Separose 4 Fast Flow beads stored in 100% isopropanol (Amersham. Bioscience) were washed four times with 1 mm HCl and twice with coupling buffer

Transcription

1 Supporting Information Materials and Methods Affinity chromatography. Three-hundred microliters of N-hydroxysuccinimideactivated Separose 4 Fast Flow beads stored in 100% isopropanol (Amersham Bioscience) were washed four times with 1 mm HCl and twice with coupling buffer containing 0.2 M NaHCO 3 and 0.5 M NaCl (ph 8.3). Beads were suspended in 400 l of coupling buffer and incubated with 0.2 mg of N3 overnight at 4 o C. N3-conjugated beads were washed five times with 1 ml of 0.1 M Tris-HCl, ph 8.5 and incubated in the 0.1 M Tris-HCl, ph 8.5 for 5 h. The beads were then sequentially washed seven times, with a buffer containing 0.1 M Tris-HCl (ph 8.5) and a buffer containing 0.1 M acetate (ph 4.5) and 0.5 M NaCl. Finally, the beads were stored in 1 ml of buffer B [20 mm HEPES, ph 7.4; 10 mm Mg(CH 3 COO) 2 ; 20% glycerol; 1 mm EGTA; 0.1% CHAPS, and proteinase inhibitor cocktail]. Membrane proteins were isolated from 5 x 10 7 PC3 cells. Harvested cells were incubated on ice for 30 min in buffer H (20 mm HEPES, ph 7.4; 10 mm Mg(CH 3 COO) 2 ; 20% glycerol; 1 mm EGTA; 250 mm sucrose), disrupted with a teflon/glass homogenizer, and centrifuged at 500 g for 10 min at 4 o C. Supernatants were centrifuged at 20,000 g for 30 min at 4 o C. The resulting pellets were dissolved in buffer B, rotated for 30 min at 4 o C, and centrifuged at 100,000 g for 60 min at 4 o C. Supernatants containing 120 g of membrane protein were incubated in IP buffer containing N3-conjugated beads for 4 h at 4 o C with slow rotation. Samples were washed with 500 l IP buffer four times and separated on 10% SDSpolyacrylamide gels. Gels were then silver stained.

2 Protein digestion and mass spectrometry. Protein bands were excised and stainstripped in 100 mmol/liter sodium thiosulfate and 30 mmol/liter potassium ferricyanide. Proteolytic peptides were recovered from the gel using overnight in-gel digestion with 12.5 ng/l of sequencing grade trypsin (Promega, Madison, WI). Trypic peptides were then dried by vacuum evaporator and analyzed by a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). A binary nanoflow HPLC system (Agilent 1100 series, Agilent) was directly coupled to an LTQ linear ion trap mass spectrometer (Thermo Electron Corp.) with automatic gain control to avoid space charge limitations. An in-house reversed-phase column (5-m C 18 material; column dimension, 75 m 13 cm) was used at a flow rate of 400 nl/min. Peptide samples were eluted over 85 min in a gradient of 90% solvent A (0.1% formic acid in H 2 O) to 40% solvent B (0.1% formic acid in acetonitrile). Tandem mass spectral data were analyzed using both the SEQUEST database search engine and the Trans-Proteomic Pipeline (TPP; Version 2.9; INTERACT, PeptideProphet TM, ProteinProphet TM ) provided by the Institute for Systems Biology. SEQUEST searches were performed against a regularly updated version of the mouse protein database from the European Bioinformatics Institute (IPI, Version 3.14, Figure Legends Figure 7. Subcellular localization of FITC-GnRH-II (FITC-GII) in prostate cancer cells. (A) Images of FITC-GnRH-II in MitoTracker-counterstained PC3, DU145, ALVA41, HeLa, CV-1, WI38, and GH3GII cells. (B) Effect of unlabelled GnRH-II analogs (GnRH-II, Trp-1, N3 and Cet) on FITC-GnRH-II in PC3 cells. Scale bars represent 10 µm.

3 Figure 8. Identification of N3-binding proteins using LC-ESI-MS/MS. (A) Silver staining of protein eluted from vehicle (CTL)- or N3-conjugated N-hydroxysuccinimide Separose 4. Multiple tandem MS spectra revealed that the p80 protein band consisted of three mitochondrial proteins: glucose regulated protein 75 (GRP75), tumor necrosis factor receptor-associated protein (TRAP-1), and hydroxyacyl-coenzyme A dehydrogenase/3-ketoacyl-coenzyme A thiolase/enoyl-coenzyme A hydratase (trifunctional protein) alpha subunit (HADHA). (B) GRP75, (C) TRAP-1, and (D) HADHA amino acid sequence. Underlined sequences were identified by multiple tandem MS spectra, which covered 32.8% of GRP75 amino acids, 24.9% of TRAP-1 amino acids, and 15.7% of HADHA amino acids.

4 Supporting information Fig 7. A PC3 DU145 ALVA41 HeLa CV-1 WI38 GH3GII Merge MitoTracker FITC-GII Merge MitoTracker FITC-GII 10 m B GnRH-II Trp-1 N3 Cet 10 m

5 Supporting information Fig 8. A C CTL N3 80kDa GRP75 TRAP1 TFE- M.W. 75 kda 80 kda 82 kda TRAP1 MARELRALLL WGRRLRPLLR APALAAVPGG KPILCPRRTT 40 AQLGPRRNPA WSLQAGRLFS TQTAEDKEEP LHSIISSTES 80 VQGSTSKHEF QAETKKLLDI VARSLYSEKE VFIRELISNA 120 SDALEKLRHK LVSDGQALPE MEIHLQTNAE KGTITIQDTG 160 IGMTQEELVS NLGTIARSGS KAFLDALQNQ AEASSKIIGQ 200 FGVGFYSAFM VADRVEVYSR SAAPGSLGYQ WLSDGSGVFE 240 IAEASGVRTG TKIIIHLKSD CKEFSSEARV RDVVTKYSNF 280 VSFPLYLNGR RMNTLQAIWM MDPKDVREWQ HEEFYRYVAQ 320 AHDKPRYTLH YKTDAPLNIR SIFYVPDMKP SMFDVSRELG 360 SSVALYSRKV LIQTKATDIL PKWLRFIRGV VDSEDIPLNL 400 SRELLQESAL IRKLRDVLQQ RLIKFFIDQS KKDAEKYAKF 440 FEDYGLFMRE GIVTATEQEV KEDIAKLLRY ESSALPSGQL 480 TSLSEYASRM RAGTRNIYYL CAPNRHLAEH SPYYEAMKKK 520 DTEVLFCFEQ FDELTLLHLR EFDKKKLISV ETDIVVDHYK 560 EEKFEDRSPA AECLSEKETE ELMAWMRNVL GSRVTNVKVT 600 LRLDTHPAMV TVLEMGAARH FLRMQQLAKT QEERAQLLQP 640 TLEINPRHAL IKKLNQLRAS EPGLAQLLVD QIYENAMIAA 680 GLVDDPRAMV GRLNELLVKA LERH 704 B D GRP75 MISASRAAAA RLVGAAASRG PTAARHQDSW NGLSHEAFRL 40 VSRRDYASEA IKGAVVGIDL GTTNSCVAVM EGKQAKVLEN 80 AEGARTTPSV VAFTADGERL VGMPAKRQAV TNPNNTFYAT 120 KRLIGRRYDD PEVQKDIKNV PFKIVRASNG DAWVEAHGKL 160 YSPSQIGAFV LMKMKETAEN YLGHTAKNAV ITVPAYFNDS 200 QRQATKDAGQ ISGLNVLRVI NEPTAAALAY GLDKSEDKVI 240 AVYDLGGGTF DISILEIQKG VFEVKSTNGD TFLGGEDFDQ 280 ALLRHIVKEF KRETGVDLTK DNMALQRVRE AAEKAKCELS 320 SSVQTDINLP YLTMDSSGPK HLNMKLTRAQ FEGIVTDLIR 360 RTIAPCQKAM QDAEVSKSDI GEVILVGGMT RMPKVQQTVQ 400 DLFGRAPSKA VNPDEAVAIG AAIQGGVLAG DVTDVLLLDV 440 TPLSLGIETL GGVFTKLINR NTTIPTKKSQ VFSTAADGQT 480 QVEIKVCQGE REMAGDNKLL GQFTLIGIPP APRGVPQIEV 520 TFDIDANGIV HVSAKDKGTG REQQIVIQSS GGLSKDDIEN 560 MVKNAEKYAE EDRRKKERVE AVNMAEGIIH DTETKMEEFK 600 DQLPADECNK LKEEISKMRE LLARKDSETG ENIRQAASSL 640 QQASLKLFEM AYKKMASERE GSGSSGTGEQ KEDQKEEKQ 679 HADHA MVACRAIGIL SRFSAFRILR SRGYICRNFT GSSALLTRTH 40 INYGVKGDVA VVRINSPNSK VNTLSKELHS EFSEVMNEIW 80 ASDQIRSAVL ISSKPGCFIA GADINMLAAC KTLQEVTQLS 120 QEAQRIVEKL EKSTKPIVAA INGSCLGGGL EVAISCQYRI 160 ATKDRKTVLG TPEVLLGALP GAGGTQRLPK MVGVPAALDM 200 MLTGRSIRAD RAKKMGLVDQ LVEPLGPGLK PPEERTIEYL 240 EEVAITFAKG LADKKISPKR DKGLVEKLTA YAMTIPFVRQ 280 QVYKKVEEKV RKQTKGLYPA PLKIIDVVKT GIEQGSDAGY 320 LCESQKFGEL VMTKESKALM GLYHGQVLCK KNKFGAPQKD 360 VKHLAILGAG LMGAGIAQVS VDKGLKTILK DATLTALDRG 400 QQQVFKGLND KVKKKALTSF ERDSIFSNLT GQLDYQGFEK 440 ADMVIEAVFE DLSLKHRVLK EVEAVIPDHC IFASNTSALP 480 ISEIAAVSKR PEKVIGMHYF SPVDKMQLLE IITTEKTSKD 520 TSASAVAVGL KQGKVIIVVK DGPGFYTTRC LAPMMSEVIR 560 ILQEGVDPKK LDSLTTSFGF PVGAATLVDE VGVDVAKHVA 600 EDLGKVFGER FGGGNPELLT QMVSKGFLGR KSGKGFYIYQ 640 EGVKRKDLNS DMDSILASLK LPPKSEVSSD EDIQFRLVTR 680 FVNEAVMCLQ EGILATPAEG DIGAVFGLGF PPCLGGPFRF 720 VDLYGAQKIV DRLKKYEAAY GKQFTPCQLL ADHANSPNKK 760 FYQ 763