roteomics 2012, 12, 938943 DOI 10.1002/pmic.

201100475 TECHNICAL BRIEF

Improving gel-based proteome analysis of soluble protein extracts by heat prefractionation
Wei Wang, Xiaolin Wu, Erhui Xiong and Fuju Tai Key Laboratory of Physiological
Ecology and Genetic Improvement of Food Crops in Henan Province, Henan Agricultural
University, China The presence of high-abundance proteins in complex protein mixtures
often masks low-
abundanceproteinsandcauseslossofresolutionof2DE.Proteinfractionationstepsconducted
prior to 2DE can enhance the detection of low-abundance proteins and improve the
resolution of 2DE. Here, we report a method to prefractionate soluble protein extracts based
on protein thermal denaturation. Soluble proteins were extracted from maize embryos and
leaves and Escherichia coli cells. Through heating at 95?C for 5 min, soluble protein extracts
were pre- fractionated as heat stable protein fraction (the supernatant) and heat labile
protein fraction (the precipitate). Our results showed that heat prefractionation enhanced
the separation of proteins in both fractions by 2DE, thereby increasing the chance of
detecting low-abundance proteins, many of which were nonvisible in unfractionated extract.
In maize embryo, 330 spots were detected in soluble protein extract, while 577 spots were
detected after prefractionation. Furthermore, this prefractionation method facilitated the
enrichment, detection, and identi- fication of de novo synthesized stress proteins. Because
of its simplicity, the one-step heat prefractionation minimizes protein loss. Finally, heat
prefractionation requires no expensive special hardware or reagents, and provides an
alternative prefractionation for increasing the resolving power of 2DE. Keywords: 2DE /
Low-abundance proteins / Prefractionation technique / Technology / Thermal denaturing
Received: September 12, 2011 Revised: December 18, 2011 Accepted: January 16, 2012
Proteomeanalysisismostcommonlyaccomplishedbyacom-
binationof2DEtoseparateandvisualizeproteinsandMSfor protein identification. 2DE-MS
strategy has proven to be a re- liable and efficient means of proteome analysis [1]. However,
questions remain concerning its ability to characterize all of the elements (especially low-
abundance proteins) of a pro- teome, because the presence of high-abundance proteins in
complex proteomes often masks low-abundance species and causes loss of resolution in
2DE [2,3]. This problem can be partially alleviated by sample prefrac- tionation using a
variety of techniques such as differential protein extraction, purification of cell organelles or
protein Correspondence: Dr. Fuju Tai, College of Life Science, Henan Agri- cultural
University, Rd. Agriculture 63, Zhengzhou 450002, China E-mail: botany2@gmail.com Fax:
+86-371-63555652 Abbreviations: trichloroacetic acid LEA, lateembryogenesisabundant;
TCA, complexes, preparative isoelectric focusing (IEF), and chro- matographic techniques
[46]. Reducing sample complex- ity through efficient fractionation allows the
comprehensive analysisofcomplexproteinmixtureswith2DE-MS.However, many of the
prefractionation methods are time-consuming, result in protein loss and often require
expensive instrumen- tation. The heat stability of proteins is a property inherent to their
structure [7,8]. In the present study, we develop a method to prefractionate soluble protein
extracts based on protein ther- mal denaturation [9]. We also design a protocol to efficiently
resolublize and recover heat labile proteins. We demonstrate that heat prefractionation can
facilitate the separation, detec- tion, and identification of proteins (especially low-
abundance species) in complex proteomes. Factors that affect the heat prefractionation are
also evaluated. The 2DE maps and other These authors contributed equally to this study. C
?2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
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Proteomics 2012, 12, 938943 939 data presented here are representative results from
three biological replicates. The source of proteins included maize embryos and leaves and
Escherichia coli cells. Sample (0.1 g fresh weight) was ground in liquid N2using a mortar
and pestle into a fine powder. The tissue powder was placed in a microcen- trifuge tube and
then washed with cold acetone containing 5 mM DTT. After vortexing thoroughly for 30 s,
the tube was centrifuged at 15 000 g for 5 min (4?C). The resultant pellet was washed once
more with cold acetone containing 5 mM DTT and air-dried. For soluble protein extraction,
the dry powder was homogenized in 2.0 mL of 0.25 M Tris-HCl, pH 6.8 in a cold mortar.
The homogenate was transferred to a 2.0-mL tube and shaken for 10 min at 240 r/min
(4?C) on an orbital shaker (AROS USA). The homogenate was centrifuged twice at 15 000
g for 10 min (4?C) to remove insoluble debris. The resulting supernatant (designated as
soluble protein extract) was pipet- ted into a microcentrifuge tube and heated at 95?C
(water bath) for 5 min. It was then cooled on ice and centrifuged (15 000 g, 10 min, 4?C).
The precipitate (containing heat labile proteins) and the supernatant (containing heat stable
proteins) were collected respectively. Proteinprecipitatefromheatdenaturingwasdifficulttobe
dissolved. We found that the basic SDS solution was effective to dissolve the heat labile
proteins. Therefore, the precipitate fraction was first suspended in 800 ?L of the basic
solution (pH 12) containing 25 mM Tris, 1% SDS, 20 mM DTT, and 5 mM EDTA. Upon
completion of the solubilization, the heat labile protein extract was buffered to pH 8.0 by
the addition of 200 ?L of 0.96 M glycine. Meanwhile, 100 ?L of 0.25 M Tris was added to
900 ?L of the heat stable pro- tein extract to increase the pH to 8.0. The pH adjustment was
essential for phenol extraction, because only in the basic condition can proteins in the
aqueous phase effectively trans- TM, Barnstead/Thermolyne, NH, fer into the phenol phase
[10, 11]. Afterwards, the extracts of heat stable and heat labile proteins and the initial solu-
ble extracts were mixed with equal volume phenol (pH 8.0, Sigma), respectively. The
mixture was thoroughly vortexed for 3 min and the phase separation was accomplished by
cen- trifugation at 12 000 g for 3 min. The phenol phase was pipetted to fresh microtubes
and precipitated as described previously [11]. Recovered heat stable proteins and heat la-
bile proteins were subjected to electrophoresis separation and gels were stained with 0.01%
Coomassie Brilliant Blue (CBB) R350. Digital images of the gels were analyzed using
PDQuest 2-D Analysis Software (Version 6.2, Bio-Rad, Bath, UK). The protein spots of
interest from maize tissues were aseptically removed under a laminar flow hood. Processing
of gel plugs, trypsin digestion, MALDI-TOF-MS/MS anal- ysis using Ettan MALDI-TOF Pro
mass spectrometer (GE Healthcare, Uppsala, Sweden) were performed as described
previously [12]. The MS and MS/MS spectra obtained were automatically matched to
proteins in NCBI databases (search date, May 6, 2011, http://www.ncbi.nlm.nih.gov/) with
the Mascot software (v2.2.03, http://www.matrixscience.com/). The following parameters
were adopted for database search: complete carbamidomethylation of cysteines and partial
ox- idation of methionines, peptide mass tolerance 1.2 Da, fragment mass tolerance 0.9
Da, and missed cleavages 2. Searches were performed in the full range of Mr and pI. No
species restriction was applied. All of the positive pro- tein identification scores were
significant (p <0.05, score >60). Functional categorization of identified proteins was
performed using annotation in NCBI and UniProtKB/Swiss- Prot
(http://www.ebi.ac.uk/swissprot/) databases. The heat prefractionation scheme was
outlined in Fig. 1A. Inbrief,solubleproteinextractwasincubatedat95?Cinorder to obtain heat
labile and heat stable proteins. Afterwards, the mixture was centrifuged and separated into
two fractions: the Figure 1. (A) Workflow for protein fractionation by heating method.
Soluble protein extract was prefrac- tionated into the supernatant and the precipitate by
heating at 95?C for 5 min and proteins in both frac- tions were recovered by phenol ex-
traction and processed for 2DE. (B) SDS-PAGEanalysistoevaluateheat prefractionation.
Lane 1, the initial soluble extract; Lane 2, the heat sta- ble protein; Lane 3, the heat labile
protein.Theloadingamountwas20 ?g protein each lane for maize em-
bryo,30?geachlaneforE.coli.Pro- teins were resolved in 12.5% SDS- PAGE gels and
visualized using CBB R 350. C ?2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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940 W. Wang et al.Proteomics 2012, 12, 938943 Figure 2. 2DE evaluation of heat
prefractionation. The image displays three representative CBB-stained gels out of a total of
nine maps from three independent experiments. Upper panel: 2DE maps of the soluble
protein extract of maize embryo. (A) Unfractionated soluble extract; (B) heat stable
proteins; (C) heat labile proteins. Proteins (450 ?g) dissolved in 250 ?L rehydration buffer
and loaded via overnight rehydration into 11 cm linear IPG strips (pH 47). IEF was
performed with an Ettan III system (GE Healthcare, USA) at 300 V for 1 h, 3000 V for 1 h,
and 6000 V for 10 h (20?C). Focused strips were resolved in 12.5% SDS/acrylamide gels.
Gels were stained using CBB R350. (D) Venn diagram displays the overlap of 2DE separated
proteins between the soluble protein extract and the fractions from maize embryos. Lower
panel: expanded regions of corresponding sections of soluble protein extract and fractions.
Spots of interest detected are indicated with names, other enriched spots in each fraction
are indicated by arrows. supernatant (containing heat stable proteins) and the precip- itate
(containing heat labile proteins). The mass ratio of the heat stable proteins to the heat labile
proteins varies with the materials. It was 70:30, 53:47, and 60:40 for maize embryos,
shoots, and root, respectively; and approximately 30:70 for E. coli cells. SDS-PAGE (Fig. 1B)
and 2DE (Fig. 2, Support- ing Information Fig. S1) analysis revealed great differences in
protein profiles among heat stable proteins, heat labile proteins, and the soluble protein
extract. After heat prefrac- tionation,themostobviouschangeisthatsomeproteinswere
enrichedinthesupernatant,whileotherswereenrichedinthe precipitate. However, spot-to-spot
comparison with PDQuest software indicated that location of related spots in gels was
sufficiently consistent among heat stable proteins, heat la- bile proteins, and the soluble
protein extract, suggesting heat prefractionation did not result in visible changes in Mr and
pI of proteins. Furthermore, heat prefractionation had high reproducibility. Spot-to-spot
comparison of two groups of 2D gels from two independent experiments showed that the
cor- responding gels shared almost the same numbers of protein spots and patterns, with
86% or 87% spots matched (Sup- porting Information Fig. 2S). The effectiveness of heat
prefractionation in reducing sample complexity was further demonstrated by comparison
ofthequalitativeandquantitativedifferencesinthe2Dgelsof heat stable proteins, heat labile
proteins, and the soluble pro- tein extract (Fig. 2). The heat labile protein fraction contained
substantially higher levels of storage proteins than either the heat stable protein fraction or
soluble protein extract, indi- cating that these high-abundant proteins could be fractioned
simplybyheatingthesample.Basedonequalproteinloading, we observed the overall
enrichment of proteins both in the heat stable protein fraction and the heat labile protein
frac- tion compared to the soluble protein extract, with substantial spots exclusive to the
heat stable protein fraction or the heat labile protein fraction. For example, many low-
abundance protein spots in a zoom-in image of a section became visible
(indicatedbyarrows)intheheatstableproteinfraction(Fig.2 I, 2 III) but not in soluble
protein extract. The enrichment was obvious for several proteins (e.g. EMB564, embryo
specific protein 5, comparison of Fig. 2A and 2B), indicating that heat prefractionation
would be useful in protein purifi- cation combined with other approaches. Moreover, after
heat fractionation, four adjacent spots in the 2DE map of soluble protein extract were,
respectively, separated into the heat stable protein fraction (two spots) and the heat labile
protein fraction (two spots) (Fig. 2, region IV, arrows), enabling pick- ing of individual
spots. As a result (Supporting Information Table S1), the four spots were identified by
MALDI-TOF as two isoforms of embryonic protein DC-8 and two isoforms of C ?2012
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Proteomics 2012, 12, 938943 941 Figure 3. Improved 2DE analysis of stress-induced
proteins in maize leaves after heat prefractionation. Maize seedlings were exposed to high
temperature and leaf-soluble proteins were extracted and prefractionated as described in
the text. Corresponding regions of control and stress samples are shown and stress-induced
spots detected upon fractionation are indicated. Proteins (450 ?g) were resolved by 2DE as
described in Fig. 2 legends. short-chain dehydrogenase/reductase (SDR). In this regard,
heat prefractionation reduced proteome complexity and improved protein separation and
identification. Another way to judge the heat fractionation quality was to look at the number
of distinct proteins detected in each fraction. Venn diagram displays the overlap of 2DE
separated proteins among the two fractions and the soluble protein ex- tract (Fig. 2D). For
maize embryo, 330 spots were detected in the soluble protein extract, while total 577 spots
were de- tected in the heat stable proteins fraction and the heat labile proteins fraction.
Clearly, there is minimal overlap between the heat stable and heat labile fractions, as about
15% of de- tected protein species appear at the same Mr/pI position in both fractions.
Therefore, heat prefractionation allows detect- ing more numbers of protein spots. In this
study, we applied CBB R350 staining for protein visualization on gels, but the use of more
sensitive silver or fluorescence staining meth- ods would allow the in-depth analysis of heat-
prefractionated proteomes. We further investigated the utility of heat prefractionation for
increasing the resolving power of 2DE-based proteome analysis in the detection of de novo
synthesized proteins. Sol- ubleproteinswereextractedfrommaizeleavesofcontroland stressed
seedlings exposed to heat stress (41?C for 11 h) and prefractionated as above. An area of
2DE gel (Fig. 3A) with typicalspotdensitywasselectedformoredetailedanalysis,in which the
expression of several proteins was significantly in- creased after heat stress (indicated by
numbers, comparison of Fig. 3B and 3C), while neighboring proteins had similar
levelsofstaining,suggestingthattheseup-regulatedproteins were modulated due to heat
stress. After heat prefractiona- tion of the soluble protein extracts, the changes in protein
abundances were further examined by comparison of each fraction. Actually, the up-
regulated proteins were heat stable (spots 110), which were enriched in the supernatant
after heat prefractionation (Fig. 3D and 3E). Besides, spots 9 and 10 appeared highly up-
regulated in abundance in the initial soluble extracts (comparing Fig. 3B with 3C), however,
the comparison of the heat stable proteins (Fig. 3D and 3E) and
theheatlabileproteins(Fig.3Fand3G)indicatedthatthough both spots were up-regulated, the
difference was not to such an extent as it was in the initial soluble extracts. Thus, heat
prefractionation provides an alternative way to further detect
denovosynthesizedstressproteinsbycomparisonofspecific protein abundance in each
fraction. Heat prefractionation was compatible with MS, and the majority of selected
protein spots were successfully identi- fied by MALDI-TOF analysis (Supporting
Information Table S1). In maize embryos, we identified several heat stable pro- teins,
including EMB564 (NP 001105429 GenBank acces- sion, the same below), two embryonic
protein DC-8 isoforms (ACG48956 and ACG37499), embryonic abundant protein 1
(ACG33377), and embryo specific protein5 (NP 001105349). In maize leaves, the identified
heat stable proteins included C ?2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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942 W. Wang et al.Proteomics 2012, 12, 938943 small heat shock proteins 16.9 (spots 2, 4,
and 6), 17.2 (spot 3) and 17.4 (spot 5), photosystem I reaction center subunit IV (spot 1),
ribosome recycling factor (spot 8), and abscisic stress ripening protein (spot 7). We
compared the amino acid compositions of heat stable proteins and heat labile proteins
identified here. Maize embryo heat stable proteins contained no Cys, but high amounts of
Glu and Gly, which was sim- ilar to a previous report [6]; however, the heat labile pro- teins
identified in maize embryos, e.g. two SDR isoforms (ACZ54904 and CAV22174), globulin 1
(ACG48473), and globulin 2 (1802402A), contained Cys and a high amount of nonpolar
amino acids (Ile, Phe, Pro, and Val) (Supporting Information Table S2). Eight maize leaf
heat stable proteins had high amounts of both polar amino acids (Glu, Lys, Ser) and
nonpolar amino acids (Val, Phe, Pro), which was some- what different from maize embryo
heat stable proteins. Leaf heat stable proteins did not contain Cys.
Weobservedthatsamplepretreatmentandextractioncom- positions greatly affected the
precipitation of heat labile pro- teins. In the presence of 5 mM EDTA, proteins were more
resistant to thermal denaturing and the amount of heat labile proteins was considerably
reduced. The effect of EDTA could be enhanced by 20 mM DTT or 15% glycerol. Grinding
sam- ple in 10% trichloroacetic acid (TCA) or 10% TCA/acetone resulted in a reduced
amount of extractable heat labile pro-
teinscomparedtoacetonealone,asmanyproteinbandswere weaker in the TCA-precipitated
samples resolved by SDS- PAGE (Supporting Information Fig. S3). This might be due to
inability of certain proteins to dissolve, suggesting that these proteins were susceptible to
irreversible denaturation by TCA. The heat stability of proteins is a property inherent to
their structures, or it might be acquired by evolution for their spe- cialized functions [8]. In
E. coli, many identified heat stable proteins are molecular chaperones functioning in the
pro- tection of other proteins against denaturation. In plants, late embryogenesis abundant
(LEA) proteins are low complexity, highly hydrophilic, unordered proteins in the hydrated
state, and heat stable after boiling [13]. Many heat stable LEA pro- teins are associated with
desiccation tolerance in radicles of Medicago truncatula seeds [7]. In this study, most
identified heat stable proteins were recognized as LEA proteins and molecular chaperones.
In Arabidopsis, a heat stable protein (AtHS1) with antimicrobial activity was found to share
high sequence identity with aspen SP1 [14], which were highly ex- pressed after exposure to
diverse abiotic stresses [15]. These resultsindicatedthattheheatstabilityofcertainproteinsmay
evolve for their specialized functions, allowing them to cope with harsh environments. In
conclusion, we have shown that this heat fractionation approach can reduce sample
complexity of soluble protein extracts and improve gel-based proteome analysis through
increasing loading amount of each fraction. The prefraction- ation method reported here
has several advantages: (i) it is simple, and no complicated techniques or equipment are
needed; (ii) the one-step prefractionation and the optimized conditions for the
solubilization and recovery of fractionated
proteinsresultinminimalproteinloss;and(iii)thisapproach combined with other purification
techniques, may also be beneficial in enriching and/or purifying specific proteins of interest
for subsequent analysis. Work in our laboratory is funded by the National Natural Sci- ence
Foundation of China (30971705), Program for Science and
TechnologyInnovationTalentsinUniversitiesofHenanProvince (2008HASTIT005) and
Innovation Scientists and Technicians Troop Construction Projects of Henan Province
(9410051003). We thank Mr. Wang Qi for providing E. coli cells. The authors have declared
no conflict of interest. References [1] Cravatt, B. F ., Simon, G. M., Yates, J. R., The
biological impact of mass-spectrometry-based proteomics. Nature 2007, 450, 9911000. [2]
Ghaemmaghami, S., Huh, W., Bower, K., Howson, R. W. et al., Global analysis of protein
expression in yeast. Nature 2003, 425, 737741. [3] Gygi, S. P ., Corthals, G. L., Zhang, Y.,
Rochon, Y. et al., Eval- uation of two-dimensional gel electrophoresis-based pro- teome
analysis technology. Proc. Natl. Acad. Sci. USA 2000, 97, 93909395. [4] Doud, M. K.,
Schmidt, M. W., Hines, D., Naumann, C. et al., Rapid prefractionation of complex protein
lysates with cen- trifugal membrane adsorber units improves the resolving power of 2D-
PAGE- based proteome analysis. BMC Ge- nomics 2004, 5, 2531. [5] Righetti, P . G.,
Castagna, A., Antonioli, P ., Boschetti, E., Pre- fractionation techniques in proteome
analysis: the mining tools of the third millennium. Electrophoresis 2005, 26, 297 319. [6]
H orth, P ., Miller, C. A., Preckel, T., Wenz, C., Efficient frac- tionation and improved
protein identification by peptide OFFGEL electrophoresis. Mol. Cell. Proteomics 2006, 5,
19681974. [7] Boudet, J., Buitink, J., Hoekstra, F . A., Rogniaux, H. et al., Comparative
analysis of the heat stable proteome of radi- cles of Medicago truncatula seeds during
germination iden- tifies late embryogenesis abundant proteins associated with desiccation
tolerance. Plant Physiol. 2006, 140, 1418 1436. [8] Kwon, S., Jung, Y., Lim, D., Proteomic
analysis of heat stable proteins in Escherichia coli. BMB Rep. 2008, 41, 108111. [9]
Kochetkov G. A., Manual for Enzymology, Vysshaya Shkola, Moscow, 1980, p. 13. [10]
Pusztai, A., Interactions of proteins with other polyelec- trolytes in a two-phase system
containing phenol and aque- ous buffers at various pH values. Biochem. J. 1966, 99, 93
101. [11] Wang, W., Scali, M., Vignani, R., Spadafora, A. et al., Protein extraction for two-
dimensional electrophoresis from olive C ?2012 WILEY-VCH Verlag GmbH & Co. KGaA,
Weinheim www.proteomics-journal.com