0 software (GE Healthcare).

CyDye Labelling, 2D-DIGE and DeCyder Analysis

The protein extracts from B. subtilis, S. cerevisiae, hu-

man brain tissue and U87 glioblastoma cell line were la-

belled with CyDyes (Table 4). Prior to labeling, pH of the all

the protein extracts was adjusted to 8.5 using 100 mM

NaOH. For each sample, 60 g of protein was labeled with

400 pmol of CyDye for 60 min on ice in dark, followed by

quenching of unlabelled dye with 10 mM lysine. Cy3, Cy5

and Cy2 labeled samples were then pooled and B. subtilis, S. cerevisiae and U87 glioblastoma cell line proteins were re-

hydrated on 24 cm IPG strip of pH 4-7, while human brain

tumor tissue sample was rehydrated on 18 cm IPG strip of

pH 4-7. The protocol followed for IEF and SDS-PAGE was

similar to the conventional 2-DE protocol described above

for B. subtilis, S. cerevisiae and U87 glioblastoma cell line.

For human brain tumor tissue, the following settings were

used: 200V for 4 h (step and hold), 500V for 1 h (step and

hold), 1000V for 1 h (step and hold), 8,000V for 13,500 Vh

(gradient) and 8,000V for 60,000 Vh (step and hold), result-

ing in a total of ~78 . 2D-DIGE gels were scanned using

Typhoon FLA9500 scanner (GE healthcare) and the images

were analyzed using DeCyder software 7.0 (GE healthcare).

In-gel Digestion and Protein Identification by MALDI-TOF/TOF

Three spots were excised from gels containing S. coeli-color, human brain tumor tissue, U87 glioblastoma cell line

and breast tissue samples, whereas 6 spots were excised from

B. subtilis and S. cerevisiae gels. In-gel digestion was carried

out using trypsin after destaining with ACN (acetonitrile)

and ABC (ammonium bicarbonate) and extracted peptides

were subjected to MS/MS analysis. 4800 MALDI-TOF/TOF

mass spectrometer (AB Sciex, Framingham, MA) linked

with 4000 series explorer software (v.3.5.3) was used for

protein identification, which was calibrated with standard

peptide mix before sample analysis. The instrument was

operated in reflectron mode using Nd:YAG 355 nm

laser, with a mass range of 800-4000 Da. MS and

MS/MS peak list was subjected to MASCOT version 2.1

(http://www.martixscience.com) search engine for protein

identification after removing the autolyzed trypsin peaks

with GPSTM

Explorer software version 3.6 (AB Sciex). The

parameters used for mascot search were as follows: trypsin

digestion, single missed cleavage, oxidation of methionine

and carbamidomethylation of cysteine as variable and fixed

TRIzol Extraction Protocol for 2-DE, DIGE and Mass Spectrometry Current Proteomics, 2013, Vol. 10, No. 4 303

modifications respectively, 75 ppm and 0.4 Da mass toler-

ances for MS and MS/MS and Swiss-Prot database. The tax-

onomy was set as B. subtilis for B. subtilis, S. coelicolor for

S. coelicolor, S. cerevisiae for S. cerevisiae and Homo sapiens for breast tissue, brain tissue and U87 glioblastoma

cell line.

RNA Extraction

During processing, the clear aqueous phase was used for

RNA extraction from B. subtilis, S. cerevisiae, human brain

tumor tissue and U87 glioblastoma cell line as per

protocol. In brief, 1 mL of isopropanol was added to

the upper aqueous layer and tubes were allowed to stand at

RT for 5 min to precipitate the RNA. Centrifugation was

performed at 7500 x g and the resulting pellet was washed

twice with 75% ethanol. Precipitated RNA was solubilized

in DEPC treated water.

RESULTS

TRIzol Protein Extraction and Two-dimensional Elec-

trophoresis

Over the past two decades, solvent precipitation methods

have been employed for protein extraction, which effectively

removes the non-protein materials. These methods are gen-

erally used to achieve higher protein yield with a larger rep-

ertoire of proteins, enabling studies involving comparative

analysis. However, frequently, solvent based protein extrac-

tion protocols need to be supplemented with desalting meth-

ods like 2-DE clean-up kits or dialysis using MWCO col-

Fig. (2). Representative images of 2-DE gels obtained from diverse samples using TRIzol extraction protocol. 2-DE gel images from (A) B.

subtilis, (B) S. coelicolor, (C) S. cerevisiae SK1 background strain, (D) Breast cancer tissue, and (E) histogram representing the number of

spots obtained from individual samples using TRIzol extraction (n=3).

304 Current Proteomics, 2013, Vol. 10, No. 4 Reddy et al.

umns, to remove the excessive salts and other non-protein

material to improve the gel quality and coverage. Employing

such strategies prove to be costly, time-intensive and involve

protein loss at every stage [20]. Though there are a few re-

ports where TRIzol protein extraction has been used for

high-throughput proteomic analysis (Table 2), its applicabil-

ity for diverse biological samples is still missing. In this

study, we have tried to address this issue and establish a

simple protein extraction protocol involving TRIzol reagent

on diverse biological samples, which could be used for

global proteome profiling applications.

As a proof-of-concept, we selected B. subtilis, S. coeli-

color, S. cerevisiae and breast cancer tissue for 2-DE, while

B. subtilis, S. cerevisiae, U87 glioblastoma cell line and hu-

man brain tissue were selected for 2D-DIGE owing to the

Fig. (3). Representative images of 2D-DIGE gels obtained from diverse samples using TRIzol extraction protocol. 2D-DIGE images from

(A) B. subtilis, (B) U87 glioblastoma cell line, (C) Human brain tissue and (D) S. cerevisiae SK1 background strain are presented. Histogram

representing the number of spots obtained from individual samples using TRIzol extraction (n=3) is shown in (E).

TRIzol Extraction Protocol for 2-DE, DIGE and Mass Spectrometry Current Proteomics, 2013, Vol. 10, No. 4 305

differences in their origin. Cell lysis was performed differ-

ently for each sample due to the diverse nature of the cell

wall and cell membrane composition, while the same TRIzol

protein extraction protocol was employed for all. Further,

comparison of triplicate gels for each sample demonstrated

good reproducibility, coverage and resolution with minimum

streaking (Fig. 2 & 3). High quality gel images were ob-

tained for all the samples. B. subtilis and yeast were consid-

ered for both 2-DE and 2D-DIGE to compare the effect of

TRIzol extraction on protein quality and CyDye labeling.

The comparison has shown a similar proteome pattern with

more spots on 2D-DIGE as expected.

Data Analysis of 2-DE and 2D-DIGE Images

B. subtilis, S. coelicolor, S. cerevisiae and breast tumor

tissue samples were subjected to 2-DE and the spot visuali-

zation was performed with PhastGelTM

Blue R (GE

healthcare). Triplicate gels from each sample were imported

into IMP7 software and analyzed using the spot detection

application and by making match sets for each sample type

separately. Initially the spot detection was done automati-

cally and then curated manually. Spot detection revealed

approximately 1500, 1500, 1200 and 1400 spots in B. sub-tilis, S. coelicolor, S. cerevisiae and breast tumor tissue re-

spectively (Table 4, Fig. 2). Most of the spots were repro-

ducibly present in all triplicate gels. Three spots showing

different intensities were excised from each sample type for

downstream MALDI-TOF/TOF analysis.

Similarly to 2-DE, four different samples, viz., B. subtilis, S. cerevisiae, U87 glioblastoma cell line and human brain

tumor tissue were selected for protein separation using 2D-

DIGE. Samples were labeled with CyDyes such as Cy3, Cy5

and Cy2 and then run in triplicate. 2D-DIGE images were

imported into DeCyder software (GE healthcare) by making

projects for each sample type separately. Differential in gel

analysis (DIA) and biological variation analysis (BVA) was

performed for each sample type. The number of spots was

seen to improve significantly over the 2-DE result for B. subtilis and S. cerevisiae. The spot number in U87 glioblas-

toma cell line and B. subtilis were approximately 2000 spots,

whereas in case of yeast and human brain tumor tissue 1950

and 1550 spots were detected respectively with good repro-

ducibility among the triplicate gels (Table 4, Fig. 3). Three

spots were excised from each sample type by running the

preparative gels for downstream MALDI analysis.

Protein Identification using MALDI-TOF/TOF

To observe the effect of TRIzol extraction on protein

identification, three spots were excised from each of the B. subtilis, S. coelicolor, breast malignant tissue and yeast 2-DE

gels with different intensities, processed for in-gel digestion

using trypsin (Promega) and analyzed using MALDI-

TOF/TOF (Absciex). The identity of the protein was estab-

lished for all samples irrespective of spot intensity, with

good score and coverage. Similarly, three differentially ex-

pressed spots, identified by 2D-DIGE analysis, were excised

from each of the B. subtilis, yeast, human brain tumor tissue

and U87 glioblastoma cell line preparative gels, and identi-

fied using MALDI-TOF/TOF analysis. Hence, a total of

three spots were analyzed each for S. coelicolor, breast ma-

lignant tissue, human brain tumor tissue and U87 glioblas-

toma cell line whereas six spots were analyzed for B. subtilis

and yeast (three spots for 2-DE and three spots for 2D-

DIGE). The sequence coverage and the score obtained for

each spot from diverse sample types was very good despite

absence of any desalting step (Table 5).

Quantitative and Qualitative Analysis of RNA

Though our major focus was on good quality protein ex-

traction, we also extracted RNA from four samples during

sequential processing. The quantity of RNA from B. subtilis,

S. cerevisiae, U87 glioblastoma cell line and human brain

tissue was examined with GeneQuant 100 (GE healthcare,

USA) by measuring 260/280 and 260/230 ratios (data not

shown). Also, the quality of RNA samples was analyzed on

1% agarose gels under denaturing conditions (Fig. 4). The

integrity and quality of the extracted RNA was found to be

good for transcriptomic studies using RT-PCR analysis.

Though many specialized methods are currently available for

the extraction of DNA, RNA and proteins separately, the

TRIzol extraction method offers sequential separation of all

three major biomolecules from a single sample in a quick

and reproducible manner. This enables genomic, transcrip-

tomic and proteomic analysis of the same cell mass and

minimizes the biological variations [21].

Fig. (4). Ascertaining RNA quality. The quality of RNA was

checked on 1% denaturing agarose gel prepared in DEPC treated

water. (A) Glioma U87 Cell line, (B) GBM tumor tissue, (C) B.

subtilis and (D) S. cerevisiae. 1-indicates 28S rRNA band in case of

glioma U87 Cell line, GBM tumor tissue and S. cerevisiae, whereas

23S rRNA band in case of B. subtilis. 2-indicates 18S rRNA band

in case of glioma U87 Cell line, GBM tumor tissue and S.

cerevisiae whereas 16S rRNA band in case of B. subtilis. 3-

indicates 5.8S/5S rRNA.

DISCUSSION

The quality of the gel directly reflects the efficiency of

protein extract, which in turn is dependent on the method

used for sample preparation. Isoelectric focusing (IEF) is

very sensitive to salts and interfering agents, which lead to

poor focusing. B. subtilis, S. coelicolor, yeast and U87

glioblastoma cell line having high quantity of salts, nucleic

acids, polysaccharides, pigments etc., whereas breast tumor

tissue and human brain tumor tissue have high content of

adipose cells which can interfere with protein separation.

Removal of these interfering agents is necessary to obtain

good quality gels for global profiling.

During TRIzol protein extraction, the guanidium salt in-

hibits activity of both proteases and RNAses, and enhances

the integrity and quality of both protein and RNA. Further,

306 Current Proteomics, 2013, Vol. 10, No. 4 Reddy et al.

Table 5. Details of 2-DE, 2D-DIGE & MALDI-TOF/TOF analysis of the spots excised from the samples.

Spot No. Uniprot ID Theoretical M.Wt/pH No. of Peptides Seq. Coverage Score

B. subtilis

1 P42415 64/5.3 24 43 1120

2 P39645 30/5.15 18 58 707

3 P80865 31.23/5.66 18 57 519

13 P30950 36.2/5.14 2 11 133

14 P17820 66/4.76 5 15 304

15 P94391 56.4/5.5 6 14 96

S. coelicolor

4 O88018 40.38/4.67 17 45 414

5 P58481 71.58/4.86 12 - 73

6 Q8CJQ8 105/9.47 9 63 177

S. cerevisiae

7 A66WL1 17.0/6.1 3 45 304

8 P34760 21.5/5 4 18 173

9 P29311 30/4.82 1 5 57

22 P10591 69.6/4.99 3 7 194

23 P04806 53.7/5.28 5 20 289

24 P60010 41.6/5.44 2 4 60

Breast tissue

10 Q15181 33/5.54 17 61 466

11 P14550 36.8/6.32 20 64 528

12 P07355 38.8/7.57 29 74 988

U87 glioblastoma cell line

16 P46108 33.3/5.44 9 35 67

17 P60174 30.7/5.65 15 61 189

18 P31949 11.7/6.56 3 35 131

Glioblastoma tissue

19 P62258 26.5/4.76 16 ------ 632

20 P07195 36.6/5.71 17 ------ 1010

21 E9PGK1 72.4/5.41 21 ------ 1080

isopropanol and ethanol precipitate the RNA and DNA re-

spectively, thus removing the chances of nucleic acid con-

tamination. The phenol/chloroform dissolves lipids, enhanc-

ing the membrane proteome coverage in soluble fraction and

also removes the polysaccharide contamination. In addition,

acetone helps to precipitate protein and removes the salt con-

tamination. Further, washing the protein pellet with guanid-

ium-HCl in 95% ethanol improves the protein quality by

removing the residual phenol content. Thus, TRIzol extrac-

tion method is able to recover RNA, DNA and protein with

high precision and quality. The quality of 2-DE and 2D-

DIGE gels obtained in our lab using protein samples ob-

tained by employing TRIzol extraction method was excellent

with minimum streaking and maximum efficiency in protein

recovery. The gels were quite comparable with those re-

ported in literature using gold standard methods for protein

extraction (Table 6).

PhastGel Blue R staining of 2-DE gels showed more than

1000 spots, whereas 2D-DIGE images showed 1500 to 2000

TRIzol Extraction Protocol for 2-DE, DIGE and Mass Spectrometry Current Proteomics, 2013, Vol. 10, No. 4 307

Table 6. Literature information for the samples analyzed in our study with different protein extraction protocol.

Sl. No. Sample

Amount of

Protein

Loaded

No. of Spots

pH of IPG

Strip/pH

Range

Length of the

IPG Strip Staining Refs.

I. Human Glioma Tumor Tissue

1 Glioma brain tumor

tissue

50 g 700 3-10 NL 24 cm Cy-Dyes [33]

2 Glioma brain tumor

tissue

NM 350 3-10 7 cm Silver stain [34]

3 Glioma brain tumor

tissue

100 g 1000 4-7 18 cm SYPRO Ruby [35]

4 Glioma Tumor tissue 185 g 500 4-7 11 cm Silver stain [36]

5 Glioma Tumor tissue 25 g 1465 (matched

spots)

3-10 NL 24 cm Cy-Dyes [37]

6 Oligodendroglioma 50 g 1458 4-7 18 cm Cy-Dyes [38]

II. Human Breast Tumor tissue

1 Breast Tissue 50-100 g NM 3-10 NL 18 cm Silver Staining [20]

2 Breast Tissue Lysate 100 g 302 7-10 11cm Sypro Ruby [39]

3 Breast Tissue (prefrac-

tionated basic protein

from 100 g Lysate)

100 g 169 7-10 11 cm Sypro Ruby [39]

4 Breast Tissue (prefrac-

tionated basic protein

from 1000 g Lysate)

1000 g 405 7-10 11 cm Sypro Ruby [39]

5 Breast Tissue 250 mg NM 4-7 11 cm CBB-250 [40]

6 Breast Tissue 150 g NM 4-7 7 cm CBB-250 [41]

III. Bacillus subtilis

1759 (HPE) 1 B. subtilis 168 200 g

1399 (Conventional 2-

DE)

4-7 18 cm LavaPurple (Fluro-

scence stain)

[42]

2 B. subtilis 50-80 g 1800 4-7 NM Silver Staining

(Analytical gel)

[43]

200 g 700 4-7

400 g 219 4–5

400 g 569 4.5–5.5

400 g 318 5–6

3 B. subtilis 168

400 g 120 5.5–6.7

18 cm Colloidal CBB,

SYPRO Ruby or

Silver Nitrate

[44]

4 B. subtilis 168 300 g NM 11 cm 3-10 Silver Stain Plus [45]

5 B. cereus 50 g 1600 24 cm 4-7 DIGE [46]

6 B. subtilis 168 50 g 1085 (matched spots) - 4-7 DIGE [47]

308 Current Proteomics, 2013, Vol. 10, No. 4 Reddy et al.

Table 6. contd….

Sl. No. Sample

Amount of

Protein

Loaded

No. of Spots

pH of IPG

Strip/pH

Range

Length of the

IPG Strip Staining Refs.

IV. Streptomyces coelicolor

1 S. coelicolor 500 g 450 4-7 18 cm CBB [48]

2 S. coelicolor

M145

200 - 290 g

782

4-7 24 cm SYPRO Ruby [49]

NM 1051 4-7

1555 4.5-5.5

913 5.5-6.7

3 S. coelicolor M145

379 6-11

18cm Silver Staining [50]

4 S. coelicolor M145 200 g 1600 (approx) 4-10 NM Silver Staining [51]

5 S. coelicolor M145 400 g NM 4-7 24 cm Lava Purple

Fluorescent staining

[52]

V. Saccharomyces cerevisiae

1 S. cerevisiae BY4741

and its isogenic potas-

sium transport double

mutant (trk1 ,2 ) strain

500 g 178-307 5-8 17 cm CBB G-250 [53]

2 S. cerevisiae

H2490

75 g 833±83 3-10NL 18 cm Silver stain [54]

3 S. cerevisiae W303-1A 350 g 1000 3-10NL 18 cm CBB [55]

4 S. cerevisiae 800 g 600 4-7 18 cm CBB [56]

5 S. cerevisiae

KNU5377 strain

1.2 mg NM 4-7 17 cm CBB R-250 [57]

6 S. cerevisiae SK1 35 g 1400 4-7 24 cm Cy-Dyes [22]

7 S. cerevisiae Wild type

strain CEN.PK113–7D

50 g NM 3-10NL 24 cm Cy-Dyes [58]

8. Yeast strains TMB

3399 and TMB 3400

50 g 3261 3-10NL 24 cm Cy-Dyes [59]

9 S. cerevisiae (SEY, Lot

No. SE-84) and

(RY, Lot No. 07078)

50 g NM 3-10 24 cm Cy-Dyes [60]

VI. Glioma Cell Lines

1 GBM cell lines (U251) 60 g NM 3-10 NL 18 cm Cy-Dyes [61]

2 A172, Hs683, T98G,

U87, and U251 human

glioma cell lines

50 g NM 3-10 24 cm Cy-Dyes [62]

3 U87 MG astrocytoma

cell line

75 g NM 3-10 NL 17 cm Cy-Dyes [63]

4 GBM cell line U251 50 g NM 4-7 24 cm Cy-Dyes [64]

5 HTB12 human

Astrocytoma cells

NM NM 3-10 13 cm Cy-Dyes [65]

NM - Not mentioned

TRIzol Extraction Protocol for 2-DE, DIGE and Mass Spectrometry Current Proteomics, 2013, Vol. 10, No. 4 309

spots with good reproducibility across triplicate gel images

for each sample. Both 2-DE and 2D-DIGE images were very

clean with minimal streaking. Though the gels obtained from

S. cerevisiae showed minor streaking, this trend was ob-

served in most of the studies on this organism reported so far

with IPG strips in the pH range of 4-7 [22]. It probably indi-

cates that most of the S. cerevisiae proteins have pH beyond

7 and are therefore unable to separate on strips of pH 4-7

range (Fig. 2). Presence of a high number of adipose cells

interferes with protein extraction from breast cancer tissue

and human brain tissue, enhancing the need for additional

steps to overcome the problems associated with focusing and

protein profiling. However, the use of TRIzol protocol re-

sulted in protein extracts, which showed good results on the

gels without employing additional clean-up methods.

Further, comparison of 2-DE and 2D-DIGE images from

B. subtilis and yeast have shown that TRIzol protein extrac-

tion has no adverse effect on CyDye labeling of the proteins.

In addition, the image quality of the 2D-DIGE gels was

found to be comparable with those reported in literature [14-

17]. The protein resolution pattern of each sample was uni-

form throughout the gel and both high as well as the low

molecular weight proteins were resolved on the gel.

On the whole, for the first time we a pro-

tein extraction protocol using TRIzol, which is applicable for

diverse samples and compatible with 2-DE, 2D-DIGE and

MALDI-TOF/TOF analysis. In our laboratory, this protocol

has consistently provided good coverage and resolution with

negligible streaking for B. subtilis, S. coelicolor, S. cere-visiae, cell line and human tissue samples. TRIzol reagent

has also been successfully used for extracting proteins from

other samples like dust mites, Haloferax volcanii, Dinoflag-ellate, A. catenella, spinal cord, kidney, liver tissues, MCF-7

and SH-SY5Y cell line as reported in literature. Based on

these results we can conclude that TRIzol extraction can

overcome the limitations posed by solvent based protein ex-

traction protocols, which need additional desalting methods.

TRIzol extraction method scores over other protocols since it

excludes the desalting or clean-up step, saving both time and

cost. In addition, this protocol performs concurrent extrac-

tion of high quality RNA, DNA and protein, and provides an

opportunity for systems level analysis even from limited

amounts of starting material.

CONFLICT OF INTEREST

The authors confirm that this article content has no

conflict of interest.

We would like to acknowledge Profs. Sarika Mehra and

Santanu K Ghosh for the culture work in their laboratories

on S. coelicolor and S. cerevisiae respectively.

FUNDING

This research was supported by a start-up grant

09IRCC007 from the IIT Bombay to SS. PJR was supported

by the IIT Bombay fellowship and RK was supported by

CSIR fellowship.

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Received: June 14, 2013 Revised: September 10, 2013 Accepted: October 01, 2013