We thank Alberto Reinders for his work in optimizing the CCMS conditions for P. aeruginosa. We also thank Pablo Manfredifor the annotation of the P. aeruginosa proteins. This work was supported by the Swiss National Science Foundation (SNF) Sinergia grant CRSII3_127433.

1. Lizat Hazırlık <ol><li> P büyütün İstenen OD LB aeruginosa hücreleri. NOT: rehberlik için: log fazı kültürleri için sabit faz kültürler için ≈ 100 ml kültür / örnek ve ≈ 500 ml cultur / örnek (OD 600Nm = 0.5) kullanın. </li><li> 5.000 x g&#39;de 20 dakika boyunca santrifüj ile pelet. </li><li> Süspanse 0.5-1 1 mi liziz tamponunda pellet g (6.7 mM MES, 6.7 mM HEPES, 200 mM NaCI, 6.7 mM kaç, DDT, 1 mM, pH 7.5) ve proteaz inhibitörü (tam mini, EDTA-içermeyen) ekleyin de DNasel olarak. </li><li> Lyse 20.000 psi bir Fransız basınç hücresi ile 3 pasajlar ile hücre, (Malzeme listesi bakınız). </li><li> Ultra santrifüj 4 ° C &#39;de, 1 saat boyunca 100,000 x g&#39;de hücre lizatı. </li><li> Süpernatant kaydedin (2. adıma gidin). </li><li> Yukarı ve aşağı pipetleme lizis tamponu 1x 1 ml pelet yıkayın. </li><li> Ultrasantrifüj işlemi, 4 ° C&#39;de 1 saat boyunca 100,000 x g&#39;de. </li><li> Pelet flaş dondurmaMembran proteinlerinin (adım 3) yakalanması için kullanılana kadar -20 ° C&#39;de, sıvı azot ve saklayın. </li></ol> C-di-GMP Ücretsiz ve Diğer Nükleotidlerinin 2. Temizleme (Çözünür Kesir Sadece) <ol><li> Soğuk lisiz tampon maddesi içinde 10 ml bir PD10 tuz giderme kolonu (bakınız Materyaller listesi) yıkayın. </li><li> Nükleotidleri gidermek amacıyla PD10 süpernatanların (≈ 1 mi) dökün. </li><li> 4 ml soğuk lisiz tamponu (500 ul aşama) ile yıkayın. </li><li> Bradford tahlili ile saptandığı haliyle en yoğun fraksiyonlar seçin ve bunları bir araya getirirler. </li></ol> 3. Pelet Tekrar Süspansiyonu ve Çözme (Membran Kesir Sadece) <ol><li> (Deterjan olmadan) 1x yakalama tamponu 500 ila 1000 ul pelletini tekrar (Tablo 1). NOT: pelet tekrar süspansiyon zordur. Kabaca iyi çözüm homojenize bir şırınga 27 G kullanmak için, sonra pelet tekrar süspansiyon bir pipet ile ilk pipet yukarı ve aşağı tavsiye edilir. </li><li> % 1 ekleyin (a / h) n-dodesil-β-D-maltopiranozit (DDİ). </li><li> Dönen bir tekerlek üzerinde, en azından 2 saat (ya da O / N), 4 ° C&#39;de inkübe edilir. </li><li> Ultrasantrifüj işlemi, 4 ° C&#39;de 1 saat boyunca 100,000 x g&#39;de. </li><li> Süpernatant Hasat. </li></ol> 4. Protein Konsantrasyon Ölçümü <ol><li> (Membran fraksiyonunun BCA deneyi ile) Bradford tahlili ile protein konsantrasyonu ölçülür. </li><li> 10 mg / ml toplam protein konsantrasyonunu belirleyin. </li></ol> 5. Yakalama <ol><li> GSYİH GTP, ATP, CTP 1 mM ve 5x yakalama tamponu (100 mM HEPES, 250 mM Kac, 50 mM MgAc,% 5 gliserol, pH 7.5) (Tablo 1) 20 ul protein 300 ug karıştırın. H 2 O ile 100 ul reaksiyon ses seviyesini ayarlama NOT: Toplam hacmi rekabet kontrol durumunda CdG-CC ve c-di-GMP seviyesini içerir (adım 5.3 ve Tablo 2). Not: tüm deneyler 200 ve gerçekleştirildi# 181; l 12-tüp PCR şeritler (Thermo Scientific). NOT: zar kısmı için, her zaman kritik misel konsantrasyonu 8 M Üre protein çözündürme aşamasına kadar (7.1 Adım) (w / v% 0.01) Yukarıdaki DDM konsantrasyonunu tutmak için emin olun. </li><li> Dönen bir tekerlek üzerinde, 30 dakika boyunca 4 ° C&#39;de inkübe edilir. </li><li> 10 uM CdG-CC (nihai konsantrasyon) eklenir. NOT: (&quot;boncuk kontrolü&quot; olarak anılacaktır) CDG-CC olmayan bir denetim ve 1 mM c-di-GMP (&quot;yarışma kontrolü&quot;) ile desteklenmiş bir denetim vardır (Tablo 2). NOT: CdG-CC konsantrasyonu 1 ile 10 uM kadar ayarlanabilir. </li><li> Karanlıkta, dönen bir tekerlek üzerinde, en az 2 saat (O / N membran fraksiyonu), 4 ° C&#39;de inkübe edilir. </li><li> Bir CaproBox kullanarak 4 dakika UV ışığı ile CDG-CC reaktif parçasının aktivasyonu ile kısa bir sıkma, çapraz bağ, sonra (kaynak = 310 nm, Işınım ≥10 mW / cm², mesafe Malzemeleri Listesi görmek λ = 2 cm). NOT: şeritleri önce çapraz bağlama kapağını kaldırın. </li><li> Hafifçe homojenize, 25 ul 5x yıkama tamponu (5 M NaCl, 250 mM Tris, pH 7.5) yeniden süspansiyon haline getirilmiş ve de streptavidin manyetik taneleri 50 ul ekle. </li><li> Dönen bir tekerlek üzerinde 1 saat süre ile 4 ° C&#39;de inkübe edilir. </li></ol> 6. Yıkama adımları NOT: (Mıknatıs: Malzeme Listesi&#39;ne bakın). Mıknatıs ile PCR şerit kapağı manyetik boncuk yakalama, ile başlayın. Ardından bir sonraki yıkama çözeltisi içeren yeni bir PCR şerit değiştirin. Mıknatısı çıkarın ve boncuklar tekrar süspansiyon ve 2 dakika inkübe. Spin ve taze kapakla kapağı değiştirin. <ol><li> Yıkama adımları (çözünebilir fraksiyon için) <ol><li> 200 ul 1x yıkama tamponu 6 kez yıkayın. </li><li> 200 ul HPLC dereceli H 2 O bir kez yıkayın </li><li> 200 ul% 80 asetonitril içinde 6 kez yıkayın. </li><li> 200 ul HPLC dereceli H 2 O 2 kez yıkayın </li></ol></li><li> Yıkama adımları (membrane fraksiyonu sadece) <ol><li> +% 0.1 DDM 200 ul 1x yıkama tamponu içinde 5 kez yıkayın. </li><li> + 0.05% DDM 200 ul 1x yıkama tamponu 2 kez yıkayın. </li><li> +% 0.025 DDM 200 ul 1x yıkama tamponu kez yıkayın. </li><li> + 0.0125% DDM 200 ul 1x yıkama tamponu kez yıkayın. </li><li> 200 ul 100 mM ABC 3 kez yıkayın (amonyum bikarbonat, NH 4 CO 3) + 2 M Üre. </li></ol></li></ol> 7. MS Numune Hazırlama <ol><li> 20 ul 100 mM ABC (membran fraksiyonu için + 8 M Üre 100 mM ABC) &#39;de (doğrudan kapakta) boncuk süspanse ve 1.5 ml tüpler içine aktarın. </li><li> Membran fraksiyonu, sadece 500 rpm&#39;de çalkalanarak, 5 dakika boyunca 60 ° C&#39;de inkübe edin. </li><li> 200 mM TCEP (tris (2-karboksietil) fosfin) ul 0.5 ekleyin ve 500 rpm &#39;de çalkalanarak 1 saat boyunca 60 ° C&#39;de inkübe edin. 25 ° C&#39;ye kadar soğutun. </li><li> Bir çalkalama, iyodoasetamid, taze hazırlanmış 400 mM, 0.5 ul ilave edilir ve 30 dakika boyunca 25 ° C&#39;de inkübe500 rpm ve karanlıkta t. </li><li> 0.5 ul 0.5 M N-asetil-sistein ilave edin ve 500 rpm&#39;de çalkalanarak, 10 dakika boyunca 25 ° C&#39;de inkübe edin. </li><li> Membran fraksiyonu, sadece: 1 ul Lys-C ekleyin ve 37 ° C, O / N inkübe edin. </li><li> 2 ug tripsin ekleyin ve 500 rpm (kurumasını önlemek için Parafilm olarak şal) sallayarak, 37 ° C&#39;de O / N kuluçkaya yatmaktadır. Not: örnekler, bu aşamada -20 ° C &#39;de muhafaza edilebilir. NOT: Membran fraksiyon sadece: tripsin eklenmesinden önce &lt;2 M&#39;ye üre konsantrasyonunu ayarlamak için 100 mM ABC ekleyin. </li><li> Kısaca tüpler aşağı spin ve mıknatıs ile boncuk toplamak. </li><li> Yeni 1.5 ml tüp içine süpernatant aktarın (boncuk bırakılırsa bu adımı tekrarlayın). </li><li> 5 ul% 5 TFA (trifloroasetik asit) + 1 ul 2 M HCI (membran fraksiyonunun 15 ul% 5 TFA + 5 ul 2 M HCI) ekleyin. </li><li> Durum C18 MicroSpin sütunları ul asetonitril 150 ile (Nest Grubu, MA, ABD) (spin 2,400 rpm&#39;de 20 sn). </li><li> Equi% 0.1 TFA (spin 20 sn, 2.400 rpm) ul C18 sütunlar, 150 ile 2 kez librate. </li><li> Örnek yükleyin ve 2 dakika, 2.000 rpm döndürün. </li><li> Akış yoluyla kolonuna yükleyin ve sıkma aşamasına (2 dakika, 2000 rpm), tekrarlayın. </li><li> % 0.1 TFA ul 150 ile 3 kez yıkanır,% 5 asetonitril (20 sn, 2400 rpm). </li><li> Yeni bir tüp ve 150 ul% 0.1 TFA ile iki kez elüte,% 50 asetonitril (2 dakika, 2000 rpm). </li><li> Hızlı Vac peptidler kurulayın. </li><li> 40 ul% 98 H2O,% 2 asetonitril,% 0.15 formik asit içinde süspanse. </li><li> Sonikasyon 20 sn (darbe döngüsü 0.5, genlik 100%; Malzeme Listesi) ve 5 sn, 12.000 rpm (tezgah üstü santrifüj) aşağı doğru döndürün. Vortex 10 sn ve 5 sn, 12.000 rpm aşağı doğru döndürün. LC-MS / MS analizi için bir HPLC vialinde aktarın. </li><li> -20 ° C&#39;de dondurun. Not: örnekler, bu aşamada -20 ° C &#39;de muhafaza edilebilir. </li></ol> 8. LC-MS / MS Analizi <ol><li> Nano-LC çalıştırın (nano-LC sistemleri) equippBir RP-HPLC kolonuna (75 mm x 37 cm) C18 reçine ile dolu olan baskı (sihirli C18 AQ 3 um),% 95 çözücü A&#39;dan (% 0.15 formik asit,% 2 asetonitril) ile% 5 solvent B bir doğrusal eğimi kullanılarak ( 0.2 ul / dk&#39;lık bir akış oranında 60 dakika boyunca% 35 çözücü B için% 98 asetonitril,% 0.15 formik asit). </li><li> (Elektropüskürtmeli bir iyon kaynağı ile bağlanmış bir çift basınç LTQ-Orbitrap Velos kütle spektrometresi), LC-MS / MS kullanılarak peptidleri analiz edin. NOT: veri toplama modu MS 20 en yoğun iyonların doğrusal iyon tuzağı MS / MS taramaları ardından 60.000 FWHM çözünürlükte kütle spektrometresi FT bölümünde tarama bir yüksek çözünürlük elde etmek için kurulmuştur. MS / MS girişimleri verimliliğini artırmak için, ücret devlet tarama modus atanmamış dışlamak için etkin ve tek iyonları şarj edildi. Haberci 100 iyon sayıları aşıldığında Hile bağlı ayrışma tetikledi. Dinamik dışlama süresi 30 saniye olarak ayarlandı. iyon birikimi süresi 300 ms (MS) ve 50 olarak ayarlandımilisaniye (MS / MS). </li></ol> 9. Veritabanı Arama <ol><li> P İndir aeruginosa NCBI-veritabanı NCBI ana aracılığı ( <a href="http://www.ncbi.nlm.nih.gov/">http://www.ncbi.nlm.nih.gov/</a> ). </li><li> MassMatrix dönüştürme aracı (kullanarak maskotu jenerik dosyalarının içine MS ham spektrumları (mgf) dönüştürün <a href="http://www.massmatrix.net/mm-cgi/downloads.py">http://www.massmatrix.net/mm-cgi/downloads.py</a> ). </li><li> P. karşı MASCOT sürüm 2.3 kullanarak bu mgf dosyasını ara İleri içeren ve ters-tuzak protein girişleri aeruginosa NCBI-veritabanı. </li><li> İki tam Triptik peptitler de bölünmelere cevapsız tolere (prolin ardından sürece) lisin ve arginin sonra siliko tripsin sindirim bir gerçekleştirin. </li><li> Değişken değişiklikler ve sabit modifikasyon gibi sistein kalıntısı carboxyamidomethylation (+57.021464 Da) olarak oksitlenmiş metioninler (15,99491 Da) izin veritabanı arama parametrelerini ayarlayın. MASCOT bizi arar İçinyüksek çözünürlüklü taramalar ing, 15 ppm habercisi kitle toleransını ayarlamak ve Da 0.6 fragmanı kitle toleransını ayarlayın. Son olarak,% 1 protein FDR ayarlayın. </li><li> P. Maskot aramalar İthalat İskele (Proteomesoftware, Versiyon 3) içine aeruginosa MTSK deneyleri,% 1 protein FDR yakın edinmek ve toplam spektral sayıları ayıklamak için parametrelerini ayarlamak. </li><li> Temsilcisi sonuçları bu çalışmada sunulan için, rekabet kontrolü ile deney karşılaştırmak için eşleştirilmiş t testi kullanıldı ve sadece 0.1 altında bir p değeri ve 2 (spektral sayıları deneme / yarışma kontrol spektral üzerinde spektral sayım oranı ile hit kabul sayımları). </li><li> Veriler daha fazla analiz için herhangi bir elektronik tablo yazılımı ihraç edilebilir. </li></ol> 10. Etiket serbest Niceleme <ol><li> Progenesis LC-MS yazılımı (Doğrusal Olmayan Dinamikleri, Sürüm 4.0) içine çiğ dosyalarını içe aktarın. </li><li> Varsayılan Setti LC-MS hizalama ve özellik algılama gerçekleştirinNGS. </li><li> Progenesis LC-MS mgf formatında veri ihracat. </li><li> NCBI P. karşı maskotu kullanan MS / MS spektrumları ara İleri içeren ve ters-tuzak protein girişleri aeruginosa veritabanı. </li><li> Progenesis LC-MS içine veritabanı arama sonuçlarını İthalat ve MS1 özellikleri peptidler kimlik haritası. </li><li> (: 0.1 altında q değeri, 2 üzeri spektral sayım oranı eşiği) veri değerlendirme (anlamlılık düzeylerinin hesaplanmasında, kat-değişim oranları) için SafeQuant ve ProteinSQAnalysis komut kullanılmıştır. </li></ol>

<ol>
	<li>Nesper, J., Reinders, A., Glatter, T., Schmidt, A., Jenal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+capture+compound+for+the+identification+and+analysis+of+cyclic+di-GMP+binding+proteins.">A novel capture compound for the identification and analysis of cyclic di-GMP binding proteins.</a> J Proteomics. 75, 4874-4878 (2012).</li><li>Hengge, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+c-di-GMP+signalling+in+bacteria.">Principles of c-di-GMP signalling in bacteria.</a> Nature reviews. Microbiology. 7, 263-273 (2009).</li><li>Sondermann, H., Shikuma, N. J., Yildiz, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=You've+come+a+long+way:+c-di-GMP+signaling.">You've come a long way: c-di-GMP signaling.</a> Curr. Opin. Microbiol. 15, 140-146 (2012).</li><li>Schirmer, T., Jenal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+mechanistic+determinants+of+c-di-GMP+signalling.">Structural and mechanistic determinants of c-di-GMP signalling.</a> Nature reviews. Microbiology. 7, 724-735 (2009).</li><li>Furukawa, S., Kuchma, S., O'Toole, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keeping+their+options+open:+acute+versus+persistent+infections.">Keeping their options open: acute versus persistent infections.</a> J. Bacteriol. 188, 1211-1217 (2006).</li><li>Christen, M., Christen, B., Folcher, M., Schauerte, A., Jenal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+cyclic+di-GMP-specific+phosphodiesterase+and+its+allosteric+control+by+GTP.">Identification and characterization of a cyclic di-GMP-specific phosphodiesterase and its allosteric control by GTP.</a> J. Biol. Chem. 280, 30829-30837 (2005).</li><li>Ryan, R. P., Fouhy, Y., Lucey, J. F., Dow, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+di-GMP+signaling+in+bacteria:+recent+advances+and+new+puzzles.">Cyclic di-GMP signaling in bacteria: recent advances and new puzzles.</a> J. Bacteriol. 188, 8327-8334 (2006).</li><li>Habazettl, J., Allan, M. G., Jenal, U., Grzesiek, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solution+structure+of+the+PilZ+domain+protein+PA4608+complex+with+cyclic+di-GMP+identifies+charge+clustering+as+molecular+readout.">Solution structure of the PilZ domain protein PA4608 complex with cyclic di-GMP identifies charge clustering as molecular readout.</a> J. Biol. Chem. 286, 14304-14314 (2011).</li><li>Fazli, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CRP/FNR+family+protein+Bcam1349+is+a+c-di-GMP+effector+that+regulates+biofilm+formation+in+the+respiratory+pathogen+Burkholderia+cenocepacia.">The CRP/FNR family protein Bcam1349 is a c-di-GMP effector that regulates biofilm formation in the respiratory pathogen Burkholderia cenocepacia.</a> Molecular Microbiology. 82, 327-341 (2011).</li><li>Hickman, J. W., Harwood, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+FleQ+from+Pseudomonas+aeruginosa+as+a+c-di-GMP-responsive+transcription+factor.">Identification of FleQ from Pseudomonas aeruginosa as a c-di-GMP-responsive transcription factor.</a> Molecular Microbiology. 69, 376-389 (2008).</li><li>Sudarsan, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Riboswitches+in+eubacteria+sense+the+second+messenger+cyclic+di-GMP.">Riboswitches in eubacteria sense the second messenger cyclic di-GMP.</a> Science. 321, 411-413 (2008).</li><li>Lenz, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+of+methyltransferases+and+other+S-adenosyl-L-homocysteine-binding+Proteins+by+Capture+Compound+Mass+Spectrometry+(CCMS).">Profiling of methyltransferases and other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS).</a> J Vis Exp. , (2010).</li><li>K&#246;ster, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capture+compound+mass+spectrometry:+a+technology+for+the+investigation+of+small+molecule+protein+interactions.">Capture compound mass spectrometry: a technology for the investigation of small molecule protein interactions.</a> Assay Drug Dev Technol. 5, 381-390 (2007).</li><li>D&#252;vel, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+proteomics+approach+to+identify+c-di-GMP+binding+proteins+in+Pseudomonas+aeruginosa.">A chemical proteomics approach to identify c-di-GMP binding proteins in Pseudomonas aeruginosa.</a> Journal of Microbiological Methods. 88, 229-236 (2012).</li><li>Christen, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allosteric+control+of+cyclic+di-GMP+signaling.">Allosteric control of cyclic di-GMP signaling.</a> J. Biol. Chem. 281, 32015-32024 (2006).</li><li>Balasubramanian, D., Mathee, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+transcriptome+analyses+of+Pseudomonas+aeruginosa.">Comparative transcriptome analyses of Pseudomonas aeruginosa.</a> Human genomics. 3, 349-361 (2009).</li><li>Smyth, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linear+models+and+empirical+bayes+methods+for+assessing+differential+expression+in+microarray+experiments.">Linear models and empirical bayes methods for assessing differential expression in microarray experiments.</a> Stat Appl Genet Mol Biol. 3 (3), (2004).</li><li>Baraquet, C., Harwood, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+diguanosine+monophosphate+represses+bacterial+flagella+synthesis+by+interacting+with+the+Walker+A+motif+of+the+enhancer-binding+protein+FleQ.">Cyclic diguanosine monophosphate represses bacterial flagella synthesis by interacting with the Walker A motif of the enhancer-binding protein FleQ.</a> Proc. Natl. Acad. Sci. U.S.A. 110, 18478-18483 (2013).</li><li>Nesper, J., Reinders, A., Glatter, T., Schmidt, A., Jenal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+capture+compound+for+the+identification+and+analysis+of+cyclic+di-GMP+binding+proteins.">A novel capture compound for the identification and analysis of cyclic di-GMP binding proteins.</a> J Proteomics. 75, 4874-4878 (2012).</li><li>Roelofs, K. G., Wang, J., Sintim, H. O., Lee, V. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+radial+capillary+action+of+ligand+assay+for+high-throughput+detection+of+protein-metabolite+interactions.">Differential radial capillary action of ligand assay for high-throughput detection of protein-metabolite interactions.</a> Proc. Natl. Acad. Sci. U.S.A. 108, 15528-15533 (2011).</li><li>DeSantis, K., Reed, A., Rahhal, R., Reinking, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+differential+scanning+fluorimetry+as+a+high-throughput+assay+to+identify+nuclear+receptor+ligands.">Use of differential scanning fluorimetry as a high-throughput assay to identify nuclear receptor ligands.</a> Nuclear receptor signaling. 10, e002 (2012).</li><li>Seidel, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+thermophoresis+quantifies+biomolecular+interactions+under+previously+challenging+conditions.">Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions.</a> Methods. 59, 301-315 (2013).</li><li>Merighi, M., Lee, V. T., Hyodo, M., Hayakawa, Y., Lory, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+second+messenger+bis-(3&#8216;-5&#8217;)-cyclic-GMP+and+its+PilZ+domain-containing+receptor+Alg44+are+required+for+alginate+biosynthesis+in+Pseudomonas+aeruginosa.">The second messenger bis-(3&#8216;-5&#8217;)-cyclic-GMP and its PilZ domain-containing receptor Alg44 are required for alginate biosynthesis in Pseudomonas aeruginosa.</a> Molecular Microbiology. 65, 876-895 (2007).</li><li>Qi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+cyclic+diguanylate+in+the+non-catalytic+EAL+domain+of+FimX+induces+a+long-range+conformational+change.">Binding of cyclic diguanylate in the non-catalytic EAL domain of FimX induces a long-range conformational change.</a> J. Biol. Chem. 286, 2910-2917 (2011).</li></ol>

The authors have nothing to disclose.

Özel dikkat protokolünün çeşitli adımlarda alınmalıdır. Protein konsantrasyonu, 10 mg bir konsantrasyonda olan kritik bir parametredir / ml hücreleri özel büyüme koşulları altında (örneğin, biyofilm veya küçük koloni varyantları) altında yetiştirilir zaman erişilmesi zor olan. Bu nedenle, pelet yeniden süspansiyon lisis tamponu düşük bir hacme yapılmalıdır. Protein konsantrasyonları, 8 mg / ml&#39;ye kadar azaltılabilir. Nesper ve diğ. 1 tarafından yayınl...

c-di-GMP onların büyüme ve davranış çeşitli yönlerini kontrol etmek için en bakteriler tarafından kullanılan bir anahtar ikinci habercidir. Örneğin, Cı-di-GMP hücre döngüsü ilerlemesini, motilite ve eksopolisakaridlerin ve yüzey adhesinler 2-4 ekspresyonunu düzenler. Bu işlemler, koordinasyonu yoluyla, c-di-GMP biyofilm oluşumunu, patojenik bakterilerin 5 bir dizi kronik enfeksiyonlar ile ilişkili olan bir süreci desteklemektedir. c-di-GMP katalitik GGDEF alanı 4 liman diguanylate siklazlar (KEGM) olarak adlandırılan enzimler tarafından sentezlenmiş edilir. Bazı KEGM aşağı c-di-GMP bağlanması üzerine siklaz aktivitesini düzenleyen bir inhibitör sitesine sahip. C-di-GMP bozulması katalitik EAL veya HD-GYP ya etki 6,7 barındıran fosfodiesteraz iki farklı sınıflarına (PDE) ile katalize edilmektedir. doğrudan, c-di-GMP bağlayan bilinen efektör proteinlerin çoğunluğu protein sadece üç sınıftan birine ait: katalitiğimüttefiki inaktif GGDEF veya EAL etki ve Pilz alanları, c-di-GMP 8 bağlanması üzerine yapısal değişikliklere uğrarlar küçük moleküler anahtarlar. KEGM, KDD ve Pilz proteinleri iyi karakterize edilir ve onların etki nispeten güvenli silico tahmin edilebilir. Belirli bir ilgi şimdi c-di-GMP efektörlerinin yeni sınıfların belirlenmesi üzerine odaklanmıştır. Farklı bağlama motifleri ile Bazı c-di-GMP efektörleri Burkholderia cenocepacia CRP / FNR protein ailesi Bcam1349 veya P. transkripsiyonel regülatör FleQ olarak son zamanlarda böyle tarif edildi aeruginosa 9,10. Buna ek olarak, c-di-cGMP-spesifik riboswitches son zamanlarda belirlenen ve bir C-di-GMP-bağımlı bir şekilde 11 içinde gen ekspresyonunu kontrol etmek için gösterilmiştir. Farklı etkileyicilerin c-di-GMP bağlama motifleri sadece kötü korunmuş tür proteinlerin biyoinformatik tahminleri zor yapıyoruz. Bu sorunu çözmek için, bir C-di-GMP SPE kullanımına dayanan bir biyokimyasal yöntemler geliştirilmiştir, spesifik yakalama Bileşik kütle spektrometresi 1,12,13 ile birlikte. Biz son zamanlarda (CDG-CC, Şekil 1) yeni bir üç değerlikli c-di-GMP Yakalama Bileşik tasarladık 1. Bu kimyasal iskele oluşmaktadır: bağlayıcı proteinleri, c-di-GMP yakalamak için yem olarak kullanılan 1), bir c-di-GMP kısım, 2) bir UV ışıkla reaktif grup bağlantı geçmek için kullanılan bağlı proteinlere CdG-CC ve 3) ise biotin streptavidin-kaplı manyetik boncuklar kullanılarak çekilen proteinleri izole etmek. CdG-CC doğrudan olarak ve özellikle hücre lizatları gibi makromoleküllerin kompleks bir karışımdan, c-di-GMP efektör yakalamak için kullanılabilir. Yakalama Bileşik bazlı ve kimyasal proteomik temelli yaklaşımlar, daha önce organizmaların geniş bir yelpazede için geçerli olduğu bildirilmiştir, örneğin Caulobacter crescentus, Salmonella enterica serovar typhimurium ve S. aeruginosa 1,14. Bu metodolojik yazıda,Biz P. özler kullanılarak MTSK prosedürü derinliği açıklamasında bir sağlamak Örneğin, aeruginosa. Bu çalışma, biyokimyasal küçük molekül sinyalizasyon dahil yeni bileşenleri tanımlamak için güçlü ve çok yönlü bir araç olarak CCMS kurar.

<table border="0" cellpadding="0" cellspacing="0" width="1179">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:236px;">caproBox</td>
			<td style="width:223px;">caprotec bioanalytics</td>
			<td style="width:207px;">1-5010-001 (220 V)</td>
			<td style="width:515px;">UV lamps coupled to a cooling 96-plate cooling block, for the photoactivation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:236px;">caproMag</td>
			<td style="width:223px;">caprotec bioanalytics</td>
			<td style="width:207px;">included in the CCMS Starter Kit</td>
			<td style="width:515px;">For easy handling of magnetic particles without pipetting</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:236px;">c-di-GMP caproKit</td>
			<td style="width:223px;">caprotec bioanalytics</td>
			<td style="width:207px;">upon request</td>
			<td style="width:515px;">The kit contains the c-di-GMP-capture compound, c-di-GMP (for the competition control), streptavidin coated magnetic beads, capture buffer, and washing buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:236px;">Disposable PD-10 Desalting Columns</td>
			<td style="width:223px;">GE Healthcare</td>
			<td style="width:207px;">17-0851-01&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">12-tube PCR strips</td>
			<td style="width:223px;">Thermo Scientific</td>
			<td style="width:207px;">AB-1114</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UIS250v sonicator with VialTweeter</td>
			<td style="width:223px;">Hielscher ultrasound technology</td>
			<td style="width:207px;">UIS250v and VialTweeter</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Miniature French Pressure Cell</td>
			<td style="width:223px;">Thermo Electron Corporation</td>
			<td style="width:207px;">FA-003</td>
			<td></td>
		</tr>
	</tbody>
</table>

capture compound mass spectrometry - a powerful tool to identify novel c-di-gmp effector proteins

Kayda değer bir ilerleme, ikinci haberci, c-di-GMP sentezi (diguanylate siklazlar) ve ayrışma (fosfodiesteraz) yer alan enzimler üzerindeki tanımı ve karakterizasyonu için doğru, son on yıl içinde yapılmıştır. Buna karşılık, az bilgi bu sinyal molekülü hücresel süreçlerin çeşitli bir yelpazede düzenleyen hangi aracılığıyla moleküler mekanizmalar ve hücresel bileşenleri ile ilgili mevcuttur. Bilinen efektör proteinlerin çoğu Pilz ailesine ait veya kataliz vazgeçmiş ve efektör fonksiyonunu benimsemiş diguanylate siklazlar veya fosfodiesterazları dejenere edilmektedir. Bu nedenle, daha iyi bir deneysel yöntemler siliko tahminler güvenilir başarısız olduğu için yeni etkenleri tanımlamak ve doğrulamak için gerekli olan bakterilerin geniş bir aralıkta hücre C-di-GMP ağ tanımlamak için kullanılır. Biz son zamanlarda güçlü bir araç olarak bir roman Yakalama Bileşik Kütle Spektrometre (MERNİS) tabanlı teknoloji geliştirdikbiyokimyasal tespit ve c-di-GMP bağlayıcı proteinler karakterize etmektedir. Bu teknik, daha önce organizmaların 1 geniş uygulanabilir olduğu bildirilmiştir. Burada biz böyle sinyal bileşenlerini prob kullanan protokol ayrıntılı bir açıklamasını verir. Bir örnek olarak, Pseudomonas aeruginosa, C-di-GMP hastalık oluşturma ve biyofilm kontrolünde önemli bir rol oynadığı bir fırsatçı patojendir kullanın. MTSK C-di-GMP ağ bilinen ya da tahmin edilen bileşenlerin% 74 (38/51) tespit edilmiştir. Bu çalışma detaylı MTSK prosedürü açıklar ve küçük molekül sinyalizasyon dahil yeni bileşenleri tanımlamak için güçlü ve çok yönlü bir araç olarak kurar.

P. roman c--di-GMP efektörler belirlemek için aeruginosa sistematik P. Çözünür ve zar fraksiyonları analiz etmek için kullanılan CCMS Bir günlük faz kültüründen aeruginosa suşu PAO1 (= 0.5 OD 600). Burada özetlemek ve bu balıkçı sefer temsili sonuçlarını tartışmak. Dört bağımsız biyolojik kopyaları kullanılmıştır. Her deney için, iki farklı CdG-CC konsantrasyonu (5 uM, 10 uM) kullanılmıştır. Özgüllük için prob için, deneyler <stro...

Watch this Scientific Journal Video about Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins at JoVE.com

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

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.

Yakalama Bileşik Kütle Spektrometre - Roman c-di-GMP Efektör Proteinler tanımlamak için güçlü bir araç

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

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Research

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Biology

11.5K Görüntüleme.  Biozentrum of the University of Basel. 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.

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

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI Numune Hazırlama: Ultra İnce Tabaka Yöntemi

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

Biyoloji

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

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

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 ...

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

Nicel Değerlendirme Elektrosprey İyonizasyon Kütle Spektrometresi kullanarak Maya Lipidome

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

Split-Ubiquitin Dayalı Membran Maya İki Hybrid (MİT) Sistemi: protein-protein etkileşimleri belirlenmesi için güçlü bir araç

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

Yapısal Kütle Spektrometresi büyük protein kompleksleri Analiz

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

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 ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

Methyltransferases, profilleme ve Diğer S Adenosil- L</subYakalama Bileşik Kütle Spektrometresi (CCMS)> homosistein bağlayıcı Proteinler

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

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 ...

Amide Hydrogen/Deuterium Exchange &amp; MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. H/D exchange on the backbone amide positions has been utilized to measure the deuteration rates of the micro-regions in a protein by mass spectrometry1,2,3. The resolution of this method depends on pepsin digestion of the deuterated protein of interest into peptides that normally range from 3-20 residues. Although the resolution of H/D exchange measured by mass spectrometry is lower than the single residue resolution measured by the Heteronuclear Single Quantum Coherence (HSQC) method of NMR, the mass spectrometry measurement in H/D exchange is not restricted by the size of the protein4. H/D exchange is carried out in an aqueous solution which maintains protein conformation. We provide a method that utilizes the MALDI-TOF for detection2, instead of a HPLC/ESI (electrospray ionization)-MS system5,6. The MALDI-TOF provides accurate mass intensity data for the peptides of the digested protein, in this case protein kinase Pak2 (also called &gamma;-Pak). Proteolysis of Pak 2 is carried out in an offline pepsin digestion. This alternative method, when the user does not have access to a HPLC and pepsin column connected to mass spectrometry, or when the pepsin column on HPLC does not result in an optimal digestion map, for example, the heavily disulfide-bonded secreted Phospholipase A2 (sPLA2). Utilizing this method, we successfully monitored changes in the deuteration level during activation of Pak2 by caspase 3 cleavage and autophosphorylation7,8,9.

MALDI-TOF mass spectrometry was successfully utilized to monitor the amide hydrogen/deuterium exchange in protein kinase Pak2 activation.

Pak2 Etkinleştirme Amid Hidrojen / Döteryum Alım Satım &amp; MALDI-TOF Kütle Spektrometre Analiz

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

İzotop Etiketleme kütle spektrometresi kullanarak Tiyol Redoks Proteom profil

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...