Breast cancer protein StarD10 identified by three-dimensional separation using free-flow electrophoresis, reversed-phase high-performance liquid chromatography, and sodium dodecyl

Peter Hoffmann1, 4

Monilola A. Olayioye2, 5

Robert L. Moritz3

Geoffrey J. Lindeman2

Jane E. Visvader2

Richard J. Simpson3

Bruce E. Kemp1

1St. Vincent’s Instituteand CSIRO HealthSciences & Nutrition,Victoria, Australia

2The Walter and Eliza HallInstitute of Medical Research,Parkville, Victoria, Australia

3Joint Protein StructureLaboratory,Ludwig Institute for CancerResearch,Victoria, Australia

4Center for Biotechnologyand Biomedicine,University Leipzig,Leipzig, Germany

5University of Stuttgart,Institute of Cell Biologyand Immunology,Stuttgart, Germany

Breast cancer protein StarD10 identified bythree-dimensional separation using free-flowelectrophoresis, reversed-phase high-performanceliquid chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis

A 35 kDa protein present in mammary tumors from Neu/ErbB2 transgenic mice wasdetected on the basis of its cross-reactivity with a phosphoserine-specific antibodyagainst the transcription factor FKHR. To isolate this protein from cytosolic extractsderived from human breast carcinoma cells, we used free-flow electrophoresis in thefirst dimension to separate proteins according to their charge, followed by reversed-phase high-performance liquid chromatography (RP-HPLC) in the second and sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the third dimen-sion. Tryptic digests of Coomassie-stained bands were analyzed by nano-spray ioni-zation-quadrupole quadrupole-time of flight-mass spectrometry identifying StarD10, aSTART domain containing protein, which cross-reacted with the anti-phospho-FKHRantibody. The site of phosphorylation was identified in immunoaffinity purified Flag-tagged StarD10 from 293T cells transiently expressing this protein. Tryptic phospho-peptides were enriched by immobilized metal affinity chromatography (IMAC) andStarD10 Ser-259-phosphate was identified by tandem mass spectrometry. Thus, free-flow electrophoresis is a powerful high-capacity complementary technique to RP-HPLC and SDS-PAGE for the purification of proteins from complex cell lysates.

Keywords: Immobilized metal ion affinity chromatography / Serine phosphorylation / STARTdomain / Tandem mass spectrometry DOI 10.1002/elps.200406197

1 Introduction

Proteomics is being widely used to identify novel cancerbiomarkers in tumor tissue, serum, whole-cell lysates,and specific cell compartments [1]. It is often difficult toisolate biomarkers in the presence of high-abundantproteins with similar physicochemical properties. Fur-thermore, because of the limited protein loading ca-pacity, the level of proteins of interest often falls belowthe detection limit. While shotgun proteomics ap-proaches [2] that rely on enzymatic digestion (typicallytrypsin) of a complex protein mixture followed byseparation of the resulting peptides via two-dimensionalchromatography can be used, the loading capacity of

this method is also limited. Furthermore, it may beessential to purify where immunodetection or otherfunctional assays are required.

Free-flow electrophoresis (FFE) has been used as acontinuous preparative separation method for proteinsfor three decades [3–6]. Due to the continuous nature ofthis technique, the loading capacity is virtually limitedonly by the sample amount. This method has been usedfor high-resolution preparative isoelectrofocusing ofcytosolic cell lysates and peroxisomal membrane pro-teins in combination with SDS-PAGE and subsequentidentification of proteins by tandem mass spectrometry[7, 8]. In this study, we used a phosphoserine-specificantibody (originally raised against pS256-FKHR) thatrecognizes a tumor cell-specific 35 kDa protein. In orderto further reduce the complexity of cell lysates after FFE,we introduced preparative RP-HPLC in the second di-mension. A three-dimensional approach comprising FFE,RP-HPLC, and SDS-PAGE, followed by peptide analysisusing tandem mass spectrometry, was developed to

Correspondence: Dr. Peter Hoffmann, Center for Biotechnologyand Biomedicine, University Leipzig, D-04103 Leipzig, GermanyE-mail: [email protected]: 149-341-9731339

Abbreviations: FFE, free-flow-electrophoresis; HPMC, hydroxy-propylmethylcellulose

Electrophoresis 2005, 26, 1029–1037 1029

2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Gen

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1030 P. Hoffmann et al. Electrophoresis 2005, 26, 1029–1037

isolate StarD10, a 35 kDa START domain protein over-expressed in breast cancer and shown to be phospho-rylated on serine 259.

2 Materials and methods

2.1 Materials

All reagents were of analytical grade and purchased fromSigma (St. Louis, MO, USA), unless otherwise indicated.Water was from a Milli-Q system (Millipore, Bedford, MA,USA). Modified trypsin was from Promega (Madison, WI,USA), formic acid was from Merck Arista (Darmstadt,Germany), methanol and propanol (HPLC-grade) werefrom Mallinckrodt Biolab Scientific (Melbourne, Australia).Carrier ampholytes Servalyte pH 3–10 were from Serva(Heidelberg, Germany) and Complete protease inhibitorswere from Roche (Basel, Switzerland). The Phosphopep-tide Isolation Kit was from Pierce (Rockford, IL, USA).Antibodies were as follows: affinity purified rabbit poly-clonal anti-phospho-FKHR (S256) antibody (New EnglandBiolabs, Beverly, MA, USA), mouse monoclonal anti-Flagantibody (Sigma), and affinity purified anti-StarD10 pep-tide antibody [9].

2.2 Cell culture

Breast epithelial cell lines and 293Tcells were maintainedin RPMI or DMEM containing 10% fetal bovine serum(FBS) (CSL, Parkville, VIC, Australia). StarD10 cDNA wascloned into the Flag-pEFrPGKpuro mammalian expres-sion vector [9]. For transient transfections, 293Tcells weretransfected with Fugene reagent (Roche) according to themanufacturer’s instructions.

2.3 Recombinant protein production

Human StarD10 cDNA was cloned into pGEX-6P(Amersham, Piscataway, NJ, USA) and transformed intoBL21 bacteria to produce a glutathione S-transferase(GST) fusion protein. Expression was induced with 0.5 M

isopropyl-b-D-1-thiogalactopyranoside for 4 h at 307C.Bacteria were harvested and resuspended in PBS con-taining 50 mg/mL lysozyme, Complete protease inhibitors(Roche), 10 mM sodium fluoride, and 20 mM b-glycero-phosphate. Triton X-100 was added to a final concentra-tion of 1% prior to two freeze-thaw cycles and sonication.GST-StarD10 was purified from clarified lysate with glu-tathione resin. GST-StarD10 was cleaved with PreScis-sion protease according to the manufacturer’s instruc-tions (Amersham). The protein concentration was esti-mated by measuring the absorbance at 260 nm.

2.4 Western blotting

Samples were mixed with SDS loading buffer and sub-jected to SDS-PAGE using 4–20% gradient gels (Novex,San Diego, CA, USA). The proteins were blotted ontopolyvinylidine difluoride membranes (Millipore). Afterblocking with 20% horse serum (Hunter, Jesmond, NSW,Australia) in PBS containing 0.1% Tween 20, filters wereprobed with specific antibodies. Proteins were visualizedwith peroxidase-coupled secondary antibody using theECL detection system (Amersham).

2.5 Protein extraction of cells

For cytosolic protein extraction, cells were lysed in hypo-tonic buffer (10 mM Hepes, pH 7.9, 133 mM sorbitol,0.5 mM sodium fluoride, 0.5 mM b-glycerophosphate plusComplete protease inhibitors), left to swell on ice for10 min, homogenized by douncing, and then centrifugedat 8006g for 10 min. The pellet was washed with hypo-tonic buffer and the supernatants were combined to yieldthe cytosolic fraction.

2.6 FFE

FFE, a continuous liquid-based isoelectric focusingmethod, was essentially performed as described in Hoff-mann et al. [7] using the Octopus apparatus (Dr. Weber,Planegg, Germany). The IEF running buffer was aqueous0.2% w/v hydroxypropylmethylcellulose (HPMC) con-taining 0.2% w/v carrier ampholytes (Servalyte pH 3–10).Electrode solutions were 100 mM H3PO4 (anode) and50 mM NaOH (cathode); the counter flow solution(0.7 L/min) was 0.2% HPMC containing 0.02 M L-arginineand 0.02 M L-lysine. Electrophoresis was performed at47C with a flow rate of 1.4 mL/min and 1250 V. The systemwas first equilibrated with IEF running buffer at 1250 Vuntil the current was stable and the performance was thentested by measuring the pH-gradient and the separationof low-molecular-mass pI markers for isoelectric focusing[10, 11]. For preparative separations, the sample wasdiluted to a final concentration of approximately 0.25 mgprotein per mL.

2.7 RP-HPLC

Analytical RP-HPLC was done using a Brownlee RP-300(10062.1 mm, 300 Å, 15 mm) column on a SMART Sys-tem (Pharmacia Biotech, Uppsala, Sweden) equippedwith a fraction collector. Proteins were eluted by a lineargradient from 0 to 100% B at a flow rate of 100 mL/mincollecting 100 mL fractions (A: 0.1% aqueous TFA, B: 60%


Electrophoresis 2005, 26, 1029–1037 3-D separation of breast cancer protein StarD10 1031

aqueous n-propanol containing 0.1% TFA). Collectedfractions were dried down in a vacuum concentrator,resuspended in SDS-sample buffer and submitted toSDS-PAGE followed by Western blotting. Preparative RP-HPLC was done on a 1090 (Hewlett Packard, Waldbronn,Germany) and fractions were collected manually in 1.5 mLEppendorf microtubes. Preparative RP-HPLC was doneusing a Brownlee RP 300 (10064.6 mm, 300 Å, 15 mm)column with a gradient described above (1 mL/min).Fractions were collected in 30 s intervals (0.5 mL) startingfrom 20 to 45 min. Aliquots of each fraction (50 mL) weredried down in a vacuum concentrator, resuspended inSDS-sample buffer and analyzed by SDS-PAGE andWestern blotting. Subsequently, 2 mL of the immunoposi-tive fractions were separated by SDS-PAGE and detectedby silver staining.

2.8 Protein precipitation and preparativeSDS-PAGE

HPLC fractions containing the 35 kDa immunoreactiveprotein were pooled and precipitated by addition of 0.5%deoxycholate and 15% TCA plus two volumes of acetoneat 2207C. After centrifugation at 16 0006g for 15 min, theprotein pellet was washed with acetone, briefly left to dry,and resuspended in sample buffer. A large 10% gel(1.5 mm615 cm) was poured according to Laemmli andelectrophoresis was performed at 150 V for 6 h. The gelwas fixed and stained with Coomassie Phast-gel Blue R(Pharmacia Biotech) or with silver nitrate [12].

2.9 In-gel digestion, desalting, and concentra-tion

Coomassie-stained bands were excised from gels andsliced into 1 mm2 pieces. The gel plugs were transferredto 1.5 mL screw cap microtubes (Quality Scientific Plas-tics, Milton, Australia) and destained with 50 mM

NH4HCO3 mixed with acetonitrile (1:1 by vol.). Cysteineswere first reduced with 1,4-dithio-DL-threitol (DTT) andthen alkylated with iodacetamide. Proteins were then in-gel digested with modified porcine trypsin [13]. The digestwas stopped with formic acid and peptides were extract-ed from the gel particles with 50 mL 25 mM NH4HCO3 for30 min in an ultrasonic bath. The supernatant was recov-ered and extraction was repeated with 50 mL acteonitrile,followed by 50 mL 5% formic acid and, finally, 50 mL ace-tonitrile. All supernatants were combined and dried downin a vacuum centrifuge and resuspended in 10 mL 5%formic acid. This solution was desalted with Zip Tips

m-C18 (Millipore) and eluted stepwise into nanospraycapillaries (MDS Proteomics, Odense, Denmark) with20%,40%, 60% aqueous methanol containing 5% formic acid.

2.10 Nano-electrospray ionizationmass-spectrometry (n-ESI-MS)

A QSTAR Pulsar i quadrupole time of flight (TOF) massspectrometer (Applied Biosystem, MDS Sciex, Toronto,Canada) equipped with a nano-electrospray ion source(MDS Proteomics) was used. TOF-MS spectra were col-lected in the “enhance all” modus and charged ions ofpeptides were chosen manually for product ion analysis.Product ion spectra (tandem MS) were acquired withcollision energies optimized for each peptide to obtain aneffective fragmentation pattern with 10% intensity of theresidual precursor ion. Argon was used as collision gasat a recorded pressure of 4.361025 Torr. Data weresearched by the tandem mass spectrum databasematching tool Mascot (http://www.matrixscience.com)[14]. The NCBInr and Swiss-Prot databases weresearched with the variable modifications deamidation,oxidation of methionine, cabarmidomethylation of cyste-ine and phosphorylation of serine and threonine. Production spectra that could not be matched were sequencedmanually.

2.11 Immunoaffinity purification of Flag-taggedStarD10

30615 cm dishes of 293T cells transiently expressingFlag-tagged StarD10 were lysed 48 h post transfection in1% NEB (50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP40,1 mM sodium orthovanadate, 10 mM sodium fluoride,20 mM b-glycerophosphate plus Complete protease in-hibitors). Lysates were clarified by centrifugation and thenpassed over anti-Flag M2 immunoaffinity resin (Sigma).The resin was washed with 1% NEB, followed by washeswith PBS. Flag-StarD10 was eluted with 100 mM glycine,pH 2.5, and fractions were neutralized with 1/10 volumeof 1 M Tris, pH 8. Fractions containing StarD10 as deter-mined by Western blotting with anti-Flag antibody werepooled and concentrated by ultrafiltration using AmiconUltra-4 columns (10 000 NMWL; Millipore). The con-centrated sample was separated on a 10% SDS-PAGEgel at 150 V for 7 h and the gel stained with CoomassiePhast Blue.

2.12 Isolation of phosphopeptides

In-gel digestion of Flag-tagged StarD10 was performedas described in Section 2.7. Gel pieces were homoge-nized to facilitate extraction of the 5 kDa peptide con-taining serine 259. Isolated peptides were diluted with5–20% acetic acid to obtain a pH of 2.5–3.5. Phospho-peptides were enriched with the SwellGel Gallium-Che-lated Disc (Phosphopeptide Isolation Kit; Pierce) accord-



ing to the manufacturer’s recommendations with slightmodifications. The incubation time for binding and elutionwas doubled and peptides were eluted three times with30 mL 0.3 M NH3. Eluates were vacuum-concentrated andresuspended in 10 mL 5% formic acid. The sample wasthen desalted on a ZipTip m-C18 (Millipore) and elutedstepwise into nano-spray capillaries (MDS Proteomics)with 20%, 40%, and 60% aqueous methanol containing5% formic acid. Then, n-ESI-MS was performed on eacheluted fraction according to Section 2.9.

3 Results and discussion

3.1 A phosphospecific FKHR antibody cross-reacts with a tumor-specific 35 kDa protein

A tumor-specific 35 kDa phosphoprotein cross-reac-tive with an antibody raised against the FKHR tran-scription factor phosphorylated on serine 256 wasinitially observed in mammary tumors derived fromMMTV-Neu transgenic mice. Transgenic expression ofNeu, the rat homolog of the receptor tyrosine kinaseErbB2/HER2, from the mouse mammary tumor viruspromoter, induces focal mammary tumors that histo-logically resemble human breast cancers [15]. Thismouse model has served to identify intracellular medi-ators of ErbB2-induced tumor development. Analysisof several breast epithelial cell lines revealed that the35 kDa protein was also present in many transformedbreast carcinoma cell lines and was absent in 184 andHBL100 immortalized breast epithelial cell lines [9].These results raised the possibility of a FKHR-relatedphosphoprotein being expressed in tumor tissue and intransformed cell lines.

3.2 FFE purification of the 35 kDa protein

The human breast carcinoma cell line SKBR3 was se-lected to purify the 35 kDa protein. The protein wasdemonstrated to localize within the cytoplasm of thesecells and could be extracted by hypotonic lysis, usingconditions that are compatible with isoelectric focusingmethods. FFE, a continuous liquid-based isoelectricfocusing method, was therefore used as the first step ofpurification. In a first small-scale approach, 26 mg cyto-solic cell extract proteins from SKBR3 cells was dilutedwith IEF running buffer to yield a final protein concentra-tion of 0.26 mg/mL and the sample was separated by FFEaccording to Section 2.3. The system was tested bymeasuring the pH-gradient and the separation of low-molecular-mass pI-markers (Fig. 1A). Approximately

900 mL of each fraction was collected over a period of50 min at a voltage of 1250 V and a current of 14 mA. Ali-quots (32 mL) of fractions 25–70 were analyzed by West-ern blotting using the anti-phospho-FKHR antibody. The35 kDa protein accumulated in fractions 46–47 and 51–52, corresponding to pIs of 5.29–5.37 and 5.93–6.04,respectively [9]. The shift towards a more acidic pI isconsistent with either phosphorylation or deamidation.Preliminary attempts to identify the 35 kDa protein wereunsuccessful because of the high abundance of malatedehydrogenase in the FFE fraction (Fig. 1B). For this rea-son a RP-HPLC step was introduced to remove themalate dehyrogenase.

3.3 Preparative 3-D separation of the 35 kDaprotein

For preparative purification of the 35 kDa protein, ap-proximately 56108 SKBR3 cells were extracted byhypotonic lysis, yielding 272 mg of cytosolic protein. Thesample was mixed in a 1:1 ratio with 1% Servalyte

pH 3–10 and 0.4% HPMC, and then further diluted withIEF running buffer to a final protein concentration of0.2 mg/mL. The sample was divided for two separateruns. They were performed at 1253 V and 11 mA on twoconsecutive days. Fractions expected to contain the35 kDa protein based on analytical FFE analysis wereexamined by Western blotting (Fig. 1C). The 35 kDaprotein was detected in fractions 47–53. The drift of onefraction of the protein towards the cathode and the lossof resolution of the potentially differentially phosphoryl-ated forms can be explained by the prolonged separa-tion time compared to the analytical run. However, inboth preparative runs, the protein appeared in the samesix fractions. The positive fractions were pooled (6.8 mg)and separated in the second dimension via preparativeRP-HPLC. Aliquots of the collected fractions were againanalyzed by Western blotting (Fig. 2A). The complexity ofthese fractions was further analyzed by silver staining(Fig. 2B), which indicated that malate dehydrogenase nolonger co-purified with fractions positive for the 35 kDaprotein, because only faint silver-stained bands weredetected at this molecular mass. Fractions 13 and 14were pooled, vacuum-concentrated, and subjected toSDS-PAGE; however, the separation of proteins ap-peared to be inhibited by the HPMC that remained afterRP-HPLC purification. To remove traces of HPMC, pro-teins from fractions 15–18 were precipitated prior toloading onto a 10% SDS-PAGE gel (see Section 2.7).Proteins could now be resolved and the gel was stainedwith Coomassie blue (Fig. 3A). Three candidate bandsmigrating at approximately 35 kDa were excised andanalyzed by tandem mass spectrometry.



Figure 1. Preparative purification of the 35 kDa protein. (A) pH-gradient and test separation of small-molecular-mass pImarkers (pI 3.9, 4.1, 5.3, 6.4, 7.4, 8.4, 10.1) by FFE in a Servalyte pH 3–10 gradient. (B) Proteins contained in fraction 46 ofSKBR3 hypotonic cell lysate (26 mg) separated by analytical FFE were precipitated and subjected to SDS-PAGE. The majorsilver-stained band migrating at 35 kDa was identified by MS to contain malate hydrogenase. (C) SKBR3 hypotonic celllysate (272 mg) was separated by two FFE runs (#1 and #2). 15 mL of fractions 45–53 was analyzed by Western blotting withanti-phospho-FKHR antibody; 10 mL of the starting material was loaded as a control.

Figure 2. Preparative RP-HPLC of the 35 kDa protein.Preparative FFE fractions 48–51 (Fig. 1C) were pooledand subjected to RP-HPLC. 2 mL aliquots of the indicatedfractions were analyzed by (A) Western blotting using anti-phospho-FKHR antibody and (B) SDS-PAGE using silverstaining.

3.5 Tandem mass spectrometry andidentification of StarD10

The individual proteins were in-gel digested with trypsinand peptides were analyzed by n-ESI-MS. Charged pep-tide ions from the TOF-MS spectrum were chosen fortandem mass spectrometry (see Section 2.8). Band #1was positively identified to contain glyceralaldehyde3-phosphate dehydrogenase, band #2 matched an entryin the TrEMBL database (Q9Y365), while band #3 wasidentified to contain inorganic pyrophosphatase (Fig. 3A).Interestingly, in the Q9Y365 (StarD10) sequence, wecould identify a motif comprising a putative serine phos-phorylation site (S259) that resembled the S256 con-sensus sequence observed in FKHR [9], making this pro-tein the most likely candidate.

The Mascot search results of peptides from band #2 arelisted with their molecular masses, amino acid sequenc-es, and Mascot scores in Table 1. Eight peptides withMascot scores indicating identity or extensive homology,with a probability higher than 95% matched the Q9Y365sequence deposited in the TrEMBL database. Uponmanual sequencing, four additional peptides with lower



Figure 3. Identification of StarD10. (A)Fractions 15–18 (Fig. 2) were pooled,precipitated, and subjected to 10%SDS-PAGE. The gel was stained withCoomassie blue and candidate bandswere excised (marked with arrows).Results of the MS identifications are

indicated. (B) Total cell lysate from 56104 MCF7 cells was subjected to SDS-PAGE. The gel was immunoblotted withStarD10-specific antibody. To estimate the amount of cellular StarD10, indicated amounts of purified recombinant StarD10protein were loaded.

Mascot scores could also be assigned to Q9Y365(Table 1). One of these peptides matched the amino acidsequence of the tryptic peptide LAASTEPQGPRPVLGRfrom Q9Y365, which could be confirmed by de novo se-quencing. De novo sequencing of the other three pep-tides revealed that they represented N-terminally trun-cated, nontryptic peptides of the same sequence. Theseresults were confirmed by a Mascot search without anenzyme specification and scores are shown in Table 1.

The molecular mass of 40 kDa of Q9Y365 was higherthan the experimentally observed mass of the 35 kDaprotein. Comparison with the highly conserved mousehomolog Q9JMD3 suggested that the 68 aa NH2-termi-nal sequence of Q9Y365 might not be translated,accounting for why we did not identify peptides corre-sponding to this sequence. This hypothesis was subse-quently confirmed when the human protein was depos-

ited in the Swiss-Prot database as a 291 amino acidprotein with a theoretical mass of 33 kDa (pI 6.7) underthe accession number Q9Y365 and described as: PCTP-like protein (PCTP-L) (StAR-related lipid transfer protein10) (StARD10) (START domain-containing protein 10)(CGI-52) (serologically defined colon cancer antigen 28)(antigen NY-CO-28). We cloned the corresponding cDNAinto a mammalian expression vector harboring an N-ter-minal Flag epitope tag. Western blotting of 293T celllysates containing the transiently expressed proteinrevealed that the phospho-FKHR antibody indeedrecognized Flag-tagged StarD10 [9].

To quantify the amount of StarD10 present in breast can-cer cell lines, lysate from MCF7 breast carcinoma cells,which express StarD10 to a similar level as that in SKBR3cells [9], was compared with purified recombinantStarD10 protein by Western blotting using a StarD10-

Table 1. Tryptic peptides identified by tandem MS from Q9Y365 (StarD10)

Mr (D mass) Peptide sequence Mascot score Rank

1986.98 (10.02 Da) SWLPMGADYIIMNYSVK 89 11661.98 (10.08 Da) AVSIQTGYLIQSTGPK 88 11548.78 (10.02 Da) AGVSVWVQAVEMDR 86 11799.98 (10.21 Da) SECa)EAEVGWNLTYSR 87 11492.76 (10.02 Da) SCa)VITYLAQVDPK 77 11542.70 (10.04 Da) LTVNADVGYYSWR 71 11564.70 (10.05 Da) WDSNVIETFDIAR 69 11433.65 (20.01 Da) ESVQVPDDQDFR 45 11647.88 (20.03 Da) LAASTEPQGPRPVLGR 33 (de novo) 11463.8 (10.02 Da) ASTEPQGPRPVLGR 13 (de novo) 31392.74 (20.01 Da) STEPQGPRPVLGR 22 (de novo) 11305.68 (20,04 Da) TEPQGPRPVLGR 14 (de novo) 1

a) Cysteine alkylated with ioda-cetamide (carbamidomethylC)de novo: peptides with Mas-cot scores lower than thefigure that indicates identityor extensive homology (p, 0.05) were manuallysequenced to confirm theresult.



specific antibody. 56104 MCF7 cells contained approxi-mately 20–50 ng of protein, indicating that StarD10 isoverexpressed to a significant level in breast cancer celllines (Fig. 3B).

3.5 Phosphorylation of human StarD10 onserine 259

Treatment of immunoprecipitated Flag-tagged StarD10with alkaline phosphatase abolished recognition by theanti-phospho-FKHR antibody, suggesting that cross-reactivity is based on phosphorylation of StarD10 [9]. Toexamine whether StarD10 was phosphorylated onserine 259, generating an epitope that resembles theone present in the FKHR protein, we immunoaffinity-purified Flag-tagged StarD10 from 293T cells transientlyexpressing the protein. StarD10 was eluted with glycineat low pH and concentrated by ultrafiltration prior toseparation by SDS-PAGE. The gel was stained withCoomassie blue and the band corresponding to

StarD10 band was excised and in-gel digested withtrypsin. Specific enrichment of phosphopetides wasachieved by immobilized metal ion affinity chromatog-raphy with gallium(III) ions. In the final fraction elutedwith 60% methanol, two [M15H1HPO3]

51-ions and a[M14H1HPO3]

41-ion were detected, the molecularmasses of which corresponded to singly phosphorylat-ed tryptic peptides T35 (229–272) and T35–36(227–272) (Fig. 4). The fragmentation patterns of the two[M15H1HPO3]

51-ions showed y-ion series up to y10,ruling out phosphorylation of serine 271 and 266. Thefragmentation spectrum of the [M14H1HPO3]

41-ionobtained the highest score in the Mascot search, andsuggested that serine 259 was phosphorylated bymatching the y14-fragment of dehydroalanine. De novosequencing demonstrated a y-ion series up to y16 andthe loss of H3PO4 of the precursor ion as well as the y14-ion of phosphoserine and dehydroalanine (Fig. 5). Thesequence of the peptides, molecular masses, andMascot scores are shown in Table 2.

Figure 4. TOF-MS spectrum of the isolated phophopeptides of StarD10. TOF-MS spectrum of phosphopeptides derivedfrom purified Flag-tagged StarD10 after in-gel digestion, IMAC enrichment, desalting, and concentration by ZipTip m-C18.The sample was eluted into a nanospray capillary and data were collected on a QSTAR pulsar i equipped with a protananano-electrospray source. The spectrum shows the tryptic peptide T36: [M15H1HPO3]

51; T35-T36 [M15H1HPO3]51 and

T36 [M14H1HPO3]41 from StarD10.



Figure 5. Phosphorylation of StarD10 on serine 259. Tandem MS spectrum of T36:[M14H1HPO3]

41: m/z 1248.7; Mr 4990.7 of StarD10 (229–272) pS 259 HLPHFKPWLHPEQSPLPSLALSELSVQHADpSLENIDESAVAESR. Data were collected on a QSTAR pulsar i equipped with aProtana nano spray source and collision energy of 30 eV. Y-ions are annotated with their matchedsequence and the loss of H3PO4 (m/z 24.5) from the precursor ion is indicated by an arrow.

Table 2. Mascot search results of phosphopeptides from Flag-tagged immunoaffinitypurified StarD10

Mr (D mass) Peptide sequence Score Charge

4990.7 (10.34 Da)T 36 (229–272)

HLPHFKPWLHPEQSPLPSLALSELSVQHADSLENIDESAVAESR (phospho ST)

32a) 41

4990.46 (10.03 Da)T 36 (229–272)

HLPHFKPWLHPEQSPLPSLALSELSVQHADSLENIDESAVAESR (phospho ST)

29a) 51

5246.46 (20.12 Da)T35–36 (227–272)

QKHLPHFKPWLHPEQSPLPSLALSELSVQHADSLENIDESAVAESR (phospho ST)

29a) 51a) Scores.28 indicate identityor

extensive homology (p,0.05).

The function of serine 259 phosphorylation of StarD10remains to be determined. In breast carcinoma cell linesStarD10 appears to be constitutively phosphorylated onthis site. The respective kinase is not only present inbreast carcinoma cell lines but also in 293Tcells and doesnot appear to require stimulation. Interestingly, analyticalFFE revealed 2 peaks, corresponding to a pI shift of 0.5 pHunits, which could be explained by the presence of asecond phosphorylation site. Whether phosphorylation ofthis site also occurs in 293Tcells and its location remain tobe determined.

4 Concluding remarks

We report a preparative 3-D method that effectivelyresolved the complexity of a cell line proteome andallowed identification of a 35 kDa breast cancer protein.Although StarD10 is overexpressed in breast cancer celllines, its isolation required several purification steps dueto masking of the protein by the high-abundant metabolicenzyme malate dehydrogenase, which has similar phys-iochemical properties. FFE overcame the limited loadingcapacity and sample loss of 2-D gel electrophoresis in the



first dimension. Coupled with RP-HPLC and SDS-PAGE ithas a very high separation capacity for complex proteinmixtures. Because the protein is maintained in its com-plete form until the very last separation step, it is possibleto collect additional information on the target protein.After each separation step, aliquots of the individualfractions can be tested not only by Western blotting asdescribed here, but also by biological activity tests or byfluorescence and radioactivity monitoring when usinglabelled proteins. The successful isolation of StarD10phosphorylated on Ser259 from cellular cytosol provesthat the 3-D separation strategy exploiting the pI, hydro-phobicity, and molecular size of a protein may be amethod of choice when complex protein mixtures areanalyzed and a high loading capacity is required.

This work was supported by the Victorian Breast CancerResearch Consortium Inc., the National Health MedicalResearch Council, Max Planck Research Award Program(BEK). Monilola A. Olayioye was supported by EMBO andHFSPO fellowships. BEK is an ARC Federation Fellow.

Received July 16, 2004

5 References

[1] Hanash, S. M., Bobek, M. P., Rickman, D. S., Williams, T., etal., Proteomics 2002, 2, 69–75.

[2] Lin, D., Tabb, D. L., Yates III, J. R., Biochim. Biophys. Acta2003, 1646, 1–10.

[3] Hannig, K., Electrophoresis 1982, 3, 235–243.

[4] Krivánková, L., Bocek, P., Electrophoresis 1998, 19, 1064–1074.

[5] Burggraf, D., Weber, G., Lottspeich, F., Electrophoresis1995, 16, 1010–1015.

[6] Weber, G., Bocek, P., Electrophoresis 1996, 17, 1906–1910.

[7] Hoffmann, P., Ji, H., Moritz, R. L., Connolly, L. M., Freckling-ton, D. F., Layton, M. J., Eddes, J. S., Simpson, R. J., Prote-omics 2001, 1, 807–818.

[8] Weber, G., Islinger, M., Weber, P., Eckerskorn, C., Völkl, A.,Electrophoresis 2004, 25, 1729–1734.

[9] Olayioye, M. A., Hoffmann, P., Pomorski, T., Armes, J.,Simpson, R. J., Kemp, B. E., Lindeman, G. J., Visvader, J. E.,Cancer Res. 2004, 64, 3538–3544.

[10] Slais, K., Friedl, Z., J. Chromatogr. A 1994, 661, 249–256.

[11] Slais, K., Friedl, Z., J. Chromatogr. A 1994, 680, 549–559.

[12] Shevchenko, A., Wilm, M., Vorm, O., Mann, M., Anal. Chem.1996, 68, 850–858.

[13] Shevchenko, A., Shevchenko, A., Anal. Biochem. 2001, 296,279–283.

[14] Perkins, D. N., Pappin, D. J., Creasy, D. M., Cottrell, J. S.,Electrophoresis 2003, 24, 3551–3567.

[15] Guy, C. T., Webster, M. A., Schaller, M., Parsons, T. J., Car-diff, R. D., Müller, W. J., Proc. Natl. Acad. Sci. USA 1992, 89,10578–10582.


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