Bu çalışma, Ulusal Sağlık Hibe GM-26.738 Enstitüleri (JAT) kısmen desteklenmektedir.

1. Pak2 Etkinleştirme <ol><li> 12 mg / ml Pak2 &#39;lik 20 ul tampon kaspaz 3 uygun önceden test edilmiş bir miktar ekleyin (pH 7.5, 50 mM Tris-HCl, 150 mM NaCl ve 2 mM DTT) ve 34 inkübe ° C 30 dakika tam tutunmaya ve etkinleştirmek Pak2. </li><li> SDS-PAGE (% 10) ve Coommassie mavi boyama parçalanma ürünleri analiz edin. </li><li> 20 ul 10 mg / ml Pak2 &#39;lik bir nihai konsantrasyonu, pH 7.5 (50 mM Tris-HCl, 150 mM NaCl, 15 mM MgCl 2, 10 mM ATP) aktivasyon tampon B bölünmüş Pak2 ekleyin ve 34 inkübe ° C&#39;de 30 dakika süreyle tam 8 siteleri otofosforilasyon tarafından Pak2 etkinleştirmek için. </li><li> SDS-PAGE 10 tarafından belirlenen P34 ve p27 parçaları göç Pak2 tam bölünme onaylayın. Otofosforilasyon koşullarının uygun doğrulama phosphopeptides tespit etmek (32 P) ATP veya kütle spektrometresi kullanarak ayrı bir deney otoradyografi yoluyla yapılır. </li></ol> 2. KimlikPak2, Pepsin-özet Fragments <ol><li> Önce agaroz boncuk kullanımı için iki kez yıkamak için pH 2.5% 0.1 Trifluroacetic Asit (TFA) 1 ml ekleyin. Sonra, boncuk kaldırmak için 15 sn 2.000 xg&#39;de örnekleri santrifüj. </li><li> % 0.1 TFA 100 ul Pak2 (150 mM NaCl 2 ul içinde 20 mg, pH 7.5, 50 mM Tris-HCl ve 2 mM DTT) ekleyin ve 5 dakika boyunca örnek pepsinojenin konjuge agaroz boncuk 30 ul inkübe . </li><li> PH 2.5,% 0.1 TFA birincil bir çözücü sistemi ile ters faz HPLC C18 kolon dengelenmesi. 0.2 ml / dak akış hızında% 80% 0.1 TFA içeren asetonitril - pepsin sindirimi (50 ul) 0 100 dk doğrusal bir degrade kullanarak kolonda sürüklenecektir. Kesirler her 2 dakikada toplanır. </li><li> (3 saat) hızlı elektrikli süpürge ile HPLC kesirler Kuru ve asetonitril (5 ul) ve pH 2.5,% 0.1 TFA (5 ul) bir çözüm örnekleri tekrar süspansiyon haline getirin. </li><li> Başlangıçta, her fraksiyon peptidler için MALDI-TOF PerSeptive Biyosistem Voyager DE STR (PE Bi kullanarak gösterildi.osystems, Foster City, CA, USA). </li><li> Peptidler içeren 16 fraksiyonları tandem kütle spektrometresi parçaları belirlemek için Q-TOF kütle spektrometresi ve bir QSTAR XL oMALDI MS / MS kullanarak tabi tutulur. </li><li> Her peptid dizisi Protein Prospector web sitesi (kullanarak Pak2 birincil dizisi dayalı teorik m / z oranları ile tandem kütle spektrometresi tarafından üretilen ürün iyonları tarafından doğrulanmadı <a href="http://prospector.ucsf.edu/">http://prospector.ucsf.edu/</a> ) 11. </li></ol> 3. Amid H / D Exchange ölçümü <ol><li> 25 H / D değişimi için gerekli tüm çözümler ve reaktifler ° C su banyosunda tutun. ATP suda çözülür ve pH kullanmadan önce 7.0 &#39;ye ayarlanır. D 2 O tampon ek 4mm ATP seyreltme sonra Pak2 ayrı ATP önlemek için. </li><li> (20 mg 2 ul ile 18 ul tamponlu D 2 O (50 mM MOPS pH 6.98, 125 mM NaCl) eklenmesi ile H / D değişimi başlatın), 150 mM içeren bir tampon olarak Pak2 NaCl, 0 ile 50 mM Tris-HCl (pH 7.5) ve 2 mM Pak2 seviyesi 10 mg / ml &#39;lik bir DTT ve pH 7.0 ° C </li><li> H / D karşılığında üç nüsha olarak 0, 0.5, 1, 3, 5 ve 10 dakika ya da 24 saat için numunelerin inkübe </li><li> H / D değişim süreci, pH 2.2, 200 ul nihai hacmi 2.5 pH getiriyor buz% 0.1 TFA 180 ul ekleyerek Quench. ATP, pH 2.2 buz 0.11% TFA 180 ul içeren numuneler için 2,5 pH söndürülür durumu korumak için kullanılır. </li><li> 30 ul aktif pepsinojenin-konjuge agaroz boncuk her söndürülür örnek bir kısım (100 ul) ekleyin. Pepsin sindirim beş dakika süreyle buz üzerinde devam etmesine izin verilir ve örnekleri her 30 saniyede bir vortekslenmiş. </li><li> 4 5.000 xg&#39;de 15 sn için örnek santrifüj sindirim Terminate ° C pepsin kaldırmak için. </li><li> Hızla en fazla 2, -80 ° C&#39;de sıvı N 2 ve mağaza örnekleri dondurmakMALDI-TOF analizi önceki gün. </li><li> MALDI için matris 5 mg / ml α-cyano-4-hydroxycinnamic asetonitril, etanol ve% 0.1 pH 2.5 TFA bir çözüm (01:01:01) çözülür ve 0 ° C&#39;de yapılan asit (CHCA) </li><li> Kısmen hızlı bir şekilde her bir örnek Çözülme ve her numunenin 1 ul ul 4 ° C MALDI hedef plakası matris ve spot 1 ile karıştırın. </li><li> MALDI-TOF analizi için 30 sn önce lekeler kuru plaka ılımlı bir vakum uygulayın. Örnek ve spektrum alma çözülme arasındaki süre yaklaşık 3 dk. </li><li> PerSeptive Biyosistem Voyager DE STR MALDI-TOF kütle spektrumları edinin. 20.000-V hızlandırma voltaj ve% 65 şebeke voltajı 180 ns darbe gecikme ile gecikmeli çıkarma kullanarak, 100.000 veri kanalları az 2 GHz örnekleme hızında veri edinin. 2 dakika içinde yüz taramaları ortalaması alınır. MALDI-TOF cihazı herhangi bir marka ya da herhangi bir özel yazılım ile sınırlı değildir. </li><li> Calcul için Data Explorer 4.0 bilgisayar programı kullanınher parçasının dötertumlanma yedik. </li><li> Arka döviz omurga amidler N-terminal amid (Şekil 1) hariç tüm olası değiştirilebilir siteleri, bölünmüş H / D değişimi 24 saat gerçek dahil deuteron sayısı oranına göre hesaplayın. </li></ol> 4. Temsilcisi Sonuçlar: Kaspaz bölünme ve otofosforilasyon SDS-PAGE Commassie boyama ile doğrulanır. İnaktif Pak2 58 kDa tek bir bant. Pak2, kaspaz bölünme tamamlandığında, ve ayrıca göç, p27 ve P34 iki parça üretir. Autophosphorylated p27 ve P34, tam fosforile p27 parçası (Şekil 2) geriliği göç nedeniyle jel bir örtüşme gösterir göç . H / D döviz deneyler, 0 ile 10 dakika (Şekil 3) arasında birden çok kez yapılmaktadır . Bir örnek olarak, m / zp içine döteryum esas zamanlı kursinaktif Pak2 EAK 1697,85 zamanla kitlesel zarfın vardiya tarafından gösterildiği gibi birden fazla izotop zirvelerinden oluşmaktadır. <img alt="Şekil 1" src="/files/ftp_upload/3602/3602fig1.jpg" /> Şekil 1 dötertumlanma hesaplanması için denklemi. <img alt="Şekil 2" src="/files/ftp_upload/3602/3602fig2.jpg" /> Şekil 2 otofosforilasyon ve Pak2 kaspaz bölünme. Kaspaz-cleaved Pak2 (sol şerit), bozulmamış inaktif Pak2 (orta şerit) ve kaspaz-cleaved autophosphorylated Pak2 (sağ şeritte), SDS-PAGE ve Coommassie mavi boyama ile analiz edildi. Hsu, YH ve ark 2 &#39;den uyarlanmıştır. <img alt="Şekil 3" src="/files/ftp_upload/3602/3602fig3.jpg" /> Şekil 3, inaktif Pak2 döteryum esas bir zaman dersin Mass Spektrum . İnaktif Pak2 H / D excha maruz kaldı0-10 dakika nge ve MALDI-TOF ile analiz. H / D değiş zaman seyri sırasında tepe m / z 1697,85 MALDI-TOF genişletilmiş izotop dağılımı bir örnek gösterilmiştir. Kırmızı noktalı çizgi, kitle zarfın ortalama kitle zarfın kayma gösterir ve bir peptid dötertumlanma gösterir.

<ol>
	<li>Anand, G. S., Hughes, C. A., Jones, J. M., Taylor, S. S., Komives, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amide+H/2H+exchange+reveals+communication+between+the+cAMP+and+catalytic+subunit-binding+sites+in+the+R(I)alpha+subunit+of+protein+kinase.">Amide H/2H exchange reveals communication between the cAMP and catalytic subunit-binding sites in the R(I)alpha subunit of protein kinase.</a> A. J. Mol. Biol. 323, 377-386 (2002).</li><li>Hsu, Y. H., Johnson, D. A., Traugh, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+conformational+changes+during+activation+of+protein+kinase+Pak2+by+amide+hydrogen/deuterium+exchange.">Analysis of conformational changes during activation of protein kinase Pak2 by amide hydrogen/deuterium exchange.</a> J. Biol. Chem. 283, 36397-36405 (2008).</li><li>Englander, S. W., Kallenbach, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+exchange+and+structural+dynamics+of+proteins+and+nucleic+acids.">Hydrogen exchange and structural dynamics of proteins and nucleic acids.</a> Q. Rev. Biophys. 16, 521-655 (1983).</li><li>Katta, V., Chait, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+changes+in+proteins+probed+by+hydrogen-exchange+electrospray-ionization+mass+spectrometry.">Conformational changes in proteins probed by hydrogen-exchange electrospray-ionization mass spectrometry.</a> Rapid Commun. Mass. Spectrom. 5, 214-217 (1991).</li><li>Dennis, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localizing+the+membrane+binding+region+of+Group+VIA+Ca2+-independent+phospholipase+A2+using+peptide+amide+hydrogen/deuterium+exchange+mass+spectrometry.">Localizing the membrane binding region of Group VIA Ca2+-independent phospholipase A2 using peptide amide hydrogen/deuterium exchange mass spectrometry.</a> J. Biol. Chem. 284, 23652-23661 (2009).</li><li>Hsu, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+binding+rigidifies+the+C2+domain+and+the+intradomain+interaction+of+GIVA+phospholipase+A2+as+revealed+by+hydrogen/deuterium+exchange+mass+spectrometry.">Calcium binding rigidifies the C2 domain and the intradomain interaction of GIVA phospholipase A2 as revealed by hydrogen/deuterium exchange mass spectrometry.</a> J. Biol. Chem. 283, 9820-9827 (2008).</li><li>Lee, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+hPAK65+by+caspase+cleavage+induces+some+of+the+morphological+and+biochemical+changes+of+apoptosis.">Activation of hPAK65 by caspase cleavage induces some of the morphological and biochemical changes of apoptosis.</a> Proc. Natl. Acad. Sci. U. S. A. 94, 13642-13647 (1997).</li><li>Rudel, T., Bokoch, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+and+morphological+changes+in+apoptotic+cells+regulated+by+caspase-mediated+activation+of+PAK2.">Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2.</a> Science. 276, 1571-1574 (1997).</li><li>Roig, J., Traugh, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytostatic+p21+G+protein-activated+protein+kinase+gamma-PAK.">Cytostatic p21 G protein-activated protein kinase gamma-PAK.</a> Vitam. Horm. 62, 167-198 (2001).</li><li>Walter, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+and+activation+of+p21-activated+protein+kinase+gamma-PAK+by+CPP32+(caspase+3).+Effects+of+autophosphorylation+on+activity.">Cleavage and activation of p21-activated protein kinase gamma-PAK by CPP32 (caspase 3). Effects of autophosphorylation on activity.</a> J. Biol. Chem. 273, 28733-28739 (1998).</li><li>Clauser, K. R., Baker, P., Burlingame, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+accurate+mass+measurement+(+/-+10+ppm)+in+protein+identification+strategies+employing+MS+or+MS/MS+and+database+searching.">Role of accurate mass measurement (+/- 10 ppm) in protein identification strategies employing MS or MS/MS and database searching.</a> Anal. Chem. 71, 2871-2882 (1999).</li></ol>

Çıkar çatışması ilan etti.

Pepsin özet Fragments Kimlik H / D değişim deney kritik bir adımdır. Peptid yanlış kimlik yanlış bir sonuca yol açabilir. Pepsin-sindirilmiş Pak2 temiz bir arka plan elde etmek için ters faz HPLC C18 kolon ile yıkandı,. Deneylerde, toplam 40 kesirler bir toplandı ve MALDI-TOF tabi. Çoklu peptidler kesirler her tespit olabilir. Peptidler dizileri tanımlamak için tandem kütle spektrometresi (Q-TOF MS / MS ve oMALDI MS / MS) tabi tutuldu. Bir peptit MS / MS spektrumları peptid kimlik doğrulamak için en az üç ürün iyonları olmalıdır. Data Explorer 4.0 bilgisayar programı (Uygulamalı Biyosistem) ilk temel düzeltme ve gürültü filtreleme yapar. Her spektrumu iki sıralı tepeleri üzerinde göre kalibre edilmelidir. 923,45 ve 1697,84 undeuterated m / z ile teorik kütle spektrumu kalibre edin. To kitle doğruluğu kalibrasyon sonra 10 sayfaya ulaşabilirsiniz. Dötertumlanma üzerine, mono-izotop daha yüksek bir kitle kayabilir. Üniter adım bu m / z oranları artar dötertumlanma ile ilişkili gelişmiş kitlelerin vermiştir. Kitle zarfın pik şiddetleri dötertumlanma düzeyini etkilemez. Ancak, belirli bir kitle zarf içinde izotopik zirvelerinden pik şiddetleri ortalama kütlesinin hesaplanması için kritik öneme sahiptir. Dahil deuteron hesaplama ilk adım döteryumlanmış örneği olmayan döteryumlanmış bir örnek peptid kütle çıkartmaktır. Diğer üç faktör, D 2 O seyreltme, yan zincirleri ve arka döviz kalan deuterons, daha fazla (Şekil 1) hesaplanmasında dikkate alınması gerekir. D 2 O seyreltme sonra D 2 O örnek karıştırma ve D 2 O tampon H / D değişimi başlatmak için seyreltme faktörü. Pepsin-digest yan zincirleri artık deuteron (% 4.5)ed peptidler çıkarılır gerekir. Arka değişim kitle ölçüm sürecinin tüm döteryumlanmış ve pepsin-sindirilmiş peptidler dötertumlanma kaçınılmaz kaybı. H / D değişimi 24 saat sonra en solvent erişilebilir peptid (m / z = 1105,60) tam bir dötertumlanma bölgede temsil eder. Peptidlerin her biri için dahil deuteron sayısı, döteryumlanmış ve non-döteryumlanmış Pak2 peptik peptidlerin ağırlık merkezi arasındaki farktır. En çok döteryumlanmış peptid m / z 1105 Pak2 H / D değişimi 24 saat iken dötertumlanma temsil etmek üzere seçildi. Geri Alım Satım Oranı peptid m / z 1105 tüm olası değiştirilebilir siteleri tarafından bölünmüş H / D değişimi 24 saat gerçek dahil deuteron sayısına göre hesaplandı.

<table border="1"></table><table border="1"><tbody><tr><td> Reaktifi Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> D 2 O </td><td> Aldrich </td><td> 7789-20-0 </td><td></td></tr><tr><td> α-cyano-4-hydroxycinnamic asit </td><td> Sigma </td><td> 28166-41-8 </td><td></td></tr><tr><td> MALDI-TOF </td><td> PE Biosystems </td><td> Voyager DE str </td><td></td></tr><tr><td> Q-TOF kütle spektrometresi </td><td> Q-TOF Ultima-Global </td><td></td><td></td></tr><tr><td> QSTAR XL oMALDI MS / MS </td><td> Applied Biosystems </td><td></td><td></td></tr><tr><td> Data Explorer 4.0 bilgisayar programı </td><td> Applied Biosystems </td><td></td><td></td></tr&gt; </tbody></table>

amide hydrogen/deuterium exchange &amp; maldi-tof mass spectrometry analysis of pak2 activation

Kütle spektrometresi ile birlikte Amid hidrojen / döteryum değişimi (Y / D değişimi), yaygın olarak,, protein-protein etkileşimleri, protein konformasyonel değişiklikler, protein dinamiği ve protein-ligand etkileşimleri arayüz analiz etmek için kullanılır olmuştur. H / D değişimi omurga amid pozisyonları, kütle spektrometresi 1,2,3, bir protein mikro-bölgeler dötertumlanma oranlarını ölçmek için kullanılmıştır. Bu yöntemin çözünürlük normalde 3-20 artıkları aralığı peptidler ilgi döteryumlanmış protein pepsin sindirim bağlıdır. Çözünürlük kütle spektrometresi ile ölçülen H / D değişimi NMR, kütle spektrometresi H / D değişim ölçüm farkı çekirdekli Tek Kuantum Tutarlılık (HSQC) yöntemi ile ölçülen tek kalıntı çözünürlüğü daha düşük olmasına rağmen boyutu ile sınırlı değildir protein 4. H / D değişimi protein konformasyonu korur sulu bir çözelti içinde yürütülmektedir. Biz bir yöntem sağlamak olduğunu utilHPLC / ESI (elektrosprey iyonizasyon)-MS sistemi 5,6 yerine 2 için algılama MALDI-TOF, izes. MALDI-TOF bu durumda protein kinaz Pak2 (γ-Pak da denir), sindirilmiş protein peptidler için doğru kütle yoğunluğu verileri sağlar. Pak 2 Proteoliz bir çevrimdışı pepsinin sindirim gerçekleştirilir. Kullanıcı erişim, kütle spektrometresi bağlı bir HPLC ve pepsin sütun veya HPLC pepsin sütununda, örneğin ağır disülfit gümrüklü salgılanan Fosfolipaz A 2, optimal bir sindirim harita sonucu değildir bulunmamış Bu alternatif bir yöntem, (SPLA 2). Bu yöntemi kullanarak, başarılı kaspaz 3 bölünme ve otofosforilasyon 7,8,9 Pak2 aktivasyonu sırasında dötertumlanma düzeyinde değişiklikler izlenir.

Watch this Scientific Journal Video about Amide Hydrogen/Deuterium Exchange & MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation at JoVE.com

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

MALDI-TOF kütle spektrometresi başarıyla protein kinaz Pak2 aktivasyon amid hidrojen / döteryum değişimini izlemek için kullanılmıştır.

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 ...

amide-hydrogendeuterium-exchange-maldi-tof-mass-spectrometry-analysis

Research

JoVE Journal

Biology

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

17.7K Görüntüleme.  Tunghai University. MALDI-TOF kütle spektrometresi başarıyla protein kinaz Pak2 aktivasyon amid hidrojen / döteryum değişimini izlemek için kullanılmıştır.

Video: Amide Hydrogen/Deuterium Exchange & MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

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

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 ...

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N2) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (&Omega;). 
Recently, IM spectrometry was coupled with mass spectrometry and a traveling-wave (T-wave) Synapt ion mobility mass spectrometer (IM-MS) was released. Integrating mass spectrometry with ion mobility enables an extra dimension of sample separation and definition, yielding a three-dimensional spectrum (mass to charge, intensity, and drift time). This separation technique allows the spectral overlap to decrease, and enables resolution of heterogeneous complexes with very similar mass, or mass-to-charge ratios, but different drift times. Moreover, the drift time measurements provide an important layer of structural information, as &Omega; is related to the overall shape and topology of the ion. The correlation between the measured drift time values and &Omega; is calculated using a calibration curve generated from calibrant proteins with defined cross-sections1.
The power of the IM-MS approach lies in its ability to define the subunit packing and overall shape of protein assemblies at micromolar concentrations, and near-physiological conditions1. Several recent IM studies of both individual proteins2,3 and non-covalent protein complexes4-9, successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available)10. We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical &Omega; values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field.

Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.

T-dalgası İyon Hareketlilik-Kütle Spektrometresi: Protein Karmaşık Analiz Temel Deneysel Prosedürler

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. ...

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

Doğrudan Atmosferik Basınç Kütle Spektrometresi Tek Hücre Analizi

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

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 ...

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment (Kartchner Caverns, AZ, USA)

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, strain levels. Commercially available and open source software tools have been developed to facilitate identification; however, no universal/standardized data analysis pipeline has been described in the literature. Here, we provide a comprehensive and detailed demonstration of bacterial identification procedures using a MALDI-TOF mass spectrometer. Mass spectra were collected from 15 diverse bacteria isolated from Kartchner Caverns, AZ, USA, and identified by 16S rDNA sequencing. Databases were constructed in BioNumerics 7.1. Follow-up analyses of mass spectra were performed, including cluster analyses, peak matching, and statistical analyses. Identification was performed using blind-coded samples randomly selected from these 15 bacteria. Two identification methods are presented: similarity coefficient-based and biomarker-based methods. Results show that both identification methods can identify the bacteria to the species level.

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment Kartchner Caverns, AZ, USA

This work details procedures for rapid identification of bacteria using MALDI-TOF MS. The identification procedures include spectrum acquisition, database construction, and follow up analyses. Two identification methods, similarity coefficient-based and biomarker-based methods, are presented.

MALDI-TOF Kütle Spektrometresi ve Özel Veritabanı kullanımı Benzersiz Mağarası Çevre (Kartchner mağaraları, AZ, ABD) Yerli Bakteriler niteleyin için

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

Mutlak Protein Niceleme Kütle Spektrometresi İzleme Seçilen Tepki

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

Kütle Spektrometresi Analizi için Peptitler içine Dokular ve Sindirim gelen Ekstrasellüler Matriks Proteinleri zenginleştirilmesi

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great potential for the detailed mass spectrometry (MS) analysis of biomolecules from biological samples. The single-probe, a miniaturized device with integrated sampling and ionization capabilities, is capable of performing both ambient MSI and in-situ SCMS analysis. For ambient MSI, the single-probe uses surface micro-extraction to continually conduct MS analysis of the sample, and this technique allows the creation of MS images with high spatial resolution (8.5 &#181;m) from biological samples such as mouse brain and kidney sections. Ambient MSI has the advantage that little to no sample preparation is needed before the analysis, which reduces the amount of potential artifacts present in data acquisition and allows a more representative analysis of the sample to be acquired. For in-situ SCMS, the single-probe tip can be directly inserted into live eukaryotic cells such as HeLa cells, due to the small sampling tip size (&lt; 10 &#181;m), and this technique is capable of detecting a wide range of metabolites inside individual cells at near real-time. SCMS enables a greater sensitivity and accuracy of chemical information to be acquired at the single cell level, which could improve our understanding of biological processes at a more fundamental level than previously possible. The single-probe device can be potentially coupled with a variety of mass spectrometers for broad ranges of MSI and SCMS studies.

Here, we present protocols to perform both ambient mass spectrometry imaging (MSI) of tissues and in-situ live single cell MS (SCMS) analysis using the single-probe, which is a miniaturized multifunctional device for MS analysis.

Tek prob Uygulamalar: Ortam Koşullarında Kütle Spektrometre Görüntüleme ve Tek Hücre Analizi

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

Histon Ekstraksiyon Gönderen Data Analizi: Aşağıdan yukarıya Kütle Spektrometre kullanarak histon post-translasyonel modifikasyonlar Analizi için komple iş akışı

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...

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

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