SW ve TMV Ulusal Sağlık Enstitüleri NCRR ve NHLBI tarafından desteklenmektedir. BSK ve HS, Ulusal Sağlık Enstitüleri NINDS ve NIGMS tarafından desteklenmektedir.

DENEYSEL PROSEDÜRLER (Şekil 2) deneysel işlemin kademeli bir şekilde aşağıda açıklanan dört bölümden oluşmaktadır. Bölüm 1: P2X2 reseptörlerinin C-terminalindeki Subcloning ve ifadesi. Biz onu bağlar beyin proteinleri tespit etmek için tam uzunlukta bakteriler P2X2 reseptörleri C-terminus dile getirdiler. <ol><li> P2X2 reseptör (Şekil 1) C-terminalindeki pGEX 4NT1 (GE Life Sciences) klonlanmış (kalıntıları 353-472) ve sıralama ile doğrulanmış, PCR ile amplifiye oldu. </li><li> Rekombinant plazmid E. dönüştü Rekombinant proteinler ifade coli (BL21). </li><li> Plazmid P2X2 CT-GST içeren bakteriler (E. coli BL21) Gliserol stoklarının steril bir pipet ucu ile kazınır ve uygun seçici marker (50 g / ml ampisilin) ​​ve 5 ml Luria-Bertani (LB) medya ekildi , 37 ° C&#39;de orbital çalkalayıcı bir gece inkübe edildi. </li><li> Gecede büyüdü kültürleri 600 nm dalga boyunda optik yoğunluk ulaşana kadar uygun bir antibiyotik ile (50 mg / ml ampisilin) ​​250 ml LB medya içine aşılanmış ve 37 ° C&#39;de 2-3 saat için 250 rpm&#39;de orbital çalkalayıcı inkübe ~~ 0.6-0.7. </li><li> Kültür IPTG 1 mM ile tetiklenen ve daha fazla protein ekspresyonu için 3 saat için 37 ° C&#39;de inkübe edildi. </li><li> 5000 g, kültür 4C (kültür 1ml ifade düzeyini analiz etmek için kaydedilir ve &#39;indüklenen&#39; olarak etiketlenmiş) 15 dakika sonra ayrıldı. Süpernatant atılır ve pelet Tuzlu Tris-EDTA (STE) tamponu (10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA pH 8.0) 20 ml yeniden süspanse edildi. </li><li> Süspansiyon, 50 ml şahin tüp içinde 15 dakika süreyle 5000 g ayrıldı. Süpernatant atılır ve yarı kuru pelet at-70C gecede donduruldu. </li><li> -70C pelet buz lizis tamponu (% 2 sarkosyl (w / v), 15 mg lizozim, 150 mM NaCl, 50 mM Tris pH7.5, 5 mM DTT ve tam proteaz inhibitörü tablet) 40 ml yeniden süspanse ve süspansiyon 30 dakika buz üzerinde inkübe edildi. Bu kuluçka sırasında, 3 ml Glutatyon-sepharose 4B boncuk Phosphate Buffer Saline (PBS) ve PBS tarafından 20 ml T-PBS (% 0.1 (v / v) PBS içinde tritonX-100) 20 ml ile yıkandı. Boncuk, 1 ml PBS içinde yeniden süspanse edildi. </li><li> Bakteriyel süspansiyon, sıvı nitrojen içinde 3 kez dondurma çözülmüş ve sonicated (1s kapalı / buz üzerinde 3 dakika süreyle 30 mikron) ve daha fazla,% 4 ile 30 dakika (v / v) triton X-100, 10 mM MgSO inkübe 4 ve 2 mM ATP buz üzerinde. </li><li> Lizat (pelet ve 1 süpernatant ml sitoplazmada ve membranlar proteinlerin miktarda test etmek için SDS sayfa için kaydedilmiş bir örnek) atılır 4C ve pelet az 15 dakika süreyle 20000 g ayrıldı. Süpernatant 1 saat (veya gece) 4C için boncuklar ile inkübe edildi. </li><li> Boncuklar, 5 dakika boyunca 500 g bükülmüş ve supernatant (örnek bağlanmamış fraksiyonu kaydedilir) atılır. Boncuk T-PBS beş kez (yıkama yıkama kontrolleri için kaydedilmiş) ile yıkandı. </li><li> Boncuk sonra% 50 (w / v) bulamaç vermek için PBS içinde yeniden süspanse ve kullanana kadar 4C buzdolabında saklanır. Protein konsantrasyonu Bradford assay (üreticinin talimatlarına göre) tarafından tahmin edilmiştir. </li></ol> Bölüm 2: tüm beyin lizatları hazırlanması P2X2 reseptörleri beyin 8 bol ifade edilmesi bilinmektedir. Mevcut analizler, sıçan tüm beyin ilginçtir (Şekil 3) P2X2 reseptörleri ile etkileşen proteinleri tespit etmek için aradı. <ol><li> Yetişkin sıçan beyin hasat (UCLA IAACUC onaylı protokole göre hayvanların ve NIH Laboratuvar Hayvanları Bakım ve Kullanım Kılavuzu göre kurban edildi) ve hemen buz PBS (3X) durulanır. </li><li> 10 ml (~ 5 kez hasat beyin ıslak ağırlık) buz lizis tamponu içeren 150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 10 mM EDTA, 1 mM EGTA, 1 mM NaF, 1 mM olmuş akyuvarlar Na 3 VO 4, 1 mM PMSF leupeptin, 5 mg / ml,% 1 (v / v) NP-40, bir proteaz inhibitörü kokteyl tablet hasat beyin eklendi. </li><li> Beyin dokusu, homojen tutarlılık bir karışım elde edilene kadar, bir bardak Dounce homojenizatör ile buz homojenize edildi. </li><li> Hücre lizat kesintisiz hücreleri çıkarmak için 5 dakika 2000 g ayrıldı. Süpernatant sağlam organelleri ve membran agrega kaldırmak için 60 dakika süreyle 29000 g de tekrar toplanır ve bükülmüş oldu. </li><li> Santrifüj hemen sonra süpernatant temiz bir tüpe transfer edildi. Tüm beyin lizat protein konsantrasyonu Bradford assay ile ölçüldü (genellikle 15 mg / ml). </li><li> Lizat aliquoted ve-70C saklanır. </li></ol> Bölüm 3: P2X2 C-kuyruk ilişkili proteinlerin İzolasyon Tüm beyin lizatları P2X2 CT-GST bağlayıcı ortakları belirlemek için tahlil aşağı çekme CT-G ile yapıldıST yem olarak glutatyon sepharose 4B boncuklar üzerinde hareketsiz. Kontroller için, boncuk bağlı yalnız GST kullanılmıştır. <ol><li> Beyin lizat 4C az 1 saat süreyle GST boncuk precleared oldu. Pelet atılır ve supernatant ileri analizler için kullanılmıştır. </li><li> GST ve P2X2 glutatyon sepharose 4 B boncuk bağlı CT-GST (10 mikrogram) Eşit miktarda lizis tamponu 4C dönme ile sıçan bütün beyin lizat precleared solubilised proteinlerin 100 g gece inkübe edildi. </li><li> Boncuk 5 dakika 1000 g bükülmüş ve supernatant atılmıştır. </li><li> Boncuk lizis tamponu ile bir kez yıkanır ve modifiye Laemli tampon kaynatılmış [4% (w / v) SDS,% 10 (v / v) 2-merkaptoetanol,% 2 (v / v) gliserol,% 0.004 (w / v) Bromophenol Mavi ve 0.125 M TrisCl pH 6.8] 5 dk. </li><li> Örnekleri 5 dakika ve% 10 SDS-PAGE (Şekil 4) ile ayrılmış süpernatant 5000 g spun. Jeller SYPRO Ruby protein leke jel (üreticinin talimatlarına göre) ve ultraviyole ışık altında görüntülendi ile boyandı. P2X2 CT ile ilişkili proteinler GST boş yem olarak kullanarak kurtarıldı ile karşılaştırıldı. Dört biyolojik (dört beyinleri ve dört bağımsız çekme çıkışlar) yapıldı çoğaltır. </li></ol> Bölüm 4: proteinlerin belirlenmesi. Jel elektroforezi ile ayrılmış Proteinler jel eksize ve kütle spektrometresi 12 tarafından belirlenen Not: Bu süreç boyunca toz olmaması keratin kirliliği azaltmak için kritik öneme sahiptir. Deneyci her zaman, saç net ve eldiven maske takınız ve eldiven kullanılmadan ilgi jel bölgeye asla dokunmayın. <ol><li> Aşağı P2X2 çekme kurtarıldı tüm görünür protein bantları, temiz bir jilet ile eksize ve doğranmış (Şekil 4). GST boş jel Sorumlu bölgelerde sokaktan aşağı çekin seviyesinin altında proteinlerin varlığını sigortalamak için bant boyama (GST boş yem için özel bantlar da eksize ve tespit edilmiştir) dikkate alınmaksızın aynı şekilde eksize edildi boyama hassasiyet cevapsız değildi. </li><li> Jel adet 200% 50 50 mM amonyum bikarbonat ul (v / v) asetonitril (ACN) ve RT 15 dakika vortekslenmiş tüpleri ile inkübe edildi. Bu yıkama adımı yineleyin. </li><li> Jel adet 200 ul asetonitril (shrink jel parçaları ve opak renkli olması gerektiğini) ekleyerek susuz. </li><li> Acetontrile kaldırılır ve jel parçaları yaklaşık 10 dakika süreyle speedvac kurutuldu. </li><li> Jel parçaları daha sonra parçaları batırmayın 30 ul taze hazırlanmış 10 mM DTT/10 mM Tris (2-karboksi-etil) yeterli hacim fosfin hidroklorür (TCEP) ile inkübe edildi. Bu 56C su banyosunda 30 dakika kaldı. </li><li> Sonraki DTT çözümü 100 ul 500% 50 asetonitril çözüm mM amonyum bikarbonat ve ammounium bikarbonat ile yıkanır jel dilimleri ile değiştirildi. </li><li> Yıkama solüsyonu aspire ve 200 ul asetonitril jeller kurutmak eklendi. </li><li> Asetonitril sonra kaldırılır ve 100 ul taze hazırlanmış 100 mM iodoacetamide çözümü serbest sülfidril alkylate eklendi. Bu adım, karanlıkta, oda sıcaklığında 25 dakika devam etti. </li><li> Iodoacetamide çözüm kaldırıldı ve vorteks sırasında jel adet 200 ul 2 dakika süreyle% 50 acetontrile (pH 8.0) 50 mM amonyum bikarbonat ile yıkandı. Bu yıkama adım tekrarlandı. </li><li> Yıkama çözeltisi çıkardıktan sonra, jel adet 200 ~ 10dk sonra speedvac tarafından kaldırıldı ul asetonitril ile inkübe edildi. </li><li> Bu aşamada jel parçaları tripsin sindirim için hazırız. Jel adet 30 ul tripsin çözüm (20 ng / mcL çalışma konsantrasyonu) inkübe ve iyi şişmiş jeller için 10 dakika boyunca buz üzerine yerleştirildi. Aşırı tripsin kaldırıldı ve 50 mM amonyum bikarbonat ile 30 ul ile kaplanmış, 37 ° C&#39;de 12 ila 16 saat boyunca devam etmesine izin sindirim boyunca dalmış tutmak için jel partikülleri rehidrate edildi. </li><li> Beş ul% 5 (v / v) formik asit, tripsin ve tutuklama sindirim devre dışı bırakmak için eklenir. </li><li> Tüpler ~ 15 dk vortekslenmiş ve kısa bir süre sonra temiz ve etiketli 0.5 ml flakon nakledildi tüpün dibine sıvı getirmek için santrifüj edildi. </li><li> Sadece kısa santrifüj ile ayrılmış iki kez karıştırın, 10-15 dakika süreyle izledi kaplıydı böylece% 0.1% 50 asetonitril Sonraki 30 ul (v / v) formik asit jel parçaları eklendi. Bu adım, adımlar arasında asetonitril formik asit yerine, bir kez tekrarlandı. </li><li> Son sıvı tek bir flakon kaldırıldı ve tüm ekstraksiyon çözeltisi kombine edildi. Bu hacim speedvac ~ 30 uL azaldı. Örnek kütle spektrometresi ile ters faz nano-sıvı kromatografisi ve protein belirlenmesi için artık hazır olduğunu. Bu özel örnekte, bu adımı ThermoF bir Orbi-trap tandem kütle spektrometre yürütülmektedirisher (yüksek kütle doğruluğu, protein tayini için hızlı görev döngüsü ve genellikle sağlam bir işletim özellikleri için seçilen), diğer modeller ve üreticilerin araçların kolayca bu noktaya kadar açıklanan yaklaşım ile birleştiğinde olabilir rağmen. </li><li> Spektrometrik kitle analizleri için detayları ve kriterler kısaca sunulmuştur. Tüm peptidler ters faz nano-LC (Eksigent) ve peptidler, 3 ul / dk akış hızında C18 ters-faz sütun üzerine yüklenen ayrıldı. Mobil faz A% 0.1 formik asit ve su içinde% 2 ACN; mobil faz B,% 0.1 formik asit ve% 20 su ACN içinde oldu. Peptitler sütundan 220 nl / dk sonra 5 dakika boyunca% 95 B, 90 dakika boyunca% 50 B% 5 B doğrusal bir degrade kullanarak bir akış hızında yıkandı, ve nihayet 5 dakika boyunca sabit% 95 B tutmak . Spectra, MS tarar ve LTQ MS / MS taramaları için kullanılan Orbi-trap ile veri bağımlı modunda elde edildi. Peptitler Bioworks yazılım paketi entegre SEQUEST algoritmasını kullanarak sıçan IPI veritabanı v.3.53 karşı spektrumları arayarak tespit edildi. Her peptid aşağıdaki ölçütler karşılanmalıdır: XCorr ≥ 2 (+1), ≥ 3 (+2), ≥ 4 (3), ve DeltaCN&gt; 0.1. Protein tanımlanması için kullanılan tüm spektrumları elle denetlenir ve proteinler iki peptidler daha az olduğu tespit edilir. Sadece proteinler GST boş çekme çıkışlar sırasında her dört P2X2 çekme çıkışlar ve yok şu mevcut ileriki analizler için kabul edildi. </li></ol><img src="/files/ftp_upload/1178/1178fig1.jpg" alt="Şekil 1" /> Şekil 1. P2X2 reseptör altbirim şematik. A. karikatür P2X2 reseptör alt biriminin topoloji göstermektedir. N ve C Termini sitozolik bir etki oluşur. P2X2 reseptör (kırmızı) C-terminalindeki aşağı çekin tahlil için yem olarak kullanılır. Bu çalışmada kullanılan P2X2 reseptör C-terminalindeki B. Amino asit dizisi. <img src="/files/ftp_upload/1178/1178fig2.jpg" alt="" /> Şekil 2. Akış şeması ve zaman çizgisi ifade için kullanılan protokol, arıtma, aşağı çekme ve proteinlerin belirlenmesi zaman çizgisi ile protokol anahat göstermektedir. Yetişkin sıçan beyin lizat aşağı çekme deneyleri için kullanılmak hemen önce taze olarak hazırlanır. <img src="/files/ftp_upload/1178/1178fig3.jpg" alt="" /> Şekil 3. Sıçan beyin P2X2 C-terminalindeki ağ ikili proteomik analiz şematik gösterimi. GST GST boncuk bağlı protein ile kaynaşmış P2X2 reseptörlerinin C-terminalindeki testleri aşağı çekme için kullanılmıştır. Beyin lizat hazırlanmış ve proteinler immobilize rekombinant proteinleri ile inkübe edildi. Bağlanmamış fraksiyonu lizis tamponu ile yıkandı. Proteinler kütle spektrometresi ile tespit edilmiştir. <img src="/files/ftp_upload/1178/Fig%204%20Singh%20et%20al.jpg" alt="" /> Şekil 4 P2X2 reseptörlerinin C-terminus bağlayıcı ortakların belirlenmesi. SYPRO boyanan jel C-terminalindeki GST (mavi kutu) ile kaynaşmış reseptörleri ile etkileşim varsayılan proteinlerin bir spektrum gösteren. Kontroller, yalnız GST tek başına (sarı kutu) ve glutatyon sepharose boncuk şeritleri de gösterilir. Oklar, daha fazla analiz için kütle spektrometresi ile eksize edildi benzersiz gruplarından örnekler göstermektedir.

<ol>
	<li>Hille, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ion channels of excitable membranes. , (2001).</li><li>Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+physiology+of+P2X+receptors+and+ATP+signalling+at+synapses.">Molecular physiology of P2X receptors and ATP signalling at synapses.</a> Nat Rev Neurosci. 2, 165-174 (2001).</li><li>Egan, T. M., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+calcium+ions+to+P2X+channel+responses.">Contribution of calcium ions to P2X channel responses.</a> J Neurosci. 24, 3413-3420 (2004).</li><li>Burnashev, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+permeability+of+ligand-gated+channels.">Calcium permeability of ligand-gated channels.</a> Cell Calcium. 24, 325-332 (1998).</li><li>Clapham, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signaling.">Calcium signaling.</a> Cell. 131, 1047-1058 (2007).</li><li>Levitan, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+protein+complexes+associated+with+neuronal+ion+channels.">Signaling protein complexes associated with neuronal ion channels.</a> Nat Neurosci. 9, 305-310 (2006).</li><li>Khakh, B. S., North, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P2X+receptors+as+cell-surface+ATP+sensors+in+health+and+disease.">P2X receptors as cell-surface ATP sensors in health and disease.</a> Nature. 442, 527-532 (2006).</li><li>Roger, S., Pelegrin, P., Surprenant, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facilitation+of+P2X7+receptor+currents+and+membrane+blebbing+via+constitutive+and+dynamic+calmodulin+binding.">Facilitation of P2X7 receptor currents and membrane blebbing via constitutive and dynamic calmodulin binding.</a> J Neurosci. 28, 6393-6401 (2008).</li><li>North, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+physiology+of+P2X+receptors.">Molecular physiology of P2X receptors.</a> Physiol Rev. 82, 1013-1067 (2002).</li><li>Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+union+of+pharmacology.+XXIV.+Current+status+of+the+nomenclature+and+properties+of+P2X+receptors+and+their+subunits..">International union of pharmacology. XXIV. Current status of the nomenclature and properties of P2X receptors and their subunits..</a> Pharmacol Rev. 53, 107-118 (2001).</li><li>Young, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+shape,+architecture+and+size+of+P2X4+receptors+determined+using+fluorescence+resonance+energy+transfer+and+electron+microscopy.">Molecular shape, architecture and size of P2X4 receptors determined using fluorescence resonance energy transfer and electron microscopy.</a> J Biol Chem. 283, 26241-26251 (2008).</li><li>Edmondson, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C+epsilon+signaling+complexes+include+metabolism-+and+transcription/translation-related+proteins:+complimentary+separation+techniques+with+LC/MS/MS.">Protein kinase C epsilon signaling complexes include metabolism- and transcription/translation-related proteins: complimentary separation techniques with LC/MS/MS.</a> Mol Cell Proteomics. 1, 421-433 (2002).</li><li>Evans, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthosteric+and+allosteric+binding+sites+of+P2X+receptors.">Orthosteric and allosteric binding sites of P2X receptors.</a> Eur Biophys J. 38, 319-327 (2009).</li></ol>

İyon kanalları büyük bir integral membran proteinleri sınıfı. Onlar kendi elektrokimyasal gradyanlar aşağı seçici plazma zarından iyonların hareketi izin su dolu gözeneklerin içerir. İyon kanalları kapısı açık ve kapalı devletler arasında. Yolluk adım p2x ligand kapılı iyon kanalları halinde vericileri (örneğin ATP) tarafından tetiklenir, ya da diğer proteinler ile etkileşimler tarafından düzenlenmiş olabilir. Son on p2x reseptörler ATP 13 bağlama nasıl anlayışımızı ...

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		<th>Comment</th>
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<td>Acetonitrile</td>
<td>Reagent</td>
<td>JT Baker</td>
<td>9829-02</td>
<td>&nbsp;</td>
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<td>Acrylamide</td>
<td>Reagent</td>
<td>BIO-RAD</td>
<td>161-0156</td>
<td>&nbsp;</td>
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<td>Ampicillin</td>
<td>Reagent</td>
<td>VWR</td>
<td>VW1507-01</td>
<td>&nbsp;</td>
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<td>Ammonium Bicarbonate</td>
<td>Reagent</td>
<td>Fluka</td>
<td>09830</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium Persulphate (APS)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A3678</td>
<td>&nbsp;</td>
<tr>
<td>Adenosine Triphosphate (ATP)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A7699</td>
<td>&nbsp;</td>
<tr>
<td>Bradford reagent</td>
<td>Reagent</td>
<td>BIO-RAD</td>
<td>500-0006</td>
<td>&nbsp;</td>
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<td>Bromophenol blue</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>B-392</td>
<td>&nbsp;</td>
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<td>Commassie blue R-250</td>
<td>Reagent</td>
<td>Santa Cruz Biotechnology</td>
<td>Sc-24972</td>
<td>&nbsp;</td>
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<td>Dithiotritol (DTT)</td>
<td>Reagent</td>
<td>EMD</td>
<td>3860</td>
<td>&nbsp;</td>
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<td>Ethylenediaminetetraacetic acid (EDTA)</td>
<td>Reagent</td>
<td>VWR</td>
<td>VW1474-01</td>
<td>&nbsp;</td>
<tr>
<td>Ethylene Glycol tetraacetic acid (EGTA)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>E8145</td>
<td>&nbsp;</td>
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<td>Formic acid</td>
<td>Reagent</td>
<td>EMD</td>
<td>11670-1</td>
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<td>Glutathione Sepharose 4B beads</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-5132-01</td>
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<td>Hydrochloric acid (HCl)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H1758</td>
<td>&nbsp;</td>
<tr>
<td>Isopropyl-beta-D-thiogalactopyranoside (IPTG)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>15502</td>
<td>&nbsp;</td>
<tr>
<td>Iodoacetamide</td>
<td>Reagent</td>
<td>Sigma</td>
<td>I1149</td>
<td>&nbsp;</td>
<tr>
<td>Luria-Bertani (LB) Media</td>
<td>Reagent</td>
<td>EMD</td>
<td>1.00547.5007</td>
<td>&nbsp;</td>
<tr>
<td>Leupeptin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>L8511</td>
<td>&nbsp;</td>
<tr>
<td>Lysozyme</td>
<td>Reagent</td>
<td>Sigma</td>
<td>62971</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium Sulphate (MgSO4)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S7653</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Chloride (NaCl)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S3014</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Flouride (NaF)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S7920</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Orthovanadate (Na3VO4)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S6508</td>
<td>&nbsp;</td>
<tr>
<td>Nonidet P40</td>
<td>Reagent</td>
<td>Fluka</td>
<td>74385</td>
<td>&nbsp;</td>
<tr>
<td>Phenylmethanesulphonylfluoride (PMSF)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P7626</td>
<td>&nbsp;</td>
<tr>
<td>Protease inhibitor tablet</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S8820</td>
<td>&nbsp;</td>
<tr>
<td>Protein standard </td>
<td>Reagent</td>
<td>BIO-RAD</td>
<td>161-0305</td>
<td>&nbsp;</td>
<tr>
<td>Sarkosyl</td>
<td>Reagent</td>
<td>Acros</td>
<td>61207</td>
<td>&nbsp;</td>
<tr>
<td>Screw top vial</td>
<td>Tool</td>
<td>Agilent Technologies</td>
<td>5182-0866</td>
<td>&nbsp;</td>
<tr>
<td>Sodium dodecyl sulfate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>L4509</td>
<td>&nbsp;</td>
<tr>
<td>SYPRO&#174; Ruby protein gel stain</td>
<td>Reagent</td>
<td>BIO-RAD</td>
<td>170-3125</td>
<td>&nbsp;</td>
<tr>
<td>N,N,N',N'-Tetramethylethylenediamine (TEMED)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T9281</td>
<td>&nbsp;</td>
<tr>
<td>Tris base</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T1503</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T9284</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>Reagent</td>
<td>Promega</td>
<td>V5111</td>
<td>&nbsp;</td>
<tr>
<td>Tween 20</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P5927</td>
<td>&nbsp;</td>
<tr>
<td>Water</td>
<td>Reagent</td>
<td>Burdick&Jackson</td>
<td>365-4</td>
<td>&nbsp;</td>
<tr>
<td>LTQ-Orbitrap tandem mass spectrometer</td>
<td>Tool</td>
<td>ThermoFisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Nano Liquid Chromatography System</td>
<td>Tool</td>
<td>Eksigent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>B-Mercaptoethanol</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M6250</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>EMD</td>
<td>GX0185-6</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

proteomics to identify proteins interacting with p2x2 ligand-gated cation channels

Ligand kapılı iyon kanalları sinir sistemi 1 sinaptik iletişim yatar. Memelilerde ligand kapılı kanallar üç aile vardır: CYS döngü, glutamat-kapılı ve p2x reseptör kanal 2. Her iki durumda da verici bağlayıcı ionlar elektrokimyasal gradyanlar aşağı akan bir gözenek açılması yol açar. Birçok ligand kapılı kanallar da downstream kanal açma süresini aşamaz rolleri sinyalizasyon 5 (örneğin gen regülasyonu) kalsiyum iyonları 3, 4, geçirgen. Böylece ligand kapılı kanallar gün birkaç milisaniye arasında değişen geniş bir zaman ölçekleri üzerinde sinyal. Ligand kapılı iyon kanalları kendilerini proteinler tarafından düzenlenir anlamak için gerekli olduğunu ve bu proteinlerin nasıl bu önemli rolleri dikkate alındığında sinyal ayar olabilir. Son çalışmalar, pek çok, hepsi değilse de, kanal protein sinyalizasyon kompleksleri 6 parçası olabilir ki öneririz . Bu yazıda, P2X2 reseptör sitozolik etki C-terminal yönlerine bağlayan proteinleri tespit etmek için nasıl. P2x reseptörler ATP açılıp kapanan katyon kanalları ve yedi alt birimden (P2X1-P2X7) oluşur. P2x reseptörler eksitatör sinaptik iletim ve nörotransmiter sürüm 7 presinaptik kolaylaştırma arabuluculuk beyin, yaygın olarak ifade edilir. P2x reseptörleri heyecanlanır ve non-Uyarılabilir hücrelerinde bulundu ve nöronal sinyalizasyon, inflamasyon ve kardiyovasküler fonksiyon 8 kilit roller arabuluculuk. P2X2 reseptörler, sinir sistemi 9 bol ve bu çalışmanın odak vardır . Her p2x altbirim ekstraselüler bölge 7 ve hücre içi N ve C Termini (Şekil 1a) 7 ile ayrılmış iki membran kapsayan kesimleri (TM1 ve TM2) sahip olduğu düşünülmektedir. P2x alt birimden 10 (P2X1-P2X7), amino asit düzeyi 11% 30-50 dizisi homoloji göstermektedir . P2x reseptörleri iyonotropik reseptörler arasında basit stokiyometri sadece üç alt birimden içerir. P2X2 C-terminalindeki 120 amino asitler oluşur (Şekil 1b) ve P2X2 reseptör sinyal komplekslerinin parçası olabileceği hipotezini destekleyen birkaç protein yerleştirme konsensüs siteleri içerir. Ancak, çeşitli işlevleri P2X2 reseptörleri 9 C-terminalindeki atfedilmiş olmasına rağmen hiçbir çalışma moleküler ortakları açıklanan bu proteinin hücre içi tarafında tam uzunlukta C-terminalindeki üzerinden çift. Bu yöntem kağıt tam uzunlukta C-terminalindeki P2X2 reseptörleri ile etkileşen proteinleri tespit etmek için bir proteomik yaklaşım açıklar.

Watch this Scientific Journal Video about Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels at JoVE.com

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Biz, ATP-kapılı P2X2 reseptörlerinin tam uzunlukta C terminus bağlamak beyin proteinleri tespit etmek için basit bir protokol açıklar. Bu yaklaşımın uzantısı ve sistematik bir uygulama tüm p2x reseptörlerine p2x reseptör sinyal daha iyi anlaşılmasına yol açması bekleniyor.

P2X2 Ligandla Gated Katyon Kanalları ile etkileşimde Proteinler tanımlamak için Proteomik

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

proteomics-to-identify-proteins-interacting-with-p2x2-ligand-gated

Research

JoVE Journal

Biology

14.6K Görüntüleme.  David Geffen School of Medicine, University of California, Los Angeles. Biz, ATP-kapılı P2X2 reseptörlerinin tam uzunlukta C terminus bağlamak beyin proteinleri tespit etmek için basit bir protokol açıklar. Bu yaklaşımın uzantısı ve sistematik bir uygulama tüm p2x reseptörlerine p2x reseptör sinyal daha iyi anlaşılmasına yol açması bekleniyor.

Video: Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

S. cerevisiae, kantitatif proteomik protein kompleksleri tanımlanması

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Biyoloji

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR-Klip - RNA Cilt Proteinler Transcriptome geniş Cilt Siteleri Belirlemek Amacıyla Bir Yöntem

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Using SCOPE to Identify Potential Regulatory Motifs in Coregulated Genes

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and motif position preference1. Each component algorithm is optimized to find a different kind of motif. By taking the best of these three approaches, SCOPE performs better than any single algorithm, even in the presence of noisy data1. In this article, we utilize a web version of SCOPE2 to examine genes that are involved in telomere maintenance. SCOPE has been incorporated into at least two other motif finding programs3,4 and has been used in other studies5-8. 
The three algorithms that comprise SCOPE are BEAM9, which finds non-degenerate motifs (ACCGGT), PRISM10, which finds degenerate motifs (ASCGWT), and SPACER11, which finds longer bipartite motifs (ACCnnnnnnnnGGT). These three algorithms have been optimized to find their corresponding type of motif. Together, they allow SCOPE to perform extremely well.
Once a gene set has been analyzed and candidate motifs identified, SCOPE can look for other genes that contain the motif which, when added to the original set, will improve the motif score. This can occur through over-representation or motif position preference. Working with partial gene sets that have biologically verified transcription factor binding sites, SCOPE was able to identify most of the rest of the genes also regulated by the given transcription factor.
Output from SCOPE shows candidate motifs, their significance, and other information both as a table and as a graphical motif map. FAQs and video tutorials are available at the SCOPE web site which also includes a "Sample Search" button that allows the user to perform a trial run. 
Scope has a very friendly user interface that enables novice users to access the algorithm's full power without having to become an expert in the bioinformatics of motif finding. As input, SCOPE can take a list of genes, or FASTA sequences. These can be entered in browser text fields, or read from a file. The output from SCOPE contains a list of all identified motifs with their scores, number of occurrences, fraction of genes containing the motif, and the algorithm used to identify the motif. For each motif, result details include a consensus representation of the motif, a sequence logo, a position weight matrix, and a list of instances for every motif occurrence (with exact positions and "strand" indicated). Results are returned in a browser window and also optionally by email. Previous papers describe the SCOPE algorithms in detail1,2,9-11.

A straight-forward and robust method to identify potential regulatory motifs in co-regulated genes is presented. SCOPE does not require any user parameters and returns motifs that represent excellent candidates for regulatory signals. The identification of such regulatory signals helps to understand the underlying biology.

Coregulated Genler Potansiyel Düzenleme Motifler belirlenmesi için KAPSAMI

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

Proteinler Peptid immunoaffiniti Zenginleştirme kullanarak Niceleme Kütle Spektrometresi ile birleştiğinde

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

Yüksek verim Kristalizasyon lipidik Bicelle Yöntemiyle Membran Proteinleri

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

Büyüme Faktörü Progranulin ile etkileşimde Proteinler tanımlamak Modifiye Maya-İki Hybrid Sistemi

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

Memeli Nükleer Reseptör kalıntıları tespit Maya İki melez Sistemi ters olduğunu Ligandlannın ve / veya antagonistleri ile Etkileşim

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that transduce the mechanical energy of sound into an electro-chemical energy processed by the peripheral and central nervous systems. Several proteomic techniques have been developed to study the cochlear inner ear, such as two-dimensional difference gel electrophoresis (2D-DIGE), antibody microarray, and mass spectrometry (MS). MS is the most comprehensive and versatile tool in proteomics and in conjunction with separation methods can provide an in-depth proteome of biological samples. Separation methods combined with MS has the ability to enrich protein samples, detect low molecular weight and hydrophobic proteins, and identify low abundant proteins by reducing the proteome dynamic range. Different digestion strategies can be applied to whole lysate or to fractionated protein lysate to enhance peptide and protein sequence coverage. Utilization of different separation techniques, including strong cation exchange (SCX), reversed-phase (RP), and gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) can be applied to reduce sample complexity prior to MS analysis for protein identification.

Proteome analysis of the cochlear sensory epithelium can be challenging due to its small size and because membrane proteins are difficult to isolate and identify. Both membrane and soluble proteins can be identified by combining multiple preparative methods and separation techniques along with high-resolution mass spectrometry.

Kapsamlı Cochlear Proteome tanımak için aşağıdan yukarıya ve Shotgun Proteomiks

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that ...

Glycopeptide Capture for Cell Surface Proteomics

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, differentiation, and proliferation. A comprehensive characterization of these proteins provides rich information for biomarker discovery, cell-type identification, and drug-target selection, as well as helping to advance our understanding of cellular biology and physiology. Surface proteins, however, pose significant analytical challenges, because of their inherently low abundance, high hydrophobicity, and heavy post-translational modifications. Taking advantage of the prevalent glycosylation on surface proteins, we introduce here a high-throughput glycopeptide-capture approach that integrates the advantages of several existing N-glycoproteomics means. Our method can enrich the glycopeptides derived from surface proteins and remove their glycans for facile proteomics using LC-MS. The resolved N-glycoproteome comprises the information of protein identity and quantity as well as their sites of glycosylation. This method has been applied to a series of studies in areas including cancer, stem cells, and drug toxicity. The limitation of the method lies in the low abundance of surface membrane proteins, such that a relatively large quantity of samples is required for this analysis compared to studies centered on cytosolic proteins.

Cell surface proteins are biologically important and widely glycosylated. We introduce here a glycopeptide-capture approach to solubilize, enrich, and deglycosylate these proteins for facile LC-MS based proteomic analyses.

Hücre Yüzey Proteomics için glikopeptiddir Yakalama

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.