MN-S Ulusal Bilim Vakfı desteği kabul RUI yazarları minnetle Thermo Fisher Scientific LTQ Orbitrap XL kütle spektrometresi kullanımına ilişkin Greensboro, Kuzey Carolina Üniversitesi&#39;nde Triad Kütle Spektrometre Tesisi kabul Che 1011859. verin. Yazarlar bu çalışmaları ile ilgili faydalı görüş ve önerileriniz için Greensboro, Kuzey Carolina Üniversitesi&#39;nden Daniel Todd, Vincent Sica ve Brandie Erhmann teşekkür ederiz.

Not: Kullanmadan önce ilgili tüm malzeme güvenlik bilgi formlarını (MSDS) danışın. Cıva klorür toksik bir kimyasaldır. Onu ve ilgili tüm çözümleri teslim sırasında Kişisel koruyucu ekipmanlar (eldivenler, emniyet gözlükleri ve laboratuvar önlüğü) giyilmelidir. Ağır metaller için belirlenen açıkça etiketlenmiş kimyasal atık şişelerde çözümler atınız. 1. Hazırlık 5 mM gazı alınmış Amonyum format tamponu, pH 7.5 <ol><li> HPLC dereceli su 450 ml amonyum format tamponu 0,1576 g çözündürülür. 1 M formik asit ve 7.5 1 M amonyum hidroksit ile, yukarıda çözeltinin pH ayarlayın. 500 ml&#39;lik bir ölçülü balona bu çözüm aktarın ve 5 mM amonyum format çözeltisinin yapılması için bir kalibrasyon hattına HPLC su ilave edilir. </li><li> Argon ile 10 dakika karıştırıldı ve tasfiye için bir vakum sistemi ile 5 mM amonyum format tamponu gaz çıkışına. İki kez tekrarlayın ve argon altında mağaza çözüm. Kullanılacağı gün, bir 0.2 mikron filtre befor ile tampon çözeltisi filtree kullanımı. </li></ol> Civa (II) klorür çözeltisi 2. Hazırlık <ol><li> 0,2375 g cıva (II) klorür tartılır. 0.035 M cıva (II) klorid solüsyonu elde etmek için 5 mM amonyum format tamponu, 25 ml içinde çözülür. </li><li> Bir 7.5 x 10 -4 M çözelti oluşturmak için 9,785 ml 5 mM amonyum format tampon 0.214 mi, 0.035 M cıva (II) klorür solüsyonu ekleyin. Argon gazı ile 7.5 x 10 -4 M cıva (II) çözeltisi battaniye. </li></ol> CGGC Stok Çözeltisinin Hazırlanması 3. <ol><li> HPLC dereceli asetonitril 0.118 ml dicysteinyl tetrapeptid, CGGC 2.0 mg, çözülür ve daha sonra 1,0647 ml 5 mM amonyum format, pH&#39;ı 5 mM CGGC stok çözeltisi oluşturacak şekilde argon gazı alınmış olan 7,5 tamponu ilave edin. </li><li> 5 mM amonyum oluşumu pH 1275 ul bir 7.5 x 10 -4 M CGGC çözelti vermek üzere 7.5 tampon, 5 mM CGGC stok çözeltisi 225 ul ekle. </li></ol> 4. HazırlıkCıva &#39;(II) ve CGGC çeşitli Reaksiyon Karışımlarının <ol><li> 1 Hazırlanması: civanın 0.5 oranı (II): CGGC çözeltisi <ol><li> 1.5 ml mikrosantrifüj tüpü içine 5 mM amonyum format, pH 7,5 tampon 255 ul koyun. Amonyum format tamponu ile bir 1.5 ml mikrosantrifüj tüpü içine 7,5 x 10 -4 M cıva (II) klorür solüsyonu 30 ul ekle. </li><li> 10 saniye için çözüm karıştırın. Sonra 1.5 ml mikrosantrifüj içine 7.5 x 10 -4 M CGGC solüsyonu 15 ul ekleyin. 10 saniye için çözüm karıştırın. Çözelti kütle spektrometresi içine enjeksiyondan önce 10 dakika boyunca bekletin. </li></ol></li><li> Cıva 1 oranında, (II): 1 hazırlanması CGGC çözeltisi <ol><li> 1.5 ml mikrosantrifüj tüpü içine 5 mM amonyum format, pH 7,5 tampon 240 ul koyun. Amonyum format tamponu ile bir 1.5 ml mikrosantrifüj tüpü içine 7,5 x 10 -4 M cıva (II) klorür solüsyonu 30 ul ekle. </li><li> 10 saniye için çözüm karıştırın. Daha sonra1,5 ml mikrosantrifüj tüpü içine 7.5 x 10 -4 M CGGC çözeltisi 30 ul ekle. Bölüm 4.1&#39;de tarif edildiği gibi benzer bir şekilde, tekrar edin. </li></ol></li><li> Cıva 2 oranında (II): 1 hazırlanması CGGC çözeltisi <ol><li> 1.5 ml mikrosantrifüj tüpü içine 5 mM amonyum format, pH 7,5 tampon 210 ul koyun. Amonyum format tamponu ile bir 1.5 ml mikrosantrifüj tüpü içine 7,5 x 10 -4 M cıva (II) klorür solüsyonu 30 ul ekle. </li><li> 10 saniye için çözüm karıştırın. Daha sonra bir 1.5 ml mikrosantrifüj tüpü içine 7.5 x 10 -4 M CGGC çözeltisi 60 ul ekle. Bölüm 4.1&#39;de tarif edildiği gibi benzer bir şekilde, tekrar edin. </li></ol></li></ol> CEEC Stok Çözeltisinin Hazırlanması 5. <ol><li> Peptidi eritmek için HPLC dereceli asetonitril 0.145 ml dicysteinyl tetrapeptid, ODAÜ 3.5 mg, eritin. Daha sonra 13,067 mi, 5 mM amonyum format ilave pH argon gazı alınmış olan 7,5 tamponu 0.5 m üretmekM CEEC çözüm. </li><li> Tüm peptid çözünene kadar çözelti vorteksleyin. Bir 7.5 x 10 5 M CEEC çözeltisi vermek üzere bir 1.5 ml mikrosantrifüj tüpü, pH 7,5 tampon çözelti, 1.125 mi CEEC 0,5 mM ilave edin ve 0,375 mi mM 5 amonyum format. Vortex karışık kadar. </li></ol> Cıva &#39;(II) ve CEEC Çözüm çeşitli Reaksiyon karışımları 6. hazırlanması <ol><li> 1 preparasyonu: cıva (II) 0,5 oranı: CEEC Çözelti <ol><li> 1.5 ml mikrosantrifüj tüpü içine 5 mM amonyum format, pH 7,5 tampon 255 ul koyun. Amonyum format tamponu ile bir 1.5 ml mikrosantrifüj tüpü içine 7,5 x 10 -4 M cıva (II) klorür solüsyonu 30 ul ekle. </li><li> 10 saniye için çözüm karıştırın. Daha sonra bir 1.5 ml mikrosantrifüj tüpü içine 7.5 x 10 -4 M CEEC çözeltisi 15 ul ekle. Bölüm 4.1&#39;de tarif edildiği gibi benzer bir şekilde, tekrar edin. </li></ol></li><li> Civa (II): 1 oranında 1 hazırlanması CEEC çözeltisi <ol><li> 240 koyun81; 1.5 ml mikrosantrifüj tüpü içine 5 mM amonyum format, pH 7.5 tamponu. Amonyum format tamponu ile bir 1.5 ml mikrosantrifüj tüpü içine 7,5 x 10 -4 M cıva (II) klorür solüsyonu 30 ul ekle. </li><li> 10 saniye için çözüm karıştırın. Daha sonra bir 1.5 ml mikrosantrifüj tüpü içine 7.5 x 10 -4 M CEEC çözeltisi 30 ul ekle. Bölüm 4.1&#39;de tarif edildiği gibi benzer bir şekilde, tekrar edin. </li></ol></li><li> Civa (II) 2 oranı: 1 hazırlanması CEEC çözeltisi <ol><li> 1.5 ml mikrosantrifüj tüpü içine 5 mM amonyum format, pH 7,5 tampon 210 ul koyun. Amonyum format tamponu ile bir 1.5 ml mikrosantrifüj tüpü içine 7,5 x 10 -4 M cıva (II) klorür solüsyonu 30 ul ekle. </li><li> 10 saniye için çözüm karıştırın. Daha sonra bir 1.5 ml mikrosantrifüj tüpü içine 7.5 x 10 -4 M CEEC çözeltisi 60 ul ekle. Bölüm 4.1&#39;de tarif edildiği gibi benzer bir şekilde, tekrar edin. </li></ol></li></ol> 7. AnalyOrbitrap ESI kütle spektrometresi ile cıva (II) ve CGGC Örneklerinde Reaksiyon karışımları hareketlendirmek <ol><li> ESI kütle spektrometresi 16 Hazırlanması <ol><li> 500 ul cam şırıngaya kalibrasyon standartları 100 ul çizin. </li><li> MS pompa enjektör beşiği şırınga yerleştirin tüp takmak ve kitle spektrofotometre içine enjekte edilir. </li><li> Dosya simgesini seçip dosya adını yazarak çalıştırmak için dosya adını ayarlayın. </li><li> Veri toplama modülü acquire veri düğmesini seçin ve 150 taramaları toplamak. </li><li> Yazılımın veri işleme modülü açarak kalibrasyon standartları doğrulamak için kromatogram analiz edin. Modülü açın, menü dosya gidin ve &quot;açık&quot; seçeneğini seçin ve iletişim kutusunda dosyayı seçin. Kromatogramda zirveleri standartları oranları şarj etmek için kitle ilişkili olduğunu doğrulayın. </li><li> 500 ul HPLC dereceli metanol yukarı çizerek 500 ul cam şırınga temizleyin ve sonra m dağıtmakBir beher içine etanol. </li><li> Cam şırınga içine HPLC dereceli metanol 500 ul çizin ve adım 7.1.2 başına sistemini yıkayın. </li><li> Parametreleri ayarlamak için yazılım yöntemi kurulum modülünü seçin. Tarama modu menüsünü seçin ve Ftms olarak analizörü tanımlamak ve sonra &quot;Tamam&quot; düğmesine tıklayın. Kılıf gaz akış hızı: 10, Kaynak sıcaklığı: 0, Kılcal gerilimi: 37 V, Tüp lensi: 95 V, Sprey gerilimi: 4.20 kV Sonra gerçek zamanlı görünümü spektrum sayfasında çeşitli ikonları tıklayarak, şu parametreleri ayarlayabilirsiniz Akış 10.00 ul / dk, Analyzer oranı: Ftms, taramalar sayısı: 150. </li></ol></li><li> ESI kütle spektrometresi üzerinde CGGC örnekleri çalıştırma <ol><li> 5 mM amonyum format, pH 7,5 tampon çalıştırın. <ol><li> 500 ul bir cam şırınga içine 5 mM amonyum format tamponu 500 ul koyun MS pompasının şırınga tutucuya yerleştirin ve boru ekleyin. </li><li> 1-2 dakika boyunca, boru içinden geçen tampon çalıştırın. </li><li> Çalıştırmak için dosya adını ayarlayındosya simgesini seçip dosya adını yazarak. </li><li> Modülünde acquire veri düğmesini seçin ve 150 taramaları toplamak. </li><li> 150 taramalar toplandıktan sonra toplama durdurmak için çalışma düğmesine tıklayın. </li><li> Veri tarayıcı modülü açın, daha sonra menüyü dosya gidin ve &quot;açık&quot; seçeneğini seçin ve iletişim kutusunda dosyayı seçin. 483, 683, 1.163 ve 1.363 m / z hiçbir zirveleri peptid veya civa (II) kompleksleri -peptid benzediğine mevcut olduğundan emin olun. </li></ol></li><li> 0,5 cıva (II): CGGC oranı çözeltisi 1 çalıştırın. <ol><li> 0,5 cıva (II): şırınga içine örnek CGGC oranı 1 250 ul koyun. </li><li> Boru ve Başbakan aparat takın, MS pompa enjektör yuvasına şırınga yerleştirin. </li><li> Dosya simgesini seçip dosya adını yazarak çalıştırmak için bir dosya adı seçin. </li><li> Veri toplama modülü acquire veri düğmesine basın ve 150 tarama toplamak ve toplama durdurmak için çalışma düğmesini tıklatın. </li><li> Modülü açın, menü dosya gidin ve &quot;açık&quot; seçeneğini seçin ve iletişim kutusunda dosyayı seçin. Kromatogram yalnız CGGC peptid için dahil olmak üzere doruklarına içerdiğini doğrulayın. </li><li> 500 ul amonyum format tamponu ile aspire ve daha sonra bir kap içine amonyum format tamponu dağıtarak şırınga yıkayın. </li><li> MS atık düğmesini seçin ve boru 500 ul amonyum format tamponu ile üç kez yıkayın. </li><li> Bir beher içine metanol dağıtma daha sonra 500 ul metanol ile aspire edilmesiyle ve şırıngayı yıkayın. </li><li> 500 ul metanol ile tüp bir kez yıkayın. </li><li> MS Seç yük dedektör düğmesine basın. </li><li> Şırıngaya amonyum format tamponu 500 ul ekle. </li><li> Boru ve Başbakan aparat takın, MS pompa enjektör yuvasına şırınga yerleştirin. </li><li> Dosya simgesini seçip dosya adını yazarak çalıştırmak tamponu için bir dosya adı seçin. </li><li> Basınacquire veri düğmesi ve 150 tarama toplamak ve ardından stop çalıştırmak düğmesini tıklatın. </li><li> Veri tarayıcı modülü açın, menü dosya gidin ve &quot;açık&quot; seçeneğini seçin ve iletişim kutusunda dosyayı seçin. CGGC run: kromatogram önceki Hg gelen zirvelerinden geçersiz olduğundan emin olun. </li></ol></li><li> 1 cıva (II): CGGC oranı çözeltisi 1 çalıştırın. <ol><li> Şırınga içine CGGC oranı örneği: 1 civa (II): 1 250 ul koyun. </li><li> 7.2.2.15 Adımları 7.2.2.2 için tarif edildiği gibi benzer bir şekilde, tekrar edin. </li></ol></li><li> 2 cıva (II): 1 başlat CGGC oranı çözümü <ol><li> Şırınga içine CGGC konsantrasyonu örneği: 2 civa (II): 1 250 ul koyun. </li><li> 7.2.2.15 Adımları 7.2.2.2 için tarif edildiği gibi benzer bir şekilde, tekrar edin. </li></ol></li></ol></li></ol> 8. Orbitrap ESI kütle spektrometresi ile Reaksiyon Merkür&#39;ün karışımlar ve CEEC Örnekleri analiz <ol><li> ESI kütle spektrometresi üzerinde CEEC örnekleri Koşu <ol><li> Tekrar analiz prosedürü (Basamak 7.1 7.2) CEEC örnekleri ve çeşitli stoikiometrik oranlarda civa (II) ve ODAÜ tepkime karışımları kullanılarak. </li></ol></li></ol>

<ol>
	<li>Clifton, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mercury+exposure+and+public+health.">Mercury exposure and public health.</a> Pediatr. Clin. N. Am. 54, 237-269 (2007).</li><li>Andersen, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+Recent+Developments+in+Chelation+Treatment+of+Metal+Intoxication.">Principles and Recent Developments in Chelation Treatment of Metal Intoxication.</a> Chem. Rev. 99, 2683-2710 (1999).</li><li>Aposhian, H. V., Maiorino, R. M., Gonzalez-Ramirez, D., Zuniga-Charles, M., Xu, Z., Hurlbut, J. M., Junco-Munoz, P., Dart, R. C., Aposhian, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mobilization+of+heavy+metals+by+newer,+therapeutically+useful+chelating+agents.">Mobilization of heavy metals by newer, therapeutically useful chelating agents.</a> Toxicology. 97, 23-38 (1995).</li><li>Flora, S. J. S., Pachauri, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chelation+in+Metal+Intoxication.">Chelation in Metal Intoxication.</a> Int. J. Environ. Res. Public Health. 7, 2745-2788 (2010).</li><li>Campbell, J. R., Clarkson, T. W., Omar, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+therapeutic+use+of+2,3-dimercaptopropane-1-sulfonate+in+two+cases+of+inorganic+mercury+poisoning.">The therapeutic use of 2,3-dimercaptopropane-1-sulfonate in two cases of inorganic mercury poisoning.</a> JAMA. 256, 3127-3130 (1986).</li><li>Rooney, J. P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+thiols,+dithiols,+nutritional+factors+and+interacting+ligands+in+the+toxicology+of+mercury.">The role of thiols, dithiols, nutritional factors and interacting ligands in the toxicology of mercury.</a> Toxicology. 234, 145-156 (2007).</li><li>Lin, X., Brooks, J., Bronson, M., Ngu-Schwemlein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evalution+of+the+association+of+mercury+(II)+with+some+dicysteinyl+tripeptides.">Evalution of the association of mercury (II) with some dicysteinyl tripeptides.</a> Bioorg. Chem. 44, 8-18 (2012).</li><li>Ngu-Schwemlein, M., Lin, X., Rudd, B., Bronson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+ESI+mass+spectrometric+analysis+of+the+association+of+mercury(II)+with+multi-cysteinyl+peptides.">Synthesis and ESI mass spectrometric analysis of the association of mercury(II) with multi-cysteinyl peptides.</a> J. Inorg. Biochem. 133, 8-23 (2014).</li><li>Winther, J. R., Thorpe, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+thiols+and+disulfides.">Quantification of thiols and disulfides.</a> Biochimica et. Biophysica Acta. 1840, 838-846 (2014).</li><li>D'Agstino, A., Colton, R., Traeger, J. C., Cantry, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Electrospray+Mass+Spectrometric+Study+of+Organomercury+(II)+and+Mercuric+Interactions+with+Peptides+Involving+Cysteinyl+Ligands.">An Electrospray Mass Spectrometric Study of Organomercury (II) and Mercuric Interactions with Peptides Involving Cysteinyl Ligands.</a> Eur. Mass Spectrom. , 273-285 (1990).</li><li>Hofstadler, S. A., Sannes-Lowery, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+ESI-MS+in+drug+discovery:+interrogation+of+noncovalent+complexes.">Applications of ESI-MS in drug discovery: interrogation of noncovalent complexes.</a> Nature Reviews Drug Discovery. 5, 585-595 (2006).</li><li>Rubino, F. M., Verduci, C., Giampiccolo, R., Pulvirenti, S., Brambilla, G., Columbi, 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+Characterization+of+Homo-+and+Heterodimeric+Mercury+(II)-bis-thiolates+of+Some+Biologically+Relevant+Thiols+by+Electrospray+Ionization+and+Triple+Quadruple+Tandem+Mass+Spectrometry.">Molecular Characterization of Homo- and Heterodimeric Mercury (II)-bis-thiolates of Some Biologically Relevant Thiols by Electrospray Ionization and Triple Quadruple Tandem Mass Spectrometry.</a> J. Am. Soc. Mass Spectrom. 15, 288-300 (2003).</li><li>Krupp, E. M., Milne, B. F., Mestrot, A., Meharg, A. A., Feldmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+into+mercury+bound+to+biothiols:+structural+identification+using+ESI-ion-trap+MS+and+introduction+of+a+method+for+their+HPLC+separation+with+simultaneous+detection+by+ICP-MS+and+and+ESI-MS.">Investigation into mercury bound to biothiols: structural identification using ESI-ion-trap MS and introduction of a method for their HPLC separation with simultaneous detection by ICP-MS and and ESI-MS.</a> Anal. Bioanal. Chem. 390, 1753-1764 (2008).</li><li>Schauml&#246;ffel, D., Tholey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+directions+of+electrospray+mass+spectrometry+for+elemental+speciation+analysis.">Recent directions of electrospray mass spectrometry for elemental speciation analysis.</a> Anal. Bioanal. Chem. 400, 1645-1652 (2011).</li><li>Patiny, L., Borel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChemCalc:+a+building+block+for+tomorrow's+chemical+infrastructure.">ChemCalc: a building block for tomorrow's chemical infrastructure.</a> J. Chem. Inf. Model. 53, 1223-1228 (2013).</li><li>Thermo Scientific. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Xcaibur Versions 2.1.0-2.3.0 Data Acquisition and Processing User Guide. Revision E. United States. , (2012).</li><li>Falcone, G., Foti, C., Gianguzza, A., Giuffr&#232;, O., Napoli, A., Pettignano, A., Piazzese, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequestering+ability+of+some+chelating+agents+towards+methylmercury(II).">Sequestering ability of some chelating agents towards methylmercury(II).</a> Anal. Bioanal. Chem. 405 (2), 881-893 (2013).</li><li>Mah, V., Jalilehvand, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutathione+Complex+Formation+with+Mercury(II)+in+Aqueous+Solution+at+Physiological+pH.">Glutathione Complex Formation with Mercury(II) in Aqueous Solution at Physiological pH.</a> Chem. Res. Toxicol. 23, 1815-1823 (2010).</li></ol>

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Hidrofobik dicysteinyl tetrapeptit CGGC (Cı 10H 18 N 4 O 5S 2 MW = 338) (Şekil 1), cıva kompleksler oluşturan (II), Şekil 2&#39;de gösterilen ve Tablo 1 Ayrıca, peptit dimerler ve trimerler de oluşturmasıyla ayrılır. aşamalı olarak, reaksiyon karışımı içindeki peptit miktarı arttıkça. M / z ilişkili dimerlerin değerleri [(2M + H) + = 677] ve trimerl...

Current clinical drugs prescribed for chelation therapy of mercury poisoning1 contain thiol group(s), which is/are responsible for binding and sequestering mercury ions2,3. However, studies have shown that these small thiol compounds [dimercaptosuccinic acid (DMSA) and dimercaptopropane-sulfonic acid (DMPS)] are not optimal for mercury chelation therapy4-6. Therefore, there is a need to understand the association and complex formation tendencies of mercury with thiols to enhance the rational drug design of thiol compounds for mercury chelation. Recently, we reported that n-alkyl and aryl dicysteinyl tripeptides with dithiol groups can serve as effective "double anchors" to accommodate the coordination sites of mercury(II) to form 1:1 mercury(II):peptide and 1:2 mercury(II):(peptide)2 complexes7. Additionally, we studied the effect of increasing cysteinyl residues on complex type formations8. In this study, we investigate the association of mercury(II) with two dicysteinyl tetrapeptides, where the cysteinyl residues are separated by two amino acid residues. In order to evaluate the effect of auxiliary binding groups for mercury, the intervening amino acids are either two glycine (unsubstituted) residues or two glutamic acid (gamma-carboxylated) residues. 
The reaction of cysteinyl peptide with mercury(II) requires conditions that will prevent the oxidation of the cysteinyl thiol groups to form disulfide bonds9. Moreover, the association of mercury(II) with cysteinyl peptides to form various types of mercury-peptide complexes is dependent on the initial ratio of mercury(II): peptide in the reaction mixture7,8. The types of mercury-peptide complexes formed in these reaction mixtures can be analyzed by soft-ionization mass spectroscopy, which is a sensitive analytical tool for determining species interactions between metal ions and peptides10-14. Accordingly, it will provide a profile of the various types of mercuriated peptide adducts that are formed under a specified electrospray ionization condition. Here, we will show how cysteinyl peptides and mercury(II) chloride solutions can be prepared in degassed ammonium formate buffer solution blanketed with argon to minimize oxidation. By reacting varying mole equivalents of mercury(II) with dicysteinyl tetrapeptides, we will show how the initial ratio of mercury(II):peptide has an effect on the types of complexes formed. We will also show how electrospray ionization (ESI) mass spectrometry can be used as a characterization tool to provide an accurate stoichiometry of mercury to peptide in the complexes formed. The associated video protocol will demonstrate the experimental conditions for preparing the mercury complexes, the procedure for analyzing the reaction mixtures under specified electrospray ionization conditions, and the characterization of the stoichiometries of mercury(II)-dicysteinyl tetrapeptide complexes, based on the distinct mercury isotope distribution patterns, by using the ChemCal program15. It is intended to assist those who are interested in using ESI orbitrap mass spectrometry to analyze various complexes formed by metal ions that exist in different isotopic forms.

<table><tbody><tr><td>Mercury(II) chloride</td><td>Sigma-Aldrich</td><td>429724</td><td>Highly toxic</td></tr><tr><td>Ammonium formate</td><td>Sigma-Aldrich</td><td>516961</td><td></td></tr><tr><td>Formic acid</td><td>Sigma-Aldrich</td><td>F0507</td><td></td></tr><tr><td>Ammonium hydroxide</td><td>Fisher</td><td>A512-P500</td><td></td></tr><tr><td>HPLC water</td><td>Fisher</td><td>W5-4</td><td></td></tr><tr><td>HPLC Acetonitrile</td><td>Fisher</td><td>BP2405-1</td><td></td></tr><tr><td>HPLC Methanol</td><td>Fisher</td><td>A452-4</td><td></td></tr></tbody></table>

a study of the complexation of mercury(ii) with dicysteinyl tetrapeptides by electrospray ionization mass spectrometry

In this study we evaluated a method for the characterization of complexes, formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptide, by electrospray ionization orbitrap mass spectrometry. This strategy is based on previous successful characterization of mercury-dicysteinyl complexes involving tripeptides by utilizing mass spectrometry among other techniques. Mercury(II) chloride and a dicysteinyl tetrapeptide were incubated in a degassed buffered medium at varying stoichiometric ratios. The complexes formed were subsequently analyzed on an electrospray mass spectrometer consisting of a hybrid linear ion- and orbi- trap mass analyzer. The electrospray ionization mass spectrometry (ESI-MS) spectra were acquired in the positive mode and the observed peaks were then analyzed for distinct mercury isotopic distribution patterns and associated monoisotopic peak. This work demonstrates that an accurate stoichiometry of mercury and peptide in the complexes formed under specified electrospray ionization conditions can be determined by using high resolution ESI MS based on distinct mercury isotopic distribution patterns.

<html> Bir çalışma ESI kütle spektrometresi ile, iki tetrapeptidlerin, CGGC ve ODAÜ (Şekil 1) için olası cıva peptit kompleksi bileşimi karakterize etmek için yapıldı. CGGC ya ODAÜ ile civa (II) kompleksleri cıva karışımları reaksiyona sokulması suretiyle araştırılmıştır (II) ve üç farklı mol oranlarında Peptid çözeltileri: 1: 0.5, 1: 1, 1: 2 ((II) civa: peptid) . Civa (II) konsantrasyonu 7.5 x 10 -6 M ve peptit konsantrasyonu bu...

Watch this Scientific Journal Video about A Study of the Complexation of MercuryII with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry at JoVE.com

A Study of the Complexation of MercuryII with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry

The characterization of complexes formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptides by electrospray ionization orbitrap mass spectrometry is presented.

Elektrosprey İyonizasyon kütle spektrometresi ile Dicysteinyl tetrapeptidlerle ile Merkür (II) kompleksinin Bir Çalışma

In this study we evaluated a method for the characterization of complexes, formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptide, ...

a-study-complexation-mercury-ii-with-dicysteinyl-tetrapeptides

Research

JoVE Journal

Chemistry

A Study of the Complexation of Mercury(II) with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry

7.8K Görüntüleme.  University of North Carolina at Greensboro. The characterization of complexes formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptides by electrospray ionization orbitrap mass spectrometry is presented.

Video: A Study of the Complexation of MercuryII with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry

Determination of the Gas-phase Acidities of Oligopeptides

Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue located at or near the N-terminus of a helix is often more acidic than that at or near the C-terminus 1-6. Although extensive experimental studies on the acid-base properties of peptides have been carried out in the condensed phase, in particular in aqueous solutions 6-8, the results are often complicated by solvent effects 7. In fact, most of the active sites in proteins are located near the interior region where solvent effects have been minimized 9,10. In order to understand intrinsic acid-base properties of peptides and proteins, it is important to perform the studies in a solvent-free environment.
We present a method to measure the acidities of oligopeptides in the gas-phase. We use a cysteine-containing oligopeptide, Ala3CysNH2 (A3CH), as the model compound. The measurements are based on the well-established extended Cooks kinetic method (Figure 1) 11-16. The experiments are carried out using a triple-quadrupole mass spectrometer interfaced with an electrospray ionization (ESI) ion source (Figure 2). For each peptide sample, several reference acids are selected. The reference acids are structurally similar organic compounds with known gas-phase acidities. A solution of the mixture of the peptide and a reference acid is introduced into the mass spectrometer, and a gas-phase proton-bound anionic cluster of peptide-reference acid is formed. The proton-bound cluster is mass isolated and subsequently fragmented via collision-induced dissociation (CID) experiments. The resulting fragment ion abundances are analyzed using a relationship between the acidities and the cluster ion dissociation kinetics. The gas-phase acidity of the peptide is then obtained by linear regression of the thermo-kinetic plots 17,18.
The method can be applied to a variety of molecular systems, including organic compounds, amino acids and their derivatives, oligonucleotides, and oligopeptides. By comparing the gas-phase acidities measured experimentally with those values calculated for different conformers, conformational effects on the acidities can be evaluated.

The determination of the gas-phase acidities of cysteine-containing oligopeptides is described. The experiments are performed using a triple quadrupole mass spectrometer. The relative acidities of the peptides are measured using collision-induced dissociation experiments, and the quantitative acidities are determined using the extended Cooks kinetic method.

Oligopeptidlerin Gaz fazlı asitliğe belirlenmesi

Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue ...

Kimya

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

Yapısal Çalışmaları üretimi disülfid stabilize Transmembran Peptit Kompleksi

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

Compact Quantum Dots for Single-molecule Imaging

Single-molecule imaging is an important tool for understanding the mechanisms of biomolecular function and for visualizing the spatial and temporal heterogeneity of molecular behaviors that underlie cellular biology 1-4. To image an individual molecule of interest, it is typically conjugated to a fluorescent tag (dye, protein, bead, or quantum dot) and observed with epifluorescence or total internal reflection fluorescence (TIRF) microscopy. While dyes and fluorescent proteins have been the mainstay of fluorescence imaging for decades, their fluorescence is unstable under high photon fluxes necessary to observe individual molecules, yielding only a few seconds of observation before complete loss of signal. Latex beads and dye-labeled beads provide improved signal stability but at the expense of drastically larger hydrodynamic size, which can deleteriously alter the diffusion and behavior of the molecule under study.
Quantum dots (QDs) offer a balance between these two problematic regimes. These nanoparticles are composed of semiconductor materials and can be engineered with a hydrodynamically compact size with exceptional resistance to photodegradation 5. Thus in recent years QDs have been instrumental in enabling long-term observation of complex macromolecular behavior on the single molecule level. However these particles have still been found to exhibit impaired diffusion in crowded molecular environments such as the cellular cytoplasm and the neuronal synaptic cleft, where their sizes are still too large 4,6,7.
Recently we have engineered the cores and surface coatings of QDs for minimized hydrodynamic size, while balancing offsets to colloidal stability, photostability, brightness, and nonspecific binding that have hindered the utility of compact QDs in the past 8,9. The goal of this article is to demonstrate the synthesis, modification, and characterization of these optimized nanocrystals, composed of an alloyed HgxCd1-xSe core coated with an insulating CdyZn1-yS shell, further coated with a multidentate polymer ligand modified with short polyethylene glycol (PEG) chains (Figure 1). Compared with conventional CdSe nanocrystals, HgxCd1-xSe alloys offer greater quantum yields of fluorescence, fluorescence at red and near-infrared wavelengths for enhanced signal-to-noise in cells, and excitation at non-cytotoxic visible wavelengths. Multidentate polymer coatings bind to the nanocrystal surface in a closed and flat conformation to minimize hydrodynamic size, and PEG neutralizes the surface charge to minimize nonspecific binding to cells and biomolecules. The end result is a brightly fluorescent nanocrystal with emission between 550-800 nm and a total hydrodynamic size near 12 nm. This is in the same size range as many soluble globular proteins in cells, and substantially smaller than conventional PEGylated QDs (25-35 nm).

We describe the preparation of colloidal quantum dots with minimized hydrodynamic size for single-molecule fluorescence imaging. Compared to conventional quantum dots, these nanoparticles are similar in size to globular proteins and are optimized for single-molecule brightness, stability against photodegradation, and resistance to nonspecific binding to proteins and cells.

Tek molekül Görüntüleme için kompakt Kuantum Noktaları

Single-molecule imaging is an important tool for  understanding the mechanisms of biomolecular function and for visualizing the  spatial and temporal ...

Mühendislik

Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique amino acid sequence, it is only realized by the folding of the polypeptide chain into a single defined three-dimensional arrangement or more commonly into an ensemble of interconverting conformations. Investigating the connection between protein conformation and its function is therefore essential for a complete understanding of how proteins are able to fulfill their great variety of tasks. One possibility to study conformational changes a protein undergoes while progressing through its functional cycle is hydrogen-1H/2H-exchange in combination with high-resolution mass spectrometry (HX-MS). HX-MS is a versatile and robust method that adds a new dimension to structural information obtained by e.g. crystallography. It is used to study protein folding and unfolding, binding of small molecule ligands, protein-protein interactions, conformational changes linked to enzyme catalysis, and allostery. In addition, HX-MS is often used when the amount of protein is very limited or crystallization of the protein is not feasible. Here we provide a general protocol for studying protein dynamics with HX-MS and describe as an example how to reveal the interaction interface of two proteins in a complex. &#160;&#160;

Protein conformation and dynamics are key to understanding the relationship between protein structure and function. Hydrogen exchange coupled with high-resolution mass spectrometry is a versatile method to study the conformational dynamics of proteins as well as characterizing protein-ligand and protein-protein interactions, including contact interfaces and allosteric effects.

Hidrojen Değişim Kütle Spektrometre Kullanarak Protein Dinamiği Analizi

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Zaman çözülmesi elektro sprey iyonizasyon Hidrojen-döteryum Değişim Kütle Spektrometre Protein Yapısı ve Dinamikleri incelenmesi için

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Biyokimya

Mass Spectrometric Approaches to Study Protein Structure and Interactions in Lyophilized Powders

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for characterizing lyophilized formulations of protein therapeutics. Labeling followed by suitable proteolytic digestion allows the protein structure and interactions to be mapped with peptide-level resolution. Since the protein structural elements are stabilized by a network of chemical bonds from the main-chains and side-chains of amino acids, specific labeling of atoms in the amino acid residues provides insight into the structure and conformation of the protein. In contrast to routine methods used to study proteins in lyophilized solids (e.g., FTIR), ssHDX-MS and ssPL-MS provide quantitative and site-specific information. The extent of deuterium incorporation and kinetic parameters can be related to rapidly and slowly exchanging amide pools (Nfast, Nslow) and directly reflects the degree of protein folding and structure in lyophilized formulations. Stable photolytic labeling does not undergo back-exchange, an advantage over ssHDX-MS. Here, we provide detailed protocols for both ssHDX-MS and ssPL-MS, using myoglobin (Mb) as a model protein in lyophilized formulations containing either trehalose or sorbitol.

Here, we present detailed protocols for solid-state amide hydrogen/deuterium exchange mass spectrometry (ssHDX-MS) and solid-state photolytic labeling mass spectrometry (ssPL-MS) for proteins in solid powders. The methods provide high-resolution information on protein conformation and interactions in the amorphous solid-state, which may be useful in formulation design.

Kitle Spektrometrik Liyofilize Toz Protein Yapısı ve Etkileşimler Eğitim Yaklaşımları

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for ...

A Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) Platform for Investigating Peptide Biosynthetic Enzymes

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for the biophysical characterization of enzyme conformational changes and enzyme-substrate interactions. Among its many benefits, HDX-MS consumes only small amounts of material, can be performed under near native conditions without the need for enzyme/substrate labeling, and can provide spatially resolved information on enzyme conformational dynamics&#8722;even for large enzymes and multiprotein complexes. The method is initiated by the dilution of the enzyme of interest into buffer prepared in D2O. This triggers the exchange of protium in peptide bond amides (N-H) with deuterium (N-D). At the desired exchange time points, reaction aliquots are quenched, the enzyme is proteolyzed into peptides, the peptides are separated by ultra-performance liquid chromatography (UPLC), and the change in mass of each peptide (due to the exchange of hydrogen for deuterium) is recorded by MS. The amount of deuterium uptake by each peptide is strongly dependent on the local hydrogen bonding environment of that peptide. Peptides present in very dynamic regions of the enzyme exchange deuterium very rapidly, whereas peptides derived from well-ordered regions undergo exchange much more slowly. In this manner, the HDX rate reports on local enzyme conformational dynamics. Perturbations to deuterium uptake levels in the presence of different ligands can then be used to map ligand binding sites, identify allosteric networks, and to understand the role of conformational dynamics in enzyme function. Here, we illustrate how we have used HDX-MS to better understand the biosynthesis of a type of peptide natural products called lanthipeptides. Lanthipeptides are genetically encoded peptides that are post-translationally modified by large, multifunctional, conformationally dynamic enzymes that are difficult to study with traditional structural biology approaches. HDX-MS provides an ideal and adaptable platform for investigating the mechanistic properties of these types of enzymes.

A Hydrogen-Deuterium Exchange Mass Spectrometry HDX-MS Platform for Investigating Peptide Biosynthetic Enzymes

Lanthipeptide synthetases catalyze multistep reactions during the biosynthesis of peptide natural products. Here, we describe a continuous, bottom-up, hydrogen-deuterium exchange mass spectrometry (HDX-MS) workflow that can be employed to study the conformational dynamics of lanthipeptide synthetases, as well as other similar enzymes involved in peptide natural product biosynthesis.

Peptit Biyosintetik Enzimleri Araştırmak için Hidrojen-Döteryum Değişim Kütle Spektrometresi (HDX-MS) Platformu

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for the biophysical characterization of enzyme conformational changes and ...

Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, metal-binding interactions, and conformational shape. Coupling ESI with ion mobility-mass spectrometry (IM-MS) provides an instrumental technique that allows for simultaneous measurement of a peptide&#8217;s mass-to-charge (m/z) and collision cross section (CCS) that relate to its stoichiometry, protonation state, and conformational shape. The overall charge of a peptide complex is controlled by the protonation of 1) the peptide&#8217;s acidic and basic sites and 2) the oxidation state of the metal ion(s). Therefore, the overall charge state of a complex is a function of the pH of the solution that affects the peptides metal ion binding affinity. For ESI-IM-MS analyses, peptide and metal ions solutions are prepared from aqueous-only solutions, with the pH adjusted with dilute aqueous acetic acid or ammonium hydroxide. This allows for pH dependence and metal ion selectivity to be determined for a specific peptide. Furthermore, the m/z and CCS of a peptide complex can be used with B3LYP/LanL2DZ molecular modeling to discern binding sites of the metal ion coordination and tertiary structure of the complex. The results show how ESI-IM-MS can characterize the selective chelating performance of a set of alternative methanobactin peptides and compare them to the copper-binding peptide methanobactin.

Ion mobility-mass spectrometry and molecular modeling techniques can characterize the selective metal chelating performance of designed metal-binding peptides and the copper-binding peptide methanobactin. Developing new classes of metal chelating peptides will help lead to therapeutics for diseases associated with metal ion misbalance.

Metal Bağlama Oligopeptidlerinin Yapısı ve Mekanizmalarının Belirlenmesinde Iyon Hareketliliği-Kütle Spektrometresi Teknikleri ve Metal Bağlayıcı Oligopeptidlerin Redoks Aktivitesi

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, ...

Analysis of Complex Molecules and Their Reactions on Surfaces by Means of Cluster-Induced Desorption/Ionization Mass Spectrometry

Desorption/Ionization Induced by Neutral SO2 Clusters (DINeC) is employed as a very soft and efficient desorption/ionization technique for mass spectrometry (MS) of complex molecules and their reactions on surfaces. DINeC is based on a beam of SO2 clusters impacting on the sample surface at low cluster energy. During cluster-surface impact, some of the surface molecules are desorbed and ionized via dissolvation in the impacting cluster; as a result of this dissolvation-mediated desorption mechanism, low cluster energy is sufficient and the desorption process is extremely soft. Both surface adsorbates and molecules of which the surface is composed of can be analyzed. Clear and fragmentation-free spectra from complex molecules such as peptides and proteins are obtained. DINeC does not require any special sample preparation, in particular no matrix has to be applied. The method yields quantitative information on the composition of the samples; molecules at a surface coverage as low as 0.1 % of a monolayer can be detected. Surface reactions such as H/D exchange or thermal decomposition can be observed in real-time and the kinetics of the reactions can be deduced. Using a pulsed nozzle for cluster beam generation, DINeC can be efficiently combined with ion trap mass spectrometry. The matrix-free and soft nature of the DINeC process in combination with the MSn capabilities of the ion trap allows for very detailed and unambiguous analysis of the chemical composition of complex organic samples and organic adsorbates on surfaces.

Neutral SO2 clusters of low kinetic energy (&#60; 0.8 eV/constituent) are used to desorb complex surface molecules such as peptides or lipids for further analysis by means of mass spectrometry using an ion trap mass spectrometer. No special sample preparation is required, and real-time observation of reactions is possible.

Kümelenme Sonucu Desorpsiyon/İyonlaşma Kütle Spektrometresi Yoluyla Karmaşık Moleküllerin ve Yüzeylerdeki Reaksiyonlarının Analizi

Desorption/Ionization Induced by Neutral SO2 Clusters (DINeC) is employed as a very soft and efficient desorption/ionization technique for mass ...

Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of protein therapeutics and the determination of the conformational transition pathways critical for biological functions, ranging from molecular recognition to enzymatic catalysis. Hydrogen/deuterium exchange (HDX) reaction coupled with top-down mass spectrometric (MS) analysis provides a means to characterize protein higher-order structures and dynamics in a conformer-specific manner. The conformational resolving power of this technique is highly dependent on the efficiencies of separating protein states at the intact protein level and minimizing the residual non-deuterated protic content during the HDX reactions. 
Here we describe a capillary electrophoresis (CE)-based variant of the HDX MS approach that aims to improve the conformational resolution. In this approach, proteins undergo HDX reactions while migrating through a deuterated background electrolyte solution (BGE) during the capillary electrophoretic separation. Different protein states or proteoforms that coexist in solution can be efficiently separated based on their differing charge-to-size ratios. The difference in electrophoretic mobility between proteins and protic solvent molecules minimizes the residual non-deuterated solvent, resulting in a nearly complete deuterating environment during the HDX process. The flow-through microvial CE-MS interface allows efficient electrospray ionization of the eluted protein species following a rapid mixing with the quenching and denaturing modifier solution at the outlet of the sprayer. The online top-down MS analysis measures the global deuteration level of the eluted intact protein species, and subsequently, the deuteration of their gas-phase fragments. This paper demonstrates this approach in differential HDX for systems, including the natural protein variants coexisting in milk.

Presented here is a protocol for a capillary electrophoresis-based hydrogen/deuterium exchange (HDX) approach coupled with top-down mass spectrometry. This approach characterizes the difference in higher-order structures between different protein species, including proteins in different states and different proteoforms, by conducting concurrent differential HDX and electrophoretic separation.

Yukarıdan Aşağıya Kütle Spektrometrisi ile Proteinlerin Konformasyonel Karakterizasyonu için Kapiler Elektroforez Bazlı Hidrojen/Döteryum Değişimi

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of ...