Name: two-dimensional gel electrophoresis coupled with mass spectrometry methods for an analysis of human pituitary adenoma tissue proteome
Uploaded: 2018-04-02T13:00:00
Description: Watch this Scientific Journal Video about Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome at JoVE.com

Bu eser Çin (Grant Hayır 81572278 ve 81272798 olarak XZ), Çin "863" planı Projesi (Grant No XZ için 2014AA020610-1), Hunan ve yetenek Giriº (XZ) Xiangya hastane fon gelen hibe Ulusal doğal Bilim Vakfı tarafından desteklenmiştir Çin'in il doğal Bilim Vakfı (XZ için Grant No. 14JJ7008). Yazarlar da Dr Dominic M. Desiderio Tennessee Üniversitesi Sağlık Bilimleri Merkezi içinde bilimsel katkısını kabul. X.Z. sürekli olarak geliştirilen ve 2DE-MS yöntemleri 2001'den başlayarak hipofiz adenom Proteom çözümlemek için kullanılan, mevcut el yazması için kavram gebe, yazdı ve el yazması revize, ilgili çalışmaları koordine ve sorumlu mali desteği ve karşılık gelen çalışma. Yl el yazması revizyonunda katıldı. Y.H başvurular topluluğu ve el yazması revizyonu'na katılmıştır. Tüm yazarlar son el yazması onayladı.

Mevcut iletişim kuralı Xiangya Hastanesi Tıbbi Etik Komitesi merkez Güney Üniversitesi, Çin kuralları izler. Cilt ve saç8keratin kirlenmesini önlemek tüm deneysel işlemin için baş bir kap ve eldiven giyilmelidir.
1. hazırlanması örnekleri
<ol><li>PA dokuları (0.2 - 0.5 mg) Nöroşirürji bölümünden toplamak. Hemen sıvı azot donma ve sonra-80 ° C depolama için transfer.</li>
	<li>Ekle 2 mL % 0,9 NaCl deiyonize distile su (GKD2O). Bu ?...

<ol>
	<li>Zhan, X., Wang, X., Cheng, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+pituitary+adenoma+proteomics:+new+progresses+and+perspectives.">Human pituitary adenoma proteomics: new progresses and perspectives.</a> Front. Endocrinol. 7 (54), (2016).</li><li>Zhan, X., Desiderio, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+proteomics+analysis+of+human+pituitary+adenomas:+Current+status+and+future+perspectives.">Comparative proteomics analysis of human pituitary adenomas: Current status and future perspectives.</a> Mass Spectrom. Rev. 24 (6), 783-813 (2005).</li><li>Wang, X., Guo, T., Peng, F., Long, Y., Mu, Y., Yang, H., Ye, N., Li, X., Zhan, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+and+functional+profiles+of+a+follicle-stimulating+hormone-positive+human+nonfunctional+pituitary+adenoma.">Proteomic and functional profiles of a follicle-stimulating hormone-positive human nonfunctional pituitary adenoma.</a> Electrophoresis. 36 (11-12), 1289-1304 (2015).</li><li>Karppinen, A., Kivipelto, L., Vehkavaara, S., Ritvonen, E., Tikkanen, E., Kivisaari, R., Hernesniemi, J., Set&#228;l&#228;, K., Schalin-J&#228;ntti, C., Niemel&#228;, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transition+from+microscopic+to+endoscopic+transsphenoidal+surgery+for+nonfunctional+pituitary+adenomas.">Transition from microscopic to endoscopic transsphenoidal surgery for nonfunctional pituitary adenomas.</a> World Neurosurg. 84 (1), 48-57 (2015).</li><li>Liu, X., Ma, S., Dai, C., Cai, F., Yao, Y., Yang, Y., Feng, M., Deng, K., Li, G., Ma, W., Xin, B., Lian, W., Xiang, G., Zhang, B., Wang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiproliferative,+antiinvasive,+and+proapoptotic+activity+of+folate+receptor+&#945;-targeted+liposomal+doxorubicin+in+nonfunctional+pituitary+adenoma+cells.">Antiproliferative, antiinvasive, and proapoptotic activity of folate receptor &#945;-targeted liposomal doxorubicin in nonfunctional pituitary adenoma cells.</a> Endocrinol. 154 (4), 1414-1423 (2013).</li><li>Zhan, X., Wang, X., Desiderio, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pituitary+adenoma+nitroproteomics:+current+status+and+perspectives.">Pituitary adenoma nitroproteomics: current status and perspectives.</a> Oxid. Med. Cell. Longev. 2013, 580710 (2013).</li><li>Zhan, X., Wang, X., Desiderio, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry+analysis+of+nitrotyrosine-containing+proteins.">Mass spectrometry analysis of nitrotyrosine-containing proteins.</a> Mass Spectrom. Rev. 34 (4), 423-448 (2015).</li><li>Zhan, X., Desiderio, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reference+map+of+a+pituitary+adenoma+proteome.">A reference map of a pituitary adenoma proteome.</a> Proteomics. 3 (5), 699-713 (2003).</li><li>Desiderio, D. M., Zhan, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+study+of+the+human+pituitary+proteome:+The+characterization+of+differentially+expressed+proteins+in+an+adenoma+compared+to+a+control.">A study of the human pituitary proteome: The characterization of differentially expressed proteins in an adenoma compared to a control.</a> Cell. Mol. Biol. 49 (5), 689-712 (2003).</li><li>Zhan, X., Desiderio, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+analysis+of+the+human+pituitary+proteome.">Heterogeneity analysis of the human pituitary proteome.</a> Clin. Chem. 49 (10), 1740-1751 (2003).</li><li>Zhan, X., Evans, C. O., Oyesiku, N. M., Desiderio, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+and+tanscriptomics+analyses+of+secretagogin+down-regulation+in+human+non-functional+pituitary+adenomas.">Proteomics and tanscriptomics analyses of secretagogin down-regulation in human non-functional pituitary adenomas.</a> Pituitary. 6 (4), 189-202 (2003).</li><li>Moreno, C. S., Evans, C. O., Zhan, X., Okor, M., Desiderio, D. M., Oyesiku, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+molecular+signaling+in+human+clinically+non-functional+pituitary+adenomas+identified+by+gene+expression+profiling+and+proetomic+analyses.">Novel molecular signaling in human clinically non-functional pituitary adenomas identified by gene expression profiling and proetomic analyses.</a> Cancer Res. 65 (22), 10214-10222 (2005).</li><li>Zhan, X., Wang, X., Long, Y., Desiderio, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+analysis+of+the+proteomes+in+clinically+nonfunctional+pituitary+adenomas.">Heterogeneity analysis of the proteomes in clinically nonfunctional pituitary adenomas.</a> BMC Med. Genomics. 7, (2014).</li><li>Zhan, X., Giorgianni, F., Desiderio, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+analysis+of+growth+hormone+isoforms+in+the+human+pituitary.">Proteomics analysis of growth hormone isoforms in the human pituitary.</a> Proteomics. 5 (5), 1228-1241 (2005).</li><li>Kohler, M., Thomas, A., P&#252;schel, K., Sch&#228;nzer, W., Thevis, M. <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+human+pituitary+growth+hormone+variants+by+mass+spectrometry.">Identification of human pituitary growth hormone variants by mass spectrometry.</a> J. Proteome Res. 8 (2), 1071-1076 (2009).</li><li>Zhan, X., Desiderio, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+pituitary+nitroproteome:+detection+of+nitrotyrosyl-proteins+with+two-dimensional+Western+blotting,+and+amino+acid+sequence+determination+with+mass+spectrometry.">The human pituitary nitroproteome: detection of nitrotyrosyl-proteins with two-dimensional Western blotting, and amino acid sequence determination with mass spectrometry.</a> Biochem. Biophys. Res. Commun. 325 (4), 1180-1186 (2004).</li><li>Zhan, X., Desiderio, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitroproteins+from+a+human+pituitary+adenoma+tissue+discovered+with+a+nitrotyrosine+affinity+column+and+tandem+mass+spectrometry.">Nitroproteins from a human pituitary adenoma tissue discovered with a nitrotyrosine affinity column and tandem mass spectrometry.</a> Anal. Biochem. 354 (2), 279-289 (2006).</li><li>Zhan, X., Desiderio, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linear+ion-trap+mass+spectrometric+characterization+of+human+pituitary+nitrotyrosine-containing+proteins.">Linear ion-trap mass spectrometric characterization of human pituitary nitrotyrosine-containing proteins.</a> Int. J. Mass Spectrom. 259, 96-104 (2007).</li><li>Zhan, X., Desiderio, D. M., Wang, X., Zhan, X., Guo, T., Li, M., Peng, F., Chen, X., Yang, H., Zhang, P., Li, X., Chen, Z. <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+the+proteomic+variations+of+invasive+relative+to+noninvasive+nonfunctional+pituitary+adenomas.">Identification of the proteomic variations of invasive relative to noninvasive nonfunctional pituitary adenomas.</a> Electrophoresis. 35 (15), 2184-2194 (2014).</li><li>Zhan, X., Desiderio, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+pathway+networks+mined+from+human+pituitary+adenoma+proteomics+data.">Signal pathway networks mined from human pituitary adenoma proteomics data.</a> BMC Med. Genomics. 3, 13 (2010).</li><li>O&#8217;Farrell, P. 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2DE 2DE-MS PMF ve 2DE-MS/MS, MS yöntemleri ile birleştiğinde, başarılı bir şekilde kullanılmıştır bizim uzun vadeli programı - insan NFPA proteomik varyasyonları ve moleküler ağ çeşitleri için moleküler mekanizmaları aydınlatma çalışmaya proteomik kullanımı ve NFPAs için etkili biyolojik keşfi. İyi tekrarlanabilirlik ile karşılaştırmalı proteomik 2DE tabanlı NFPA proteomik varyasyonları9,10,11...

PA insan endokrin sisteminde önemli roller oynar hipotalamus-hipofiz-hedef organ eksen sistemleri insan hipofiz bezinde oluşan ortak bir tümördür. PA klinik olarak fonksiyonel ve işlevsiz PAs (odak ve NFPA)1,2içerir. NFPA erken aşamada tanı ve tedavisi nedeniyle sadece biraz yüksek hormon düzeyleri (örneğin, LH ve FSH) kan kan3,4 karşılık gelen hormon düzeyleri önemli ölçüde artmıştır odak göre zordur ,5. Moleküler mekanizmalar açıklama ve etkili biyolojik keşfi tanı, tedavi ve NFPA Prognozunda önemli klinik öneme sahip. Uzun vadeli hedefimiz geliştirmek ve NFPA onun moleküler mekanizmaları netleştirmek için güvenilir biyolojik keşfi için çalışmaya proteomik yöntemleri kullanın ve tanılama ve prognostik işaretleri yanı sıra etkili tedavi hedefleri tanımak olduğunu. 2-DE MS yöntemleri ile birleştiğinde kapsamlı bir şekilde kullanıldığını Proteom başvuru kurulması da dahil olmak üzere insan PA Proteom1,2,6,7, ilgili uzun vadeli araştırma programımızda 3,8, analiz differentially ifade protein profilleri9,10,11,12,13, hormon türevleri14 haritalar ,15, fosforilasyon14 ve tirozin nitration16,17,18gibi translasyonel modifikasyon, göreli olarak invazif proteomik varyasyonu invaziv olmayan NFPAs19ve birden çok önemli yolu (mitokondrial disfonksiyon, hücre döngüsü bozukluk, oksidatif stres ve ağları MAPK sinyal keşfi yol açan NFPA alt türlerinden13, proteomik heterojen Sistem anormallik) NFPA13,19,20dakikaya değişmiş.
2DE ayıran proteinler isoelectric noktası (PI) (isoelectric odaklanarak, IEF) ve molekül ağırlığı (yolu ile Sodyum Lauryl Sülfat polyacrylamide Jel Elektroforez, SDS-sayfa) göre1,2,3, 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23. bu ortak ve klasik ayırma proteomik, alanında Proteom ve proteomik içinde 199524kavramlarını giriş beri bir tekniktir. MS 2DE ayrılmış proteinlerin PMF ve MS/MS stratejileri de dahil olmak üzere kimlik bulmak için çok önemli bir tekniktir. MS aletleri, özellikle de algılama hassasiyeti ve çözünürlük, LC sistemi, geliştirilmesi ile birlikte yönleri çok hızlı gelişimi büyük ölçüde düşük veya çok düşük bereket proteinler arasında en üst düzeye çıkarmak için bir Proteom kimlik geliştirir bir Proteom kapsama. Bu da bizim geleneksel kavramları yalnızca bir meydan okuyor veya iki protein 2B jel karmaşık insan dokusu Proteom analizinde bir nokta mevcut ve birden çok protein 2B jel karmaşık insan dokusu analizinde bir nokta belirlemek için bir fırsat sağlar Proteom ve NFPA Proteom kapsama en üst düzeye çıkarmak.
Burada 2DE-maldı MS PMF, 2DE-maldı MS detaylı protokolleri açıklamak/MS ve 2DE-LC-MS/başarıyla insan NFPA Proteom analizinde kullanılan MS. İletişim kuralları hazırlık içerir örnekleri, ilk ikinci boyut (isoelectric odaklanarak, IEF), (SDS-sayfa), görselleştirme (Gümüş boyama ve Coomassie mavi boyama) proteinlerin boyut, görüntü analiz 2D jel, jel tripsin sindirim, Arıtma tryptic peptidler, PMF, MS/MS ve3,8,25,26yakıcı veritabanı. Ayrıca, bu iletişim kuralı kolayca diğer insan dokusu proteomes analiz için çevirir.

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			<td height="61" style="height:61px;width:207px;">Ettan IPGphor 3&#160;</td>
			<td style="width:264px;">GE Healthcare</td>
			<td style="width:76px;"></td>
			<td style="width:160px;">isoelectric focusing system.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Ettan DALTsix multigel caster</td>
			<td style="width:264px;">Amersham Pharmacia Biotech, Piscataway, NJ, USA</td>
			<td style="width:76px;"></td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="59">
			<td height="59" style="height:59px;width:207px;">Ettan DALT II System</td>
			<td style="width:264px;">Amersham Pharmacia Biotech, Piscataway, NJ, USA</td>
			<td style="width:76px;"></td>
			<td style="width:160px;">The vertical electrophoresis system</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Ettan IPGphor strip holder</td>
			<td style="width:264px;">Amersham Pharmacia Biotech, Piscataway, NJ, USA</td>
			<td style="width:76px;"></td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Ettan DALTsix multigel caster</td>
			<td style="width:264px;">Amersham Pharmacia Biotech, Piscataway, NJ, USA</td>
			<td style="width:76px;"></td>
			<td style="width:160px;">Multigel caster</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Voyager DE STR MALDI-TOF MS&#160;</td>
			<td style="width:264px;">ABI, Foster City,CA</td>
			<td style="width:76px;"></td>
			<td style="width:160px;">MALDI-MS PMF</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">MALDI-TOF-TOF</td>
			<td style="width:264px;">Autoflex III, Bruker</td>
			<td style="width:76px;"></td>
			<td style="width:160px;">MALDI-MS/MS mass spectrometer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">LTQ-OrbiTrap Velos Pro ETD</td>
			<td style="width:264px;">Thermo Scientific, Waltham, MA, USA</td>
			<td style="width:76px;"></td>
			<td style="width:160px;">ESI-MS/MS mass spectrometer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">EASY-nano LC system&#160;</td>
			<td style="width:264px;">Proxeon Biosystems, Odense, Denmark</td>
			<td style="width:76px;"></td>
			<td style="width:160px;">High performance liquid chromatography system</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">PepMap C18 trap column&#160;</td>
			<td style="width:264px;">300 &#956;m i.d. &#215; 5 mm length; Dionex Corp., Sunnyvale, CA, USA</td>
			<td style="width:76px;"></td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:207px;">RP C18 column</td>
			<td style="width:264px;">75 &#956;m i.d., 15 cm length; Dionex Corp., Sunnyvale, CA, USA</td>
			<td style="width:76px;"></td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:207px;">KimWipe</td>
			<td style="width:264px;">Kimvipe&#160;</td>
			<td style="width:76px;"></td>
			<td style="width:160px;">Insoluble paper towel</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:207px;">Watter</td>
			<td style="width:264px;">Made by PURELAB flex instrument</td>
			<td style="width:76px;"></td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:207px;">Polytron Model P710/35 homogenizer</td>
			<td style="width:264px;">Brinkmann Instruments, Westbury, NY</td>
			<td style="width:76px;"></td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:207px;">PDQuest</td>
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			<td style="width:76px;"></td>
			<td style="width:160px;">PMF-based protein searching software&#160;&#160;</td>
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		<tr height="47">
			<td height="47" style="height:47px;width:207px;">Mascot software</td>
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			<td style="width:76px;"></td>
			<td style="width:160px;">MS/MS-based protein searching software</td>
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		<tr height="47">
			<td height="47" style="height:47px;width:207px;">Proteome Discoverer software v.1.3 beta</td>
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			<td height="47" style="height:47px;width:207px;">Xcalibur software v.2.1</td>
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			<td style="width:76px;"></td>
			<td style="width:160px;">MS/MS data-acquired management software&#160;</td>
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			<td style="width:160px;">MS/MS-based protein searching software I</td>
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		<tr height="47">
			<td height="47" style="height:47px;width:207px;">MASCOT (version 2.3.02)&#160;</td>
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			<td style="width:160px;">MS/MS-based protein searching software II</td>
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			<td style="width:264px;">AMRESCO</td>
			<td style="width:76px;">0172</td>
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			<td height="40" style="height:40px;width:207px;">ACRYLAMIDE</td>
			<td style="width:264px;">AMRESCO</td>
			<td style="width:76px;">0341</td>
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			<td height="40" style="height:40px;width:207px;">DTT</td>
			<td style="width:264px;">Sigma-Aldrich</td>
			<td style="width:76px;">D0632</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Thiourea</td>
			<td style="width:264px;">Sigma-Aldrich</td>
			<td style="width:76px;">T8656</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Urea</td>
			<td style="width:264px;">VETEC</td>
			<td style="width:76px;">V900119</td>
			<td style="width:160px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:207px;">SDS</td>
			<td style="width:264px;">AMRESCO</td>
			<td style="width:76px;">0227</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">CHAPS</td>
			<td style="width:264px;">AMRESCO</td>
			<td style="width:76px;">0465</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">TEMED</td>
			<td style="width:264px;">AMRESCO</td>
			<td style="width:76px;">M146</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Ammonium Persulfate</td>
			<td style="width:264px;">AMRESCO</td>
			<td style="width:76px;">M133</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Trypsin</td>
			<td style="width:264px;">Promega, Madison, WI, USA</td>
			<td style="width:76px;">V5111</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">IPG buffer pH 3-10, NL</td>
			<td style="width:264px;">GE Healthcare</td>
			<td style="width:76px;">17-6000-87</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Immobiline Dry Strip pH 3-10NL,18cm</td>
			<td style="width:264px;">GE Healthcare</td>
			<td style="width:76px;">17-1235-01</td>
			<td style="width:160px;"></td>
		</tr>
	</tbody>
</table>

two-dimensional gel electrophoresis coupled with mass spectrometry methods for an analysis of human pituitary adenoma tissue proteome

İnsan hipofiz adenom (PA) insan hipofiz bezi hipotalamus-hipofiz-hedef organ eksen sisteminde oluşur ve klinik olarak işlev veya işlevsiz PA (odak ve NFPA) sınıflandırılabilir ortak bir tümördür. NFPA erken aşamada tanı ve tedavi ancak odak için karşılaştırıldığında kan hormon yükselen nedeniyle zordur. Uzun vadeli hedefimiz güvenilir biyolojik açıklama PA moleküler mekanizmaları ve etkili tanı, prognostik işaretleri ve tedavi hedefleri tanınması için keşfetmek için proteomik yöntemleri kullanmaktır. Kütle spektrometresi (MS) yöntemlerle birleştiğinde etkili iki boyutlu Jel Elektroforez (2DE) buraya örnekleri, 2D Jel Elektroforez, protein görselleştirme, görüntü analizi, jel hazırlanması da dahil olmak üzere insan PA proteomes analiz sunuldu Tripsin sindirim, peptid kitle parmak izi (PMF) ve tandem spektrometresi (MS/MS) kitle. 2-boyutlu Jel Elektroforez matris yardımlı lazer desorpsiyon/iyonlaşma kütle spektrometresi PMF (2DE-maldı MS PMF), 2DE-maldı MS/MS ve 2DE-sıvı Kromatografi (LC) MS/MS yordamları başarıyla uygulanan NFPA Proteom analizinde bir. Bir yüksek-duyarlı Kütle Spektrometre kullanımıyla, birçok protein ile her 2D jel insan PA Proteom kapsamını maksimize etmek için karmaşık PA doku analizinde bir spot 2DE-LC-MS/MS yönteminde tespit edilmiştir.

1. 2DE-maldı MS PMF: yukarıda açıklanan deneysel bir işlem ile toplam 150 µg proteinlerin NFPA FSH ifade edilen dokulardan elde (erkek; 50 yıl yaşlı, ACTH (-), GH (-), PRL (-), LH (-), FSH (+) ve TSH (-)) ve bir 18 cm ile dizilmiş IPG Şerit (pH 3-10 NL) ve bir geniş formatlı SDS-sayfa jeli, gümüş boyama ile görüntülenir. NFPA doku Proteom (şekil 1), bir ortalama konum sapma 1,98 ± IEF yönde 0,75 mm ile v...

Watch this Scientific Journal Video about Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome at JoVE.com

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

İnsan hipofiz adenom doku Proteom analizi için kütle spektrometresi yöntemleri ile birleştiğinde iki boyutlu Jel Elektroforez

Burada, kütle spektrometresi (ayırmak ve bir iyi ve tekrarlanabilir 2DE desen sunar insan hipofiz adenom doku Proteom tanımlamak için MS) ile birleştiğinde bir iki boyutlu Jel Elektroforez (2DE) mevcut. Çoğu protein her 2DE yerde karmaşık kanser Proteom yüksek-duyarlı MS kullanımı ile analiz ederken gözlenir.

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may ...

Research

JoVE Journal

Cancer Research

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Jel Hibridizasyonunu Kullanarak Telomer Uzunluğunu Nicellemek İçin Modifiye Terminal Restriction Fragment Analysis

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Vitro ve In Vivo'da Fare Dendritik Hücrelerini Hedeflemek için Tam Uzunlukta Proteinli Saflaştırılmış DEC-205 Yönlendirilmiş Antikorun Kimyasal Konjugasyonu

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Sphingolipids are cellular components that have well-established roles in human metabolism and disease. Mass spectrometry can be used to determine whether ...

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