Bu çalışma NCI Klinik Proteomik Teknoloji Değerlendirme Merkezi (CPTAC) hibe (# U24 CA126476) yanı sıra Eğlence Endüstrisi Vakfı (EIF) ve AYF Kadınlar Kullanıcı Kanser Araştırma Fonu Meme Kanseri Biyomarker Discovery Konsorsiyumu bir hibe tarafından finanse edilen, ve oldu Keck Vakfı, Kanarya Vakfı, Paul G. Allen Aile Vakfı cömert hediyeler.

Deneysel İşlemler: Tahlil sentetik peptidler ve anti-peptid antikorları gerektirir. Seçilen peptidler ilgi protein benzersiz olması, 8 ila 22 amino asitleri içeren ve bilinen hiçbir post-translasyonel modifikasyonlar olmalıdır. Methionine artıkları genellikle kaçınılması ve çift bazlı amino asitler (örneğin, KK, KR, RR) içeren peptidler arzu edilmez. Bu teknik için, (13 C ve 15 N), C-peptid terminus (yani K veya R etiketli) amino asitler etiketli ağır içeren kararlı izotop etiketli peptidler, iç standartlar gibi kullanmak yaygındır . Aşağıdaki protokol Epitomics A.Ş. (Burlingame, CA) ve New England Peptid (Gardner, MA) sentetik peptidler elde edilen anti-peptid antikorları kullanarak, fare protein osteopontin, peptid GDSLAYGLR ölçmek için geliştirilen bir test açıklamaktadır. 1) peptidler zenginleştirilmesi 3) kütle spektrometresi ile analizi karmaşık protein karışımı, 2 tripsin sindirim): Bu protokol, üç temel aşamadan oluşmaktadır (Şekil 1). Bu fare osteopontin protein ile çivili bir insan plazma örnek gösterilecektir. 1. Tripsin enzimatik sindirim ve temizlik <ol><li> Islak buz üzerinde 10 mcL düzgün plazma kısım çözülme. </li><li> BCA yöntemi ile total protein konsantrasyonu belirlemek ve askıda katı madde kaldırmak için örnek santrifüj. </li><li> 1000 mcL derin kuyucuklu plaka delmek mümkün film ile kaplayın ve depolama tüp pipetleyin 10 mcL kısım. </li><li> Her bir numune üre / 30mm dithiothreitol (DTT) (son konsantrasyon 6M üre / 20mm DTT) taze 9M 20 mcL ekleyin. </li><li> 37 az 30 dakika boyunca inkübe ° C </li><li> Her bir numune için taze 500 mM iodoacetamide (son IAM 50mm) 3 mcL ekleyin. </li><li> Oda sıcaklığında, karanlıkta 30 dakika inkübe edin. </li><li> 100 mM Tris (pH 8) (~ 0.6M sulandırır üre) 257 mcL ekleyin. </li><li> Tripsin stok solüsyonu (;: 01:50 enzim substrat oranı 1 mikrogram / mcL) 10 mcL ekleyin. </li><li> Inkübe 37 ° C gece (12-16 saat). </li><li> Düzgün formik asit (% 1 final konsantrasyon) 3 mcL ekleyin. </li><li> Stabil izotop standart (çoğullanır tahlil yapmak, genellikle bu 10 mcL standart isotopically etiketli peptid içeren 50-100 fmol hakkında ise birden fazla standartları eklenir) ekleyin. </li><li> Oasis kartuş plaka ile% 80 asetonitril atarak akışı ile% 0.1 formik asit 500 mcL yıkayın. Bu 3 kez tekrarlayın. </li><li> 500 mcL% 0.1 formik asit su ekleyerek kartuşu plaka dengelenmesi ve flow-through atın. Bu 4 kez tekrarlayın. </li><li> Kartuş plaka sindirmek numuneleri yükleyin ve akışı çok yavaş bu yüzden vakum ayarlayabilirsiniz. </li><li> 500 mcL% 0.1 formik asit su ile yıkayın ve flow-through atın. Bu 3 kez tekrarlayın. </li><li> 1000 ul derin kuyu plakasına% 80 asetonitril% 0.1 formik asit, 2 x 500 mcL ekleyerek Zehir peptidler (flow-through atmayın). </li><li> Eluat kuruyacak kadar (ya da speedvac) Lyophilize. (Liyofilizasyon tercih edilen yöntem) </li><li> Sulandırın 50 mcL PBS +% 0.03 ahbap ekleyerek peptidler kurutulur. </li></ol> 2. Peptid immünoafinite zenginleştirme <ol><li> Standart Kingfisher 96 kuyucuğu örnek aktarın. </li><li> 1 mg antikor ve ortalama 1.5 mcL hedef Protein-G kaplı manyetik bilye (yanı sıra boncuklar önce antikor çapraz isteğe bağlıdır) ekleyin. Emin olun boncuk iyi sallayarak veya vorteks askıya alınır. </li><li> Plaka folyo ile örtün. </li><li> Nazik boncuk askıya sağlamak için yuvarlanan (12-16 saat) gece inkübe edin. </li><li> Folyo yüzeyi herhangi bir sıvı kaldırmak için 5 saniye boyunca 32 xg&#39;de plaka santrifüjleyin. </li><li> Folyo kapağını çıkarın. </li><li> Yıkama ve elüsyon boncuklar elle ya da otomatik bir şekilde yapılabilir. Bu prosedür Kingfisher bir platform üzerinde otomatik adımları açıklar. </li><li> 200 mcL PBS +% 0.03 ahbap (1 dakika Yıkama başına) boncuk, 2 kez yıkayın. </li><li> PBS 200 mcL 1:10 seyreltme +% 0.03 ahbap (1 dakika) ile boncuk, 1 kez yıkayın. </li><li> % 5 asetik asit +% 0.03 ahbap 25 uL peptidler kolonda sürüklenecektir. </li><li> Bir mıknatıs elüsyon plaka koyun ve artık boncuk aktarmak için özen, 96 plaka eluat transfer. </li><li> Sızdırmazlık mat plaka Kapak. </li><li> Eluat içeren plaka, analiz için üçlü quadrapole kütle spektrometresi teslim edilir. </li></ol> 3. Birden fazla reaksiyon izleme Analizi - kütle spektrometresi <ol><li> SRM / MRM analizi için Geçişler 0.5 mcL / dak kütle spektrometresi% 30 acetonitrile/0.1% formik asit peptid standart mikrolitrelik çözüm başına 1 picomole beslerken tarafından seçilen ve optimize edilebilir. Istikrarlı sprey elde edilir, bir MS / MS spektrum toplamak. </li><li> Geçişler thre belirleyerek MS / MS spektrum seçilene biraz gürültü ile yelpazenin bir bölgede bol parçası iyonları. Çarpın ücret peptid iyonları için, y-iyon parçaları, z&gt; m / s tespit habercisi m / z genellikle SRM / MRM testinin geliştirilmesi için en iyisidir. Şekil 2 476,3 MS / MS spektrumu ve seçilen geçiş iyonlar&gt; 508,3, 579,3 peptid GDSLAYGLR 692,4 (ağır stabil izotop standart 481,3 geçişler&gt; 518,3, 589,3, 702,4 gösterilir). </li><li> Kullanılan kütle spektrometresi marka ve modeline bağlı olarak, her geçiş için optimize edilebilir birden çok parametre vardır. Burada seçilen her geçiş çarpışma enerjisi çarpışma enerjisi Ramping ve sinyal seviyesi izleme optimize edilmiş. Her geçiş için 25 bir çarpışma enerjisi kullanıldı. </li><li> Kısaca, aşağıdaki gibi SRM / MRM analizi için tipik bir yapılandırma: Mobil faz (A)% 0.1 formik asit (B)% 90 Asetonitril /% 0.1 formik asit, 0,3 x 5 mm C18 tuzak sütun, 75 mikron ID x 15 cm C18 analitik kolon (Reprosil-Pur C18 AQ, 120 A ° gözenek). Enjeksiyon hacmi 10 mcL ve örnek toplam akış oranı% 3 B 5 dakika yüklü 3 mcL / dk ve 3 doğrusal bir degrade tarafından yıkandı,% 45 - 300 B - 10 dakika içinde 400 nL / dak. 4000 QTRAP (ABSCIEX, Foster City, CA) Koşullar sprey gerilim 2.3kV, iyon kaynağı sıcaklığı 150 ° C, 12 GS1 ve perde gaz 15. </li><li> Örnek, numune 10 mcL enjekte edilerek SRM / MRM tarafından analiz edilir. Pik alanları program Skyline kullanarak hafif ve ağır peptidler için entegre 14, bu durumda, arka plan gürültü ücretsiz en bol bulunan bir geçiş kullanarak her bir örnek etiketsiz / etiketli peptid pik alan oranı (PAR), Y5 geçiş hesaplayın. (ışık peptid 476,3 579,3, ağır standart peptid 481,3 589,3). </li></ol> 4. Temsilcisi Sonuçlar: Ölçülen pik alan oranları (çivili ağır isotopically etiketli peptid ışık endojen peptid akrabası) hedef peptid nicel bir ölçüsüdür. Şekil 3, hafif ve ağır bir SISCAPA zenginleştirilmiş örnek peptidlerin bir örnek kromatogram gösterir . Aynı zamanda ve çoklu geçişler Zehir hafif ve ağır peptidler peptid kimliğini teyit etmek için her için takip edilebilir unutmayın. <img src="/files/ftp_upload/2812/2812fig1.jpg" alt="Şekil 1"/> Şekil 1 SISCAPA süreci şematik bir bakış. Karmaşık bir protein karışımı peptidlerin içine sindirilir. Hedeflenen peptid analitler (endojen analit ve çivili bir kararlı izotop etiketli internal standart) Protein G-kaplı manyetik parçacıklar üzerinde immobilize anti-peptid antikorları kullanarak zenginleştirilmiştir. Izolasyonundan sonra, hedeflenen peptidler manyetik partiküller yıkandı, ve iç standart göre kantitatif için kütle spektrometresi ile analiz. <img src="/files/ftp_upload/2812/2812fig2.jpg" alt="Şekil 2"/> Şekil 2 SRM / MRM geçişler için üç parça seçimi gösteren peptid GDSLAYGLR MS / MS spektrumu. <img src="/files/ftp_upload/2812/2812fig3.jpg" alt="Şekil 3"/> Şekil 3, hafif bir peptid analitin tepe profili (kırmızı) ve ağır stabil izotop etiketli internal standart (mavi) gösteren örnek kromatogramlar . Kromatografi sistemi Zehir gibi ışık peptid (476,3 579,3) ve ağır stabil izotop etiketli Y5 geçiş için Kromatogramlar standart (481,3 589,3) zamanla çizilir.

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Çıkar çatışması ilan etti.

Tarif edildiği gibi, protokolde en kritik adım boncuk kuluçka dönemi boyunca iyi karıştırılmış kalır sağlanmasıdır. Boncuk iyi / flakon altına yerleşmek için izin artan değişkenlik neden olacaktır. Aynı zamanda önemli bir kuluçka dönemi sonrasında iyi / flakon üstüne kalır herhangi bir sıvı aşağı spin. Tekrarlanabilir tripsin sindirim da önemlidir. Ancak, sindirim muhtemel alternatif yöntemleri, belirli bir hedef proteinlerin için optimize edilmiş olabilir 15 Elüsyon ücretsiz antikor tespiti ile karışmaz; sindirim için prosedür SISCAPA ile birlikte laboratuvarda yaygın olarak kullanılmıştır Burada anlatılan antikor peptidler daha sonra sütun veya elutes hariç, ama antikor büyük deneylerde ya da serbest antikor varsa afinite zenginleştirme önce Protein G boncuk çapraz olabilir çünkü büyük olasılıkla peptid, bir sorun olur. Biz de herhangi bir kalıntı boncuk veya parçacıklar kaldırmak için örnek otosampler plakası gibi, akış ters yönde tuzak sütun yıkama altında bir mıknatıs (yükleme karşılaştırıldığında) yerleştirmek için yararlı bulduk. Açıklanan manyetik boncuk yaklaşıma ek olarak, tekniği de bir sütun biçiminde (yani afinite kromatografisi) adapte olabilir 12, 16 Kullanıcı genel bir protokol ile rahat sonra, bu teknik, aynı zamanda genel analiz geliştirmek bazı değişiklikler için uygun. İlk 11, birden fazla analitler zenginleştirme adım (yani çoklama) antikorlar birleştirerek tek bir tahlil analiz etmek mümkündür. Kütle spektrometresi, aynı anda çok sayıda analitlerin analiz yeteneğine sahiptir. İkinci olarak, orijinal örnek hacminin artırılması testin duyarlılığını artırır.

<table border="1"><tbody><tr><th> Malzemenin Adı </th><th> Şirket </th><th> Katalog numarası </th></tr><tr><td> 1000ul Derin kuyu 96-kuyu tabak </td><td> Eppendorf </td><td> 951032786 </td></tr><tr><td> Oasis HLB 1 cc kartuş plaka, Eyl-pak, 40 mg 96 plaka </td><td> Waters </td><td> 186003966 </td></tr><tr><td> Kingfisher 96 plaka </td><td> Thermo Fisher Scientific </td><td> 97002540 </td></tr><tr><td> 96, beyaz duvar tabağı temizleyin </td><td> Bio-Rad </td><td> HSP9601 </td></tr><tr><td> 96 plaka için kapak Folyo </td><td> Excelscientific </td><td> 12-169 </td></tr><tr><td> 96 plaka Axymat Sızdırmazlık mat </td><td> Axygen </td><td> 521-01-151 </td></tr><tr><td> X delmek film </td><td> Sigma-Aldrich </td><td> Z722502 </td></tr><tr><td> Kingfisher PCR mıknatıs kafa ile manyetik boncuk işlemci </td><td> Thermo Fisher Scientific </td><td> HSP 9601 </td></tr></tbody></table> Tablo 1: Malzeme <table border="1"><tbody><tr><th> Malzemenin Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar (isteğe bağlı) </th></tr><tr><td> Üre </td><td> Sigma Aldrich </td><td> U6031 </td><td></td></tr><tr><td> Trizma baz </td><td> Sigma Aldrich </td><td> T1503 </td><td></td></tr><tr><td> Dithiothreitol (DTT) </td><td> Delmek </td><td> 20291 </td><td> &quot;Dithiothreitol no-tartmak&quot; </td></tr><tr><td> Iodoacetamide (IAM) </td><td> Sigma Aldrich </td><td> I1149 </td><td></td></tr><tr><td> Tripsin Altın </td><td> Promega </td><td> V5280 </td><td></td></tr><tr><td> Protein G manyetik boncuk, 2.8um </td><td> Invitrogen </td><td> 10004D </td><td></td></tr><tr><td> Fosfat tamponlu salin (PBS) </td><td> Thermo Fisher Scientific </td><td> L5401 </td><td></td></tr><tr><td> Chaps </td><td> Thermo Fisher Scientific </td><td> 28300 </td><td></td></tr><tr><td> Formik asit </td><td> EMD </td><td> 11670 </td><td></td></tr><tr><td> Asetonitril </td><td> Thermo Fisher Scientific </td><td> A998-1 </td><td></td></tr><tr><td> Asetik asit </td><td> Sigma Aldrich </td><td> 242853 </td><td></td></tr></tbody></table> Tablo 2: Reaktifler <table border="1"><tbody><tr><th> Çözüm </th><th> Yorumlar (isteğe bağlı) </th></tr><tr><td> 100 mM Tris pH: 8 </td><td></td></tr><tr><td> 9 M, 100 mM Tris (pH 8) üre / 30 mM DTT </td><td> Her zaman taze hazırlanmış olmalıdır </td></tr><tr><td> 100 mM Tris 500 mM IAM (pH 8) </td><td> Her zaman taze hazırlanmış olmalıdır </td></tr><tr><td> 100 mM Tris, pH 8 tripsin 1mg/ml </td><td></td></tr><tr><td> 100 mM Tris, pH 8 tripsin inhibitörü 1mg/ml </td><td></td></tr><tr><td> Kararlı izotop standart Mastermix </td><td></td></tr><tr><td> Anti-peptid antikor Mastermix </td><td></td></tr></tbody></table> Tablo 3: Çözümler hazırlanacak

quantification of proteins using peptide immunoaffinity enrichment coupled with mass spectrometry

Proteinlerin ölçümünde kantitatif testleri için büyük bir ihtiyaç vardır. Ölçümde büyük ölçüde altın standart olarak kabul Geleneksel sandviç immunoassay, yüksek maliyetli, uzun teslim süresi ile ilişkilidir ve sakıncaları (örneğin antikorlar, otoantikor girişim, &#39;kanca etkisi&#39;) ile doludur. 1 alternatif bir tekniktir zenginleştirme yakınlığına . peptidlerin kantitatif kütle spektrometresi ile birleştiğinde, genellikle SISCAPA (Anti-Peptid Antikorlar Kararlı İzotop Standartları ve Capture) olarak adlandırılan bu teknikte 2 / çoklu seçilen reaksiyon izleme kütle spektrometresi ile stabil izotop seyreltme ve algılama ile peptitler afinite zenginleştirme ( SRM / MRM-MS), peptidler, kendi proteinler vekilleri olarak kantitatif ölçüm sağlar. SRM / MRM-MS, küçük moleküllerin 3, 4 doğru kantitatif için kurulan ve daha yakın zamanda, plazma ve hücre lizatları proteinlerinin düzeyini ölçmek için adapte edilmiştir. Proteinlerin kantitatif ulaşmak için 5-7, bu büyük moleküllerin sindirilir bileşeni peptidler gibi tripsin gibi bir enzim kullanarak. Dizisi bu türün hedef protein (yani &quot;proteotypic&quot; peptitler) özgü bir veya daha fazla seçilmiş peptidler daha sonra anti-peptid antikorları kullanarak örnek zenginleştirilmiş ve kantitatif stokiyometrik vekilleri örnek protein konsantrasyonu olarak ölçülür. Bu nedenle, stabil izotop seyreltme (SID) yöntemleri (yani bir çivili-peptid standart etiketli stabil izotop), SRM / MRM karmaşık biyolojik matrisler proteinlerin ölçümü için vekilleri gibi proteotypic peptidlerin konsantrasyonlarını ölçmek için kullanılan olabilir. Akuple Deneyleri geleneksel immunoassay göre bazı avantajları vardır. Reaktifler oluşturmak için nispeten daha az pahalı, özgüllüğü analit için mükemmel, testlerin son derece çoğullanır zenginleştirme düzgün plazma (tükenmesi gerekli) yapılan ve tekniği, proteinler ya da değişiklikler geniş bir yelpazeye uygun olabilir bir manyetik boncuk platformuna adapte olarak bu video ilgi. 8-13 temel protokol göstermektedir.

Watch this Scientific Journal Video about Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry at JoVE.com

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

Kararlı İzotop Standartlar ve Anti-Peptid Antikorlar (SISCAPA) kendi proteinler için vekilleri olarak peptidler kantitatif ölçüm sağlamak için kararlı izotop dilüsyon kütle spektrometresi (MRM-MS) ile peptitler çiftler yakınlık zenginleştirme yakalayın. Burada kısmen otomatik bir biçimde manyetik parçacıklar kullanarak protokol açıklar.

Proteinler Peptid immunoaffiniti Zenginleştirme kullanarak Niceleme Kütle Spektrometresi ile birleştiğinde

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

quantification-proteins-using-peptide-immunoaffinity-enrichment

Research

JoVE Journal

Biology

22.7K Görüntüleme.  Fred Hutchinson Cancer Research Center - FHCRC. Kararlı İzotop Standartlar ve Anti-Peptid Antikorlar (SISCAPA) kendi proteinler için vekilleri olarak peptidler kantitatif ölçüm sağlamak için kararlı izotop dilüsyon kütle spektrometresi (MRM-MS) ile peptitler çiftler yakınlık zenginleştirme yakalayın. Burada kısmen otomatik bir biçimde manyetik parçacıklar kullanarak protokol açıklar.

Video: Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

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

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

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

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

Nicel Değerlendirme Elektrosprey İyonizasyon Kütle Spektrometresi kullanarak Maya Lipidome

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

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 ...

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

Işıksal Çözülmüş İki foton Mikroskop kullanarak G Protein Coupled Reseptör Etkileşmeleri in vivo Niceleme

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

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

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

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

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

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

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

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 ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Protein Kompleksi belirlenmesi<em&gt; Escherichia coli</em&gt; Tandem Kütle Spektrometresi ile Kombine Sıralı Peptid Affinity Arıtma kullanarak

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

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

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

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

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

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

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 ...