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 Page 2 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 www.proteomics-journal.com Page 3 940 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 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com Page 4 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 www.proteomics-journal.com Page 5 942 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. 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