Analytical Biochemistry 385 (2009) 208–214

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Analytical Biochemistry

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Analysis of ultra acidic proteins by the use of anodic acidic gels

Kristina Hempel a, Ran Rosen a,b,1, Dörte Becher a, Knut Büttner a, Michael Hecker a, Eliora Z. Ron b,*

a Institut für Mikrobiologie, Ernst Moritz Arndt Universität Greifswald, D-17487 Greifswald, Germanyb Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, 69978 Tel Aviv, Israel

a r t i c l e i n f o a b s t r a c t

Article history:Received 21 July 2008Available online 19 November 2008

Keywords:Acidic proteinsTwo-dimensional gel electrophoresisPosttranslational modifications

0003-2697/$ - see front matter � 2008 Elsevier Inc. Adoi:10.1016/j.ab.2008.11.013

* Corresponding author. Fax: +972 3 6414138.E-mail address: eliora@post.tau.ac.il (E.Z. Ron).

1 Current address: Agentek (1987), 61580 Tel Aviv, I2 Abbreviations used: IPG, immobilized pH gradien

electrophoresis; MDLC, multidimensional liquid chromametal ion affinity chromatography; IMAEP, immobilizesis; SDS–PAGE, sodium dodecyl sulfate–polyacrylamideBroth; EDTA, ethylenediaminetetraacetic acid; PMSF, pIEF, isoelectric focusing; DTT, dithiothreitol; TEMED, Ndiamine; DTAB, decyltrimethylammonium bromide; MMS, liquid chromatography–tandem MS; MALDI–TOF/desorption/ionization–tandem time-of-flight MS; BMM

Ultra acidic proteins, generated by posttranslational modifications, are becoming increasingly importantdue to recent evidence showing their function as regulatory elements or as intermediates in degradationpathways in bacteria. Such proteins are important in neurodegenerative diseases and embryonic devel-opment, and they include the Alzheimer-related tau (s) protein (resulting from posttranslational modi-fications) and the phosphor-storage embryonic proteins. The ultra acidic proteins are difficult to studybecause standard two-dimensional gel electrophoresis is inadequate for their analysis. Here we describea novel electrophoresis system of anodic acidic gels that can replace isoelectric focusing as the firstdimension of separation in two-dimensional electrophoresis. The system is based on a sodium acetatebuffer (pH 4.6), is compatible with traditional stains (e.g., Coomassie blue) as well as novel fluorescentdyes (e.g., Pro-Q Diamond), and is quantitative for the analysis of ultra acidic proteins. The anodic acidicgels were used for the functional classification of the ultra acidic part of the Bacillus subtilis proteome,showing significant improvement over traditional two-dimensional electrophoresis.

� 2008 Elsevier Inc. All rights reserved.

Proteins made ultra acidic as a result of multiple phosphoryla-tions have been shown in several eukaryotic systems. The tau (s)protein in its hyperphosphorylated form is associated with neuro-degenerative disorders (for a review, see Ref. [1]), and the egg yolkprotein phosvitin, which is crucial for embryonic development,serves as a phosphate storage metal chelator with antioxidationactivity [2–4]. We have shown [5] the existence of a novel groupof highly phosphorylated proteins that appear to have physiologi-cal importance. These proteins accumulate in bacteria duringstress and may be important in the degradation process. These pro-teins have an apparent pI of less than 2.5 and reach the acidic endof any immobilized pH gradient (IPG)2 strip that was tested in a tra-ditional two-dimensional gel electrophoresis (2-DE). It has also beendemonstrated that polyphosphorylated bacterial ribosomal proteinsare better substrates for proteases than the nonphosphorylated form[6].

ll rights reserved.

srael.t; 2-DE, two-dimensional geltography; IMAC, immobilizedd metal affinity electrophore-gel electrophoresis; LB, Luria

henylmethylsulfonyl fluoride;,N,N0 ,N0-tetramethylethylene-

S, mass spectrometry; LC–MS/TOF MS, matrix-assisted laser, Bacillus Minimal Medium.

Thus, the presence of physiologically important ultra acidicproteins appears to be a general phenomenon of eukaryotic and pro-karyotic cells. This group of proteins has not been studied exten-sively even now, when proteomic methods are widely used inresearch, because their separation and purification are not trivial.There are several potential widely accepted methods for studyingphosphoproteins. These include methods such as multidimensionalliquid chromatography (MDLC) [7], immobilized metal ion affinitychromatography (IMAC) [8], and immobilized metal affinity electro-phoresis (IMAEP) [9]. Our attempts to separate the bacterial ultraacidic proteins with the first two methods were unsuccessful. There-fore, it is important to develop separation tools for analyzing thecomplement of ultra acidic proteome.

Here we show several limitations of the standard 2-DE for theanalysis of highly acidic proteins that prevent its use in a quantita-tive manner. In addition, because the acidic proteins comprise asmall fraction of the cellular proteins, a large overload of IPG stripsis necessary for their visualization under most growth conditions.In this article, we describe a novel procedure for separating andanalyzing highly acidic proteins. The method is based on an anodicacidic gel system, which is similar in concept to the cathodic acidicgels that were used in the past for separation of highly alkalineproteins, such as ribosomal proteins, from other cellular proteins[10,11]. In an analogous manner, the anodic acidic gel system al-lows a high load of protein sample for separation of highly acidicproteins from all of the other proteins. We further developed thesystem to a two-dimensional separation system because theseacidic anodic gels can be used as a substitute for a standard first

Analysis of ultra acidic proteins / K. Hempel et al. / Anal. Biochem. 385 (2009) 208–214 209

dimension prior to separation by sodium dodecyl sulfate–poly-acrylamide gel electrophoresis (SDS–PAGE) in the second dimen-sion [12]. This novel electrophoresis system is compatible withstandard stains, such as colloidal Coomassie blue and silver, as wellas with fluorescent dyes, such as Pro-Q Diamond.

Materials and methods

Bacteria, media, and growth conditions

Escherichia coli strain MG1655 (K12 wild type), Bacillus subtilisstrain 168 (wild type), and B. subtilis strain PS37 (DcodY) wereused. Cultures of B. subtilis were grown in minimal medium as de-scribed previously (BMM) or in Luria Broth (LB, Difco, Detroit, MI,USA) [13]. Cultures of E. coli were grown at 30 �C in Davis andMingioli minimal medium [14] supplemented with 0.2% glucose,Mops minimal salts medium [15] supplemented with 0.2% glucose,or LB.

Protein extraction

Bacterial cultures were collected and quickly cooled to 0 �C. Thecells were centrifuged and washed twice with cold TE–PMSF(10 mM Tris [pH 7.5], 1 mM ethylenediaminetetraacetic acid[EDTA], and 1.4 mM phenylmethylsulfonyl fluoride [PMSF]). Thewashed cells were resuspended in 0.5 ml of TE–PMSF and dis-rupted by sonication. Cell debris and protein aggregates were re-moved by centrifugation at 20,000g for 30 min at 4 �C. Theprotein concentration was determined using the Bradford method[16] with the RotiNanoquant Kit (Roth, Karlsruhe, Germany). Thesupernatants containing the proteins were lyophilized.

Two-dimensional PAGE

Isoelectric focusing (IEF) was performed with commerciallyavailable IPG strips (18 cm, pH 4.0–7.0, Amersham Biosciences, Upp-sala, Sweden). Samples were loaded by rehydration for 24 h in a solu-tion containing 8 M urea, 2 M thiourea, 1% (w/v) Chaps, 20 mMdithiothreitol (DTT), and 0.5% (v/v) Pharmalytes 3-10. The IEF was

Fig. 1. Behavior of ultra acidic proteins in a standard two-dimensional gel. Acidic proteinof the electrode paper during the two early steps of the IEF process results in a complete lfollowing separation of total cellular extracts on an IPG strip (pH 4.0–7.0) with electrosubmitted to SDS–PAGE, and stained with silver nitrate. The arrow points to the acidic

performed with the MultiphorII unit (Amersham Biosciences)employing the following voltage profile: linear increase from 0 to500 V for 500 Vh, 500 V for 2500 Vh, linear increase from 500 to3500 V for 10,000 Vh, and a final phase of 3500 V for 35,000 Vh. Afterconsecutive equilibration of the gels in solutions containing DTT andiodoacetamide [17], the separation in the second dimension wasperformed in polyacrylamide gels of 12.5% T and 2.6% C on the Inves-tigator 2-D Electrophoresis System (Genomic Solutions, Ann Arbor,MI, USA) with approximately 2 W per gel. Analytical and preparativegels were loaded with 100 to 300 lg of crude protein extract, and thegels were stained with silver nitrate [18], colloidal Coomassie, orPro-Q Diamond (Molecular Probes, Eugene, OR, USA).

Anodic acidic gels

The anodic acidic gels at the end of the protocol developmentwere as follows. A 12.5% T and 4% C polyacrylamide gel containinga solution of 0.1 M potassium acetate (pH 4.6), 0.1% b-D-dodecylmaltoside, 8 M urea, 0.05% tetrabutylammonium hydrogen sulfate,0.25% N,N,N0,N0-tetramethylethylenediamine (TEMED), and 0.125%ammonium peroxodisulfate (APS) was polymerized at 37 �C for1 h. Here 0.2 M potassium acetate (pH 4.6) and 0.03% b-D-dodecylmaltoside was used as running buffer and 0.2 M potassium acetate(pH 5.5), 2.7% b-D-dodecyl maltoside, 0.3% decyltrimethylammoni-um bromide (DTAB), 10% glycerol, 6 M urea, and 10% b-mercap-toethanol was used as sample buffer. For sample loading, theprotein extracts were lyophilized and dissolved in 20 ll (formini-gel format) or 60 ll (for large-gel format) of sample bufferfor 30 min at room temperature prior to sample application intothe gel slots. The samples were separated with a constant currentof 20 mA for 24 h at room temperature in a mini-gel apparatus(Bio-Rad, Hercules, CA, USA) of two parallel anodic acid gels or,alternatively, with a constant current of 200 mA for 12 h at 18 �Cin a large format apparatus of two parallel anodic acid gels.

Equilibration prior to application on a slab SDS–PAGE for seconddimension was performed in a 10-fold gel volume of a solution of4% SDS, 20% glycerol, and 125 mM Tris–HCl (pH 6.8) for 15 to20 min. Following this equilibration step, the anodic acidic gels

s (marked with an arrow in panel A) accumulate during heat shock. Three exchangesoss of the ultra acidic proteins (panel B). Panel C shows the anodic ends of IPG stripsde paper (upper strip) and without it (lower strip). The strips were equilibrated,

band.

Fig. 2. Anodic acidic gels can separate the ultra acidic proteins from high amountsof total bacterial protein extracts. Samples of 50 lg egg yolk phosvitin and of 1000,3000, and 5000 lg total cellular extracts from stationary B. subtilis strain 168cultures were separated in lanes 1, 2, 3, and 4, respectively. The gels were stained byPro-Q Diamond (A), scanned, destained, and stained again with colloidal Coomassieblue (B), and scanned, destained, and stained again with silver (C).

210 Analysis of ultra acidic proteins / K. Hempel et al. / Anal. Biochem. 385 (2009) 208–214

were loaded on a polyacrylamide slab gel consisting of a stackinggel (3.7% T and 2.5% C) and a separating gel (12.5% T and 2.6% C).

Fig. 3. Optimization of the sample buffer. For selection of a reducing agent for the anstationary E. coli cultures (lanes 1–3) or 5 lg egg yolk phosvitin (lanes 4–6) was separateglycerol, and 6 M urea) containing no reducing agent (lanes 1 and 4), 10% b-mercaptoetmarker (B), samples of 5 lg egg yolk phosvitin (lanes 1 and 10) or 400 lg total cellular extand 10: light green SF yellowish (C37H34N2O9S3Na2); lane 2: bromophenol blue (C1

(C14H14N3O3SNa); lane 6: orange G (C16H10N2O7S2Na2); lane 7: methyl red (C15H15N3O2);The arrow marks the location of the egg yolk phosvitin front, as detected by a Pro-Q DiaLight Green SF Yellowish based on Ref. [24]. (For interpretation of the references to colo

Protein identification

The identification was performed with protein spots isolatedfrom colloidal Coomassie blue-stained gels. It was accomplishedby mass spectrometry (MS) according to established protocols[19,20]. Peptide masses were determined in the positive ion reflec-tor mode in a 4700 Proteomics Analyzer (Applied Biosystems, Fos-ter City, CA, USA) with internal calibration using trypsin autodigestproducts. Database searches were performed by Mascot (MatrixScience, London, UK) and MS-Fit (http://prospector.ucsf.edu/ucsfhtml4.0/msfit.htm) software. The proteins listed in the articlewere identified by at least two peptide sequences when Mascotwas used or with a sequence coverage of 20% and a four-peptideminimum when MS-Fit was used.

Protein classification

Protein classification was performed by PANDORA (ProteinAnnotation Diagram Oriented Analysis, version 2, http://www.pan-dora.cs.huji.ac.il). The user set was defined by all of the identifiedproteins (as Swiss–Prot accession numbers), and the resolutionwas set as 0.01 (default resolution) [21].

Results

Analysis of ultra acidic proteins by IPG strips

In proteomes of bacteria separated by 2-DE, it is possible to showaccumulation of ultra acidic proteins under various growth condi-tions. These proteins, which reach the acidic end of the gel(Fig. 1A), have an isoelectric point beyond the range of the commer-cially available IPG strips (and low pH [2.5–5.0] and homemadeIPGs) and, therefore, do not focus to any point on the strip. This re-sults in major sample variations depending on the protein and salt

odic acidic gel sample buffer (A), samples of 1600 lg total cellular extracts fromd with sample buffer (0.2 M potassium acetate [pH 5.5], 3% dodecyl maltoside, 10%hanol (lanes 2 and 5), or 20 mM DTT (lanes 3 and 6). For selection of a front colorracts from stationary E. coli (lanes 2–8) were separated with various dyes. Lanes 1, 3,9H9Br4O5SNa); lane 4: trypan blue (C34H24N6O14S4Na4); lane 5: methyl orangelane 8: Congo red (C32H22N6O6S2Na2); lane 9: acid fuchsin (C20H17N3O9S3Na2) [24].mond and colloidal Coomassie blue double stain. Panel C presents the structure ofur in this figure legend, the reader is referred to the web version of this article.)

Analysis of ultra acidic proteins / K. Hempel et al. / Anal. Biochem. 385 (2009) 208–214 211

concentrations. It was hypothesized that the ultra acidic proteinscan migrate out of the gel into the electrode paper that separates be-tween the gels and the electrodes or can be lost due to precipitationsat the acidic end of the gel. Indeed, Figs. 1A and 1B indicate that whenmimicking a sample with low salt content (also similar to high pro-tein concentration) by several exchanges of the electrode paper (re-peated removal of salt), the group of the highly acidic proteinsbecame undetectable, apparently lost in the electrode paper. It isalso shown (Fig. 1C) that the electrode paper is essential for preven-tion of insoluble precipitates of highly acidic proteins and salt; there-fore, it cannot be eliminated from the system.

Anodic acidic gel system

In 1992, Heukeshoven and Dernick [10] described a native hor-izontal cathodic electrophoresis system for protein separation un-der acidic conditions. Attempts to use this previously describedsystem for separation of ultra acidic proteins from total bacterialprotein extracts by reversing the polarity were not successful.Therefore, several changes were made to adapt the system for ultraacidic proteins. These included development of a homogeneousbuffer system, changes in the sample buffer composition, andchanges in the polymerization temperature. In addition, the sys-tem described below is a slab gel system instead of a thin horizon-tal gel system so as to increase the capacity of the gel.

The separation system that resulted in quantitative separationof ultra acidic proteins from high loads (up to 5 mg/lane) of totalbacterial extracts is based on a potassium acetate (pH 4.6) buffer

Fig. 4. Anodic acidic gel separation of proteins from various biological origins.Proteins were extracted from E. coli (left lane), B. subtilis grown on minimal medium(BMM, middle lane), and B. subtilis grown on LB medium (right lane).

system both in gel and as running buffer. The proteins were sepa-rated in a denatured form due to the sample buffer composition(described below) and the supplementation of dodecyl maltosideto the gel and running buffer. The gels were prepared as describedin Materials and Methods and were used to separate variousamounts of total cellular protein extracts of B. subtilis strain 168culture (Fig. 2). The results indicated that the separation is quanti-tative given that a linear correlation was found between theamount of total cellular extract and the intensity of the stain withcolloidal Coomassie blue and a specific phosphoprotein stain. Thecomparison of the Pro-Q Diamond stain and the nonspecific pro-tein stains suggests that most of the visible proteins in the gelare highly phosphorylated and, therefore, ultra acidic. Further sup-port for the extreme acidity of these proteins is the comparison ofthe migration distance with that of the highly phosphorylated eggyolk phosvitin, which was used as a standard protein and migratedslower than the majority of B. subtilis ultra acidic proteins.

The sample buffer composition was found to have a major effecton protein precipitation in the gel slots and, hence, on the amountsof protein in gel. A major component of the sample buffer wasfound to be the reducing agent b-mercaptoethanol (Fig. 3A). Theoptimized sample buffer composition was found to be a solutionof 0.2 M potassium acetate (pH 5.5), 2.7% b-D-dodecyl maltoside,0.3% DTAB, 10% glycerol, 6 M urea, and 10% b-mercaptoethanol.

Additional adaptation was necessary to obtain an appropriatemarker for the separation front. Several acidic dyes were comparedwith bromophenol blue, and light green SF yellowish was found tomigrate slightly faster than egg yolk phosvitin and the bacterial ul-tra acidic proteins (Fig. 3B and C).

A similar protein signature was obtained for B. subtilis growingunder different conditions, namely, in rich LB medium and mini-mal medium (BMM). In contrast, the acidic protein signature of

Fig. 5. Two-dimensional separation of ultra acidic proteins using anodic acidic gelsas the first dimension. Here 400 lg of total cellular extracts from exponentiallygrowing DcodY B. subtilis was separated by an anodic acidic gel; the lane was cut,equilibrated as described, and separated by SDS–PAGE as the second dimension;and the gel was stained with colloidal Coomassie G-250. This gel section representsthe top part of the anodic acidic gels and, therefore, represents the less acidicproteins (left panel). A global image of two-dimensional separation of ultra acidicproteins using anodic acidic gels stained by Pro-Q Diamond is shown in the rightpanel. The top of the anodic acidic gel was placed on the left side of the second-dimension gel.

Table 1Identified ultra acidic proteins of DcodY B. subtilis.

Protein Function Accessionnumbera

Functional classificationb Mrc pId

RpoC RNA polymerase (beta0 subunit) BG10729 RNA synthesis–elongation 133920 8.86RpoB/

YcgNRNA polymerase (beta subunit) BG10728 RNA synthesis–elongation 133597 4.90

MetS Methionyl-tRNA synthetase BG10101 Aminoacyl-tRNA synthetases 76188 5.14Tkt Transketolase BG11247 Main glycolytic pathways 72344 4.99OppA Oligopeptide ABC transporter (binding protein) (initiation of sporulation,

competence development)BG10771 Transport/Binding proteins and lipoproteins 61491 5.83

IlvD Dihydroxy acid dehydratase BG11532 Metabolism of amino acids and related molecules 59548 5.44PckA Phosphoenolpyruvate carboxykinase BG11841 Main glycolytic pathways 58300 5.26GroEL Class I heat shock protein (molecular chaperonin) BG10423 Protein folding 57424 4.73LeuA 2-Isopropylmalate synthase BG11948 Metabolism of amino acids and related molecules 56912 5.73RocA Pyrroline-5-carboxylate dehydrogenase BG10622 Metabolism of amino acids and related molecules 56320 5.67GltX Glutamyl-tRNA synthetase BG10154 Aminoacyl-tRNA synthetases 55555 5.02AtpA ATP synthase (subunit alpha) BG10819 Membrane bioenergetics (electron transport chain

and ATP synthase)54598 5.22

MmsA(IolA)

Methylmalonate-semialdehyde dehydrogenase BG11117 Metabolism of amino acids and related molecules 53453 5.31

GatA/YerM

Glutamyl-tRNA(Gln) amidotransferase (subunit A) BG12839 Aminoacyl-tRNA synthetases 52664 5.39

AtpD ATP synthase (subunit beta) BG10821 Membrane bioenergetics (electron transport chainand ATP synthase)

51420 4.80

CtpA Carboxy-terminal processing protease BG11794 Metabolism of amino acids and related molecules 51149 8.44PdhD Dihydrolipoamide dehydrogenase E3 subunit (pyruvate dehydrogenase and

2-oxoglutarate dehydrogenase)BG10210 Main glycolytic pathways 49733 4.95

YurX Similar to hypothetical proteins BG14010 Similar to unknown proteins–from otherorganisms

48293 5.12

Eno Enolase BG10899 Main glycolytic pathways 46581 4.68Icd/CitC Isocitrate dehydrogenase BG10856 Tricarboxylic acid cycle 46418 5.03OdhB 2-Oxoglutarate dehydrogenase (dihydrolipoamide transsuccinylase, E2

subunit)BG10273 Tricarboxylic acid cycle 45827 4.86

YkrT/MtnK

YkrT BG13279 Similar to unknown proteins–from B. subtilis 45347 5.04

YxbF Alternate gene name: YxaT BG11356 Similar to unknown proteins–from B. subtilis 44283 6.82RocD Ornithine aminotransferase BG10722 Metabolism of amino acids and related molecules 43762 5.07TufA Elongation factor Tu BG11056 Protein synthesis–elongation 43593 4.92PrfB Peptide chain release factor 2 BG10742 Protein synthesis–termination 42073 5.03CitZ Citrate synthase II BG10855 Tricarboxylic acid cycle 41729 5.55PdhA Pyruvate dehydrogenase (E1 alpha subunit) BG10207 Main glycolytic pathways 41548 5.91SucC Succinyl-CoA synthetase (beta subunit) BG12680 Tricarboxylic acid cycle 41372 5.04LeuB 3-Isopropylmalate dehydrogenase BG10675 Metabolism of amino acids and related molecules 39945 4.93Ald l-Alanine dehydrogenase BG10468 Metabolism of amino acids and related molecules 39683 5.28IlvC Ketol acid reductoisomerase (acetohydroxy acid isomeroreductase) BG10672 Metabolism of amino acids and related molecules 37458 5.49PdhB Pyruvate dehydrogenase (E1 beta subunit) BG10208 Main glycolytic pathways 35474 4.74YfmC Similar to ferrichrome ABC transporter (binding protein) BG12954 Transport/Binding proteins and lipoproteins 35021 7.69Prs Phosphoribosyl pyrophosphate synthetase BG10114 Metabolism of nucleotides and nucleic acids 34868 5.94CitH/

MdhSecondary transporter of divalent metal ions/citrate complexes BG11146 Transport/Binding proteins and lipoproteins 33644 4.92

IolH Alternate gene name: YxdG�myo-inositol catabolism BG11123 Metabolism of carbohydrates and relatedmolecules–specific pathways

33524 5.16

GtaB UTP-glucose-1-phosphate uridylyltransferase BG10402 Cell wall 33070 5.10CysK Cysteine synthetase A BG10136 Metabolism of amino acids and related molecules 32820 5.64Tsf Elongation factor Ts BG19025 Protein synthesis–elongation 32354 5.17RocF Arginase BG10932 Metabolism of amino acids and related molecules 32154 5.10YaaD Similar to hypothetical proteins BG10075 Detoxification 31612 5.26FbaA Fructose-1,6-bisphosphate aldolase BG10412 Main glycolytic pathways 30401 5.19RplB Ribosomal protein L2 (BL2) BG11217 Ribosomal proteins 30332 10.54YktC/

SuhBSimilar to myo-inositol-1(or 4)-monophosphatase BG11818 Metabolism of carbohydrates and related

molecules–specific pathways29760 5.24

RpsB Ribosomal protein S2 BG19004 Ribosomal proteins 27967 6.27YutF Similar to N-acetyl-glucosamine catabolism BG14042 Metabolism of carbohydrates and related

molecules–specific pathways27957 4.74

RplA Ribosomal protein L1 (BL1) BG10164 Ribosomal proteins 24923 9.30RpsD Ribosomal protein S4 (BS4) BG10372 Ribosomal proteins 22835 9.88RplC Ribosomal protein L3 (BL3) BG11218 Ribosomal proteins 22683 9.83RplD Ribosomal protein L4 BG11219 Ribosomal proteins 22391 10.03

AhpC Alkyl hydroperoxide reductase (small subunit) BG11385 Detoxification 20627 4.48YpuI Similar to hypothetical proteins–from B. subtilis BG10526 Similar to unknown proteins–from B. subtilis 20351 5.59RplE Ribosomal protein L5 (BL6) BG10760 Ribosomal proteins 20148 9.54RplF Ribosomal protein L6 (BL8) BG11408 Ribosomal proteins 19509 9.49YetL Similar to transcriptional regulator (MarR family) BG12868 RNA synthesis–regulation 19218 7.15RpsG Ribosomal protein S7 (BS7) BG19006 Ribosomal proteins 17893 10.03RpsE Ribosomal protein S5 BG10442 Ribosomal proteins 17623 9.92YwoH Similar to transcriptional regulator (MarR family) BG12495 RNA synthesis–regulation 16424 7.83RplM Ribosomal protein L13 BG11970 Ribosomal proteins 16292 9.80

212 Analysis of ultra acidic proteins / K. Hempel et al. / Anal. Biochem. 385 (2009) 208–214

Table 1 (continued)

Protein Function Accession numbera Functional classificationb Mrc pId

RplP Ribosomal protein L16 BG10755 Ribosomal proteins 16190 10.41RplO Ribosomal protein L15 BG10444 Ribosomal proteins 15383 10.28RpsL Ribosomal protein S12 (BS12) BG19009 Ribosomal proteins 15183 11.72RpsH Ribosomal protein S8 (BS8) BG10762 Ribosomal proteins 14703 10.30RpsI Ribosomal protein S9 BG19007 Ribosomal proteins 14168 11.01RpsK Ribosomal protein S11 (BS11) BG10731 Ribosomal proteins 13925 11.63GsiB General stress protein BG10826 Adaptation to atypical conditions 13798 5.32RpsM Ribosomal protein S13 BG10730 Ribosomal proteins 13661 11.55RplQ Ribosomal protein L17 (BL15) BG11041 Ribosomal proteins 13610 10.27RplS Ribosomal protein L19 BG12667 Ribosomal proteins 13608 11.29RplR Ribosomal protein L18 BG11409 Ribosomal proteins 12878 10.46RpsJ Ribosomal protein S10 BG19008 Ribosomal proteins 11527 10.21RplU Ribosomal protein L21 (BL20) BG10333 Ribosomal proteins 11137 10.16RplW Ribosomal protein L23 BG11221 Ribosomal proteins 10790 10.09RpsS Ribosomal protein S19 (BS19) BG19011 Ribosomal proteins 10583 10.12RpsO Ribosomal protein S15 (BS18) BG19010 Ribosomal proteins 10435 10.98RpsP Ribosomal protein S16 (BS17) BG10831 Ribosomal proteins 9997 10.66Hbs Nonspecific DNA binding protein HBsu BG10276 DNA packaging and segregation 9884 8.96RpmC Ribosomal protein L29 BG10756 Ribosomal proteins 7577 10.63RpsU Ribosomal protein S21 BG11648 Ribosomal proteins 6694 11.58

Note. Here 400 lg of a total protein extract of exponentially growing DcodY B. subtilis was separated in anodic acidic gels as a first dimension and by SDS–PAGE as a seconddimension. The protein bands were cut out and identified by MALDI–TOF MS. Proteins that were also identified from classical 2-DE are marked with bold letters.

a SubtiList accession number (http://bioinfo.hku.hk/SubtiList).b SubtiList functional classification (http://bioinfo.hku.hk/SubtiList).c Theoretical molecular weight of the unmodified protein as calculated by the ExPASy compute pI/Mw tool (http://au.expasy.org/tools/pi_tool.html).d Theoretical pI of the unmodified protein as calculated by the ExPASy compute pI/Mw tool (http://au.expasy.org/tools/pi_tool.html).

Analysis of ultra acidic proteins / K. Hempel et al. / Anal. Biochem. 385 (2009) 208–214 213

E. coli was significantly different from that of B. subtilis (Fig. 4).These results further demonstrate that this method reflects thephysiological signature of acidic proteins.

As of now, the gels are vertical, nonsupported, and 0.8 to 1 mmthick. These characteristics make them somewhat difficult to han-dle. Yet it was possible to stain them as described above and totransfer them to the second dimension, as described bellow.

SDS–PAGE for separation in the second dimension

The resolution of the above-described anodic acidic gels is low.These are suitable for separation of the highly acidic proteins fromother cellular proteins, but not for separation among themselves.Another reason for the need of further separation of the proteinsby SDS–PAGE is the incompatibility of the anodic acidic gels withMS for protein identification. Several attempts at protein identifi-cation by liquid chromatography–tandem MS (LC–MS/MS) andmatrix-assisted laser desorption/ionization–tandem time-of-flightMS (MALDI–TOF/TOF MS) were not successful.

To increase the resolution, a second dimension of separationaccording to the molecular weight of the proteins was accom-plished by SDS–PAGE [12]. The transfer of the proteins from theanodic acidic gels to SDS gels requires an equilibration step inwhich the b-D-dodecyl maltoside is exchanged by SDS. Unlike theequilibration step of IPG strips [17] where the proteins, at thebeginning of the equilibration process, are not charged as beingin their isoelectric point and so have relatively low solubility, theproteins in the anodic acidic gel system seem to be highly soluble.Consequently, long equilibration periods of more than 40 min re-sulted in complete loss of the proteins from the gel. Sufficientequilibration conditions were found to be 15 to 20 min incubationin a 10-fold gel volume of a solution of 4% SDS, 20% glycerol, and125 mM Tris–HCl (pH 6.8). Following this equilibration step, theanodic acidic gels were loaded on a polyacrylamide slab gel. Thissecond dimension of size-dependent separation resulted in distinctprotein bands (Fig. 5). Protein bands that were excised from suchgels after the second dimension were analyzed by MALDI–TOF/TOF MS. Using a combination of peptide mass fingerprinting andMS/MS data analysis, it was possible to identify one to four pro-teins in each band (Table 1). It should be noted that despite the

similarity between the concentration of the acrylamide monomersin the anodic acidic gels and the SDS–PAGE separating gels, theydiffer in their pore size due to the difference in the pH during poly-merization. Acrylamide polymerization under acidic conditions re-sults in higher pore size in comparison with polymerization of thesame monomer mixture at alkaline pH [10]. The relatively largepore size of the anodic acidic gels may be an additional reasonfor protein loss during prolonged equilibration.

Anodic acidic gels for functional analysis of the ultra acidic proteomeof B. subtilis

The ultra acidic proteins of exponentially growing DcodY B.subtilis (chosen due to relatively high quantities of ultra acidic pro-teins) were separated as described above (Fig. 5). Following thetwo-dimensional separation, protein bands were cut out of colloidalCoomassie-stained gels and identified (Table 1). The 80 identifiedproteins (only 7 of which could be identified following separationof the same extract in a standard two-dimensional gel) were classi-fied by PANDORA software according to their function and structure(functional classification according to GO:Molecular function; struc-tural classification according to SCOP:class, SCOP:fold, Inter-Pro:Domain, and InterPro:Family functions) [21]. The resultsindicate that the ultra acidic proteins can be classified into severaldistinct functional groups but have no common structural featuresapart from their acidic nature that results (at least in many of them)from a phosphate-containing modification. The most commongroups of proteins consist of the translation apparatus and abundantenzymes involved in basic metabolic pathways such as glycolysisand amino acid biosynthesis.

Discussion

Analysis of proteomes by 2-DE is a widespread analytical meth-od. Although this method has many advantages for proteomicstudies, such as high resolving power that enables separation ofsubpopulations of the same gene product, it is limited in the anal-ysis of several groups of proteins such as high molecular weightproteins and membrane proteins [22,23]. Recently, a novel group

214 Analysis of ultra acidic proteins / K. Hempel et al. / Anal. Biochem. 385 (2009) 208–214

of highly acidic modified proteins was discovered by standard 2-DEanalysis [5]. Although, this group of proteins is detectable by clas-sical 2-DE, the method has several limitations in the analysis ofsuch proteins and the results are inconsistent. The pI of these pro-teins is outside of the range represented in IPG strips; thus, theymigrate like inorganic salts toward the anodic electrode paper.

The novel anodic acidic gel electrophoresis method describedhere provides a new research tool for separation and enrichmentof highly acidic proteins in gel. The separation in gel can be com-bined with various specific staining methods that can provideessential data on the nature of the highly acidic modification(e.g., Pro-Q Diamond for phosphorylation, Pro-Q Emerald for glyco-proteins). For testing the method, we used as a model system thehighly phosphorylated proteins that accumulate during variousphysiological conditions in bacteria. Identification of the highlyacidic proteins from an exponentially growing DcodY B. subtilisindicated that this group consists of approximately 80 proteinsthat take part in various cellular processes. The structural analysisby PANDORA and the functional analysis according to the SubtiListdatabase (Table 1) and by PANDORA (data not shown) showed thatthe proteins do not fit into specific structural groups but rather be-long to a limited number of functional groups. These results sug-gest physiological importance for the highly acidic proteins inbacteria that apparently play a role in protein degradation [5]. Sim-ilar analysis with standard 2-DE was not possible due to the lownumber of identified proteins.

The presence and physiological importance of the highly phos-phorylated proteins in eukaryotic and bacterial cells raise interestin tools for comparative proteomic studies of such proteins. Thetraditional 2-DE tools are incomplete for their analysis, and prote-ome analysis by LC coupled to MS is usually limited to small partsof the protein (i.e., single peptides) and, therefore, is not suitablefor analysis of proteins that carry posttranslational modifications.We failed to separate the ultra acidic proteins with standard meth-ods such as IMAC and MDLC. Therefore, we developed the use ofanodic acidic gels described here, which provide a quantitative toolfor analysis of ultra acidic proteins in combination with mass spec-trometric compatibility for the analysis of their modifications.

Acknowledgments

We thank Dirk Albrecht for his help in MALDI–TOF/TOF proteinidentification. This work was partially supported by a grant fromthe German–Israeli Foundation (R.R., M.H., and E.Z.R.), the AlfreidKrupp Kolleg Greifswald Foundation and the Alfreid Krup von Boh-len und Halbach Fellowship for Research Fellows (R.R.), the Peikov-sky Valachi Postdoctoral Fellowship (R.R.), and the Manja andMorris Leigh Chair for Biophysics and Biotechnology (E.Z.R.).

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