Name: analyzing large protein complexes by structural mass spectrometry
Uploaded: 2010-06-19T17:00:00
Duration: 15 min 35 s
Description: Watch this Scientific Journal Video about Analyzing Large Protein Complexes by Structural Mass Spectrometry at JoVE.com

लेखकों उनके महत्वपूर्ण समीक्षा के लिए, और पांडुलिपि के लिए अपने योगदान के लिए शेरोन समूह के सदस्यों को धन्यवाद देता हूं. हम Morasha और Bikura प्रोग्राम्स, इसराइल विज्ञान फाउंडेशन (अनुदान नग 1823-1807 और 378/08), जोसेफ Cohn मिनर्वा Biomembrane अनुसंधान के लिए केंद्र, नई वैज्ञानिकों के लिए Chais परिवार फैलो प्रोग्राम, इब्राहीम के समर्थन के लिए आभारी हैं और सोनिया Rochlin फाउंडेशन, वोल्फसन परिवार चैरिटेबल ट्रस्ट, हेलेन और मिल्टन ए Kimmelman केंद्र Biomolecular संरचना और विधानसभा के लिए, Shlomo और Sabine Beirzwinsky की संपत्ति, Meil ​​डे Botton Aynsley, और करेन Siem, ब्रिटेन. हम दान Tawfik और Itamar Yadid हमें लेक्टिन संस्करण नमूना देने के लिए आभारी हैं.

<ol>
	<li>Alberts, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+as+a+collection+of+protein+machines:+preparing+the+next+generation+of+molecular+biologists.">The cell as a collection of protein machines: preparing the next generation of molecular biologists.</a> Cell. 92 (3), 291-294 (1998).</li><li>Benesch, J. L., Ruotolo, B. T., Simmons, D. A., Robinson, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+complexes+in+the+gas+phase:+technology+for+structural+genomics+and+proteomics.">Protein complexes in the gas phase: technology for structural genomics and proteomics.</a> Chem Rev. 107 (8), 3544-3567 (2007).</li><li>Heck, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Native+mass+spectrometry:+a+bridge+between+interactomics+and+structural+biology.">Native mass spectrometry: a bridge between interactomics and structural biology.</a> Nat Methods. 5 (11), 927-933 (2008).</li><li>Sharon, M., Robinson, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mass+spectrometry+in+structure+elucidation+of+dynamic+protein+complexes.">The role of mass spectrometry in structure elucidation of dynamic protein complexes.</a> Annu Rev Biochem. 76, 167-193 (2007).</li><li>Hernandez, H., Robinson, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+the+stoichiometry+and+interactions+of+macromolecular+assemblies+from+mass+spectrometry.">Determining the stoichiometry and interactions of macromolecular assemblies from mass spectrometry.</a> Nat Protoc. 2 (3), 715-726 (2007).</li><li>Wilm, M., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+properties+of+the+nanoelectrospray+ion+source.">Analytical properties of the nanoelectrospray ion source.</a> Analytical Chemistry. 68 (1), 1-8 (1996).</li><li>Sobott, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tandem+mass+spectrometer+for+improved+transmission+and+analysis+of+large+macromolecular+assemblies.">A tandem mass spectrometer for improved transmission and analysis of large macromolecular assemblies.</a> Anal Chem. 74 (6), 1402-1407 (2002).</li><li>van den Heuvel, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+the+performance+of+a+quadrupole+time-of-flight+instrument+for+macromolecular+mass+spectrometry.">Improving the performance of a quadrupole time-of-flight instrument for macromolecular mass spectrometry.</a> Anal Chem. 78 (21), 7473-7483 (2006).</li><li>Chernushevich, I. V., Thomson, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collisional+cooling+of+large+ions+in+electrospray+mass+spectrometry.">Collisional cooling of large ions in electrospray mass spectrometry.</a> Anal Chem. 76 (6), 1754-1760 (2004).</li><li>Mora, d. e. l. a., J, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospray+ionization+of+large+multiply+charged+species+proceeds+via+Dole+s+charged+residue+model.">Electrospray ionization of large multiply charged species proceeds via Dole s charged residue model.</a> Anal Chim Acta. 406, 93-104 (2000).</li><li>Lorenzen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+macromolecular+tandem+mass+spectrometry+of+large+non-covalent+complexes+using+heavy+collision+gases.">Optimizing macromolecular tandem mass spectrometry of large non-covalent complexes using heavy collision gases.</a> Int J Mass Spectrom. 268, 198-206 (2007).</li><li>Benesch, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quadrupole-time-of-flight+mass+spectrometer+modified+for+higher-energy+dissociation+reduces+protein+assemblies+to+peptide+fragments.">Quadrupole-time-of-flight mass spectrometer modified for higher-energy dissociation reduces protein assemblies to peptide fragments.</a> Anal Chem. 81 (3), 1270-1274 (2009).</li><li>Taverner, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subunit+architecture+of+intact+protein+complexes+from+mass+spectrometry+and+homology+modeling.">Subunit architecture of intact protein complexes from mass spectrometry and homology modeling.</a> Acc Chem Res. 41 (5), 617-627 (2008).</li><li>Ferrige, A. G., Seddon, M. J., Skilling, J., Ordsmith, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+application+of+MaxEnt+to+high+resolution+mass+spectrometry.">The application of MaxEnt to high resolution mass spectrometry.</a> Mass Spectrom. 6, 765-770 (1992).</li><li>van Breukelen, B., Barendregt, A., Heck, A. J., van den Heuvel, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+stoichiometries+and+oligomeric+states+of+glutamate+synthase+protein+complexes+with+curve+fitting+and+simulation+of+electrospray+mass+spectra.">Resolving stoichiometries and oligomeric states of glutamate synthase protein complexes with curve fitting and simulation of electrospray mass spectra.</a> Rapid Commun Mass Spectrom. 20 (16), 2490-2496 (2006).</li><li>Sharon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symmetrical+modularity+of+the+COP9+signalosome+complex+suggests+its+multifunctionality.">Symmetrical modularity of the COP9 signalosome complex suggests its multifunctionality.</a> Structure. 17 (1), 31-40 (2009).</li><li>Hernandez, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subunit+architecture+of+multimeric+complexes+isolated+directly+from+cells.">Subunit architecture of multimeric complexes isolated directly from cells.</a> EMBO Rep. 7 (6), 605-610 (2006).</li><li>Yadid, I., Tawfik, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+functional+beta-propeller+lectins+via+homo-oligomeric+assembly+of+shorter+fragments.">Reconstruction of functional beta-propeller lectins via homo-oligomeric assembly of shorter fragments.</a> J Mol Biol. 365 (1), 10-17 (2007).</li><li>Videler, H. <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+of+intact+ribosomes.">Mass spectrometry of intact ribosomes.</a> FEBS Lett. 579 (4), 943-947 (2005).</li><li>Uetrecht, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+mass+spectrometry+of+viral+assemblies:+molecular+composition+and+stability+of+dimorphic+hepatitis+B+virus+capsids.">High-resolution mass spectrometry of viral assemblies: molecular composition and stability of dimorphic hepatitis B virus capsids.</a> Proc Natl Acad Sci U S A. 105 (27), 9216-9220 (2008).</li><li>Uetrecht, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+and+shape+of+hepatitis+B+virus+capsids+in+vacuo.">Stability and shape of hepatitis B virus capsids in vacuo.</a> Angew Chem Int Ed Engl. 47 (33), 6247-6251 (2008).</li><li>Morton, V. L., Stockley, P. G., Stonehouse, N. J., Ashcroft, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+virus+capsid+assembly+from+non-covalent+mass+spectrometry.">Insights into virus capsid assembly from non-covalent mass spectrometry.</a> Mass Spectrom Rev. 27 (6), 575-595 (2008).</li><li>Sharon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=20S+proteasomes+have+the+potential+to+keep+substrates+in+store+for+continual+degradation.">20S proteasomes have the potential to keep substrates in store for continual degradation.</a> J Biol Chem. 281 (14), 9569-9575 (2006).</li><li>van Duijn, E., Heck, A. J., Vies, S. M. v. a. n. d. e. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inter-ring+communication+allows+the+GroEL+chaperonin+complex+to+distinguish+between+different+substrates.">Inter-ring communication allows the GroEL chaperonin complex to distinguish between different substrates.</a> Protein Sci. 16 (5), 956-965 (2007).</li><li>van Duijn, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tandem+mass+spectrometry+of+intact+GroEL-substrate+complexes+reveals+substrate-specific+conformational+changes+in+the+trans+ring.">Tandem mass spectrometry of intact GroEL-substrate complexes reveals substrate-specific conformational changes in the trans ring.</a> J Am Chem Soc. 128 (14), 4694-4702 (2006).</li><li>Synowsky, S. A., van Wijk, M., Raijmakers, R., Heck, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+multiplexed+mass+spectrometric+analyses+of+endogenously+expressed+yeast+nuclear+and+cytoplasmic+exosomes.">Comparative multiplexed mass spectrometric analyses of endogenously expressed yeast nuclear and cytoplasmic exosomes.</a> J Mol Biol. 385 (4), 1300-1313 (2009).</li><li>Sharon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+organization+of+the+19S+proteasome+lid:+insights+from+MS+of+intact+complexes.">Structural organization of the 19S proteasome lid: insights from MS of intact complexes.</a> PLoS Biol. 4 (8), e267-e267 (2006).</li><li>Zhou, 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+reveals+modularity+and+a+complete+subunit+interaction+map+of+the+eukaryotic+translation+factor+eIF3.">Mass spectrometry reveals modularity and a complete subunit interaction map of the eukaryotic translation factor eIF3.</a> Proc Natl Acad Sci U S A. 105 (47), 18139-18144 (2008).</li></ol>

ब्याज की कोई संघर्ष की घोषणा की.

अधिग्रहण के लिए उच्च गुणवत्ता स्पेक्ट्रा नमूना तैयार कदम है, जो नमूना एकाग्रता और बफर विनिमय शामिल करने के लिए ध्यान दिया जाना चाहिए. पतला नमूने ओवर कम संकेत उपज, जबकि अत्यधिक ध्यान केंद्रित नमूने बल्कि चिपचिपा हो सकता है, और electrospray सुई ब्लॉक. इसके अलावा, लवण, ग्लिसरॉल, डिटर्जेंट, धातु आयनों, और कम करने एजेंट (डीटीटी या β-mercaptoethanol) जैसे समाधान additives, प्रोटीन की बाहरी सतह का पालन करते हैं, और चोटियों के व्यापक बनाने के कारण. इसलिए, क्रम में अच्छी तरह से हल चोटियों को प्राप्त करने के लिए, इन घटकों को न्यूनतम संभव सांद्रता में जोड़ा जाना चाहिए. एक अन्य महत्वपूर्ण पैरामीटर केशिका nanoflow की स्थिति, मास स्पेक्ट्रोमीटर छिद्र करने के लिए सापेक्ष है. &quot;मीठी हाजिर&quot; ढूँढना अनुभवहीन उपयोगकर्ताओं के लिए चुनौतीपूर्ण हो सकता है है, फिर भी, यह काफी स्पेक्ट्रा की गुणवत्ता को प्रभावित करती है. यह भी महत्वपूर्ण है की जांच करने के लिए स्प्रे की दीक्षा nanoflow पहले केशिका. छोटी बूंद के आकार टिप व्यास, जो 1μm ~ होना चाहिए की एक समारोह है. छोटी बूंदों अधिक कुशल ionization के लिए सीसा, और इसलिए लाभप्रद होगा. करने के लिए मान्य है कि वहाँ केशिका के भीतर कोई हवाई बुलबुले कि प्रवाह को ब्लॉक कर सकते हैं कर रहे हैं यह भी महत्वपूर्ण है, और है कि अधिग्रहण के दौरान सोने की कोटिंग केशिका से छीन नहीं है, यदि हां, तो आगे टिप ट्रिम. ध्यान रखें कि नमूने के एक अत्यधिक राशि अनुकूलन केशिका वोल्टेज, तेजी voltages, दबाव, और टक्कर ऊर्जा जैसे एमएस शर्तों की संभावना में वृद्धि होगी. कुल मिलाकर, प्रोटोकॉल में समझाया प्रक्रियाओं संरचना, stoichiometry और कई प्रोटीन परिसरों की वास्तुकला (2,3,4 समीक्षा) को निर्धारित करने के लिए इस्तेमाल किया गया है. Ribosome 19 जैसे बड़े एमडीए परिसरों और अत्यधिक आदेश दिया वायरस capsids 20-22, के विश्लेषण से आणविक मशीनों 23-25, या सबयूनिट बातचीत नेटवर्क के लक्षण वर्णन के लिए बाध्य सब्सट्रेट परिभाषित करने में 26,17,16,17,27,28 के रूप में सेवा लेकिन इस दृष्टिकोण के मूल्य के कुछ उदाहरण हैं.

नमूना आवश्यकताओं: <table border="1"><tr><td> नमूना </td><td> आवश्यकताएँ </td><td> टिप्पणियाँ </td></tr><tr><td> आयतन </td><td> 1-2 μL </td><td> केशिका प्रति </td></tr><tr><td> एकाग्रता </td><td> 1 - 20μM </td><td> जटिल प्रति </td></tr><tr><td> बफर </td><td> Aquanos पीएच में अमोनियम एसीटेट जैसे अस्थिर बफर = 6 - 8 </td><td> 5 आमतौर पर मिमी 1M </td></tr><tr><td> डिटर्जेंट </td><td> मिनिमल </td><td> डिटर्जेंट अणुओं के क्लस्टर व्यापक और अनसुलझे चोटियों का उत्पादन </td></tr><tr><td> ग्लिसरॉल </td><td> मिनिमल (5% अप करने के लिए) </td><td> Nonspecifically प्रोटीन का पालन करता है और, फलस्वरूप, व्यापक चोटियों मनाया जाता है </td></tr><tr><td> कार्बनिक सॉल्वैंट्स </td><td> 50% </td><td> प्रोटीन परिसरों denature शायद </td></tr><tr><td> एसिड </td><td> 4% </td><td> Denature प्रोटीन परिसरों </td></tr><tr><td> साल्ट </td><td> मिनिमल </td><td> नमक adducts व्यापक और अनसुलझे चोटियों के लिए नेतृत्व </td></tr><tr><td> डीटीटी </td><td> मिनिमल </td><td> 1-2 सुक्ष्ममापी उपस्थित किया जा सकता है </td></tr><tr><td> Chelating एजेंट </td><td> मिनिमल </td><td> 250 सुक्ष्ममापी ऊपर व्यापक adduct गठन के लिए नेतृत्व </td></tr></table>

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.

Watch this Scientific Journal Video about Analyzing Large Protein Complexes by Structural Mass Spectrometry at JoVE.com

Analyzing Large Protein Complexes by Structural Mass Spectrometry

मास स्पेक्ट्रोमेट्री बड़े प्रोटीन परिसरों का विश्लेषण करने के लिए एक महत्वपूर्ण उपकरण साबित हो गया है. इस विधि बहु - सबयूनिट विधानसभाओं की रचना stoichiometry, और समग्र वास्तुकला में अंतर्दृष्टि को सक्षम बनाता है. यहाँ हम का वर्णन है,, कदम - कदम के द्वारा, कैसे एक संरचनात्मक मास स्पेक्ट्रोमेट्री विश्लेषण प्रदर्शन करने के लिए, और macromolecular संरचनाओं विशेषताएँ.

स्ट्रक्चरल मास स्पेक्ट्रोमेट्री द्वारा बड़े प्रोटीन परिसरों विश्लेषण

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

analyzing-large-protein-complexes-by-structural-mass-spectrometry

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Biology

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 नमूना तैयार: अल्ट्रा पतली परत मेथड

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

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.

टी लहर आयन गतिशीलता मास स्पेक्ट्रोमेट्री प्रोटीन परिसर विश्लेषण के लिए मूल प्रायोगिक प्रक्रिया

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. ...

Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like experimental conditions. Laser ablation electrospray ionization (LAESI) is one such development and is particularly well-suited for the investigation of water-containing specimens. LAESI utilizes a mid-infrared laser beam (2.94 &mu;m wavelength) to excite the water molecules of the sample. When the ablation fluence threshold is exceeded, the sample material is expelled in the form of particulate matter and these projectiles travel to tens of millimeters above the sample surface. In LAESI, this ablation plume is intercepted by highly charged droplets to capture a fraction of the ejected sample material and convert its chemical constituents into gas-phase ions. A mass spectrometer equipped with an atmospheric-pressure ion source interface is employed to analyze and record the composition of the released ions originating from the probed area (pixel) of the sample. A systematic interrogation over an array of pixels opens a way for molecular imaging in the microprobe analysis mode. A unique aspect of LAESI mass spectrometric imaging is depth profiling that, in combination with lateral imaging, enables three-dimensional (3D) molecular imaging. With current lateral and depth resolutions of ~100 &mu;m and ~40 &mu;m, respectively, LAESI mass spectrometric imaging helps to explore the molecular structure of biological tissues. Herein, we review the major elements of a LAESI system and provide guidelines for a successful imaging experiment.

Laser ablation electrospray ionization (LAESI) is an atmospheric-pressure ion source for mass spectrometry. In the imaging mode, a mid-infrared laser probes the distributions of molecules across a tissue section or a biofilm. This technique presents a new approach for diverse bioanalytical studies carried out under native experimental conditions.

वायुमंडलीय दबाव LAESI मास स्पेक्ट्रोमेट्री द्वारा जैविक ऊतकों और Biofilms के आण्विक इमेजिंग

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like ...

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.

वायुमंडलीय दबाव में मास स्पेक्ट्रोमेट्री से एकल कक्ष के प्रत्यक्ष विश्लेषण

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.

Thiol redox आइसोटोप टैगिंग मास स्पेक्ट्रोमेट्री proteome रूपरेखा

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.

में प्रोटीन परिसरों की पहचान<em&gt; Escherichia कोलाई</em&gt; संयोजन में अग्रानुक्रम मास स्पेक्ट्रोमेट्री के साथ अनुक्रमिक पेप्टाइड संबंध शुद्धीकरण का उपयोग

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

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

प्रोटीन, प्रोटीन बातचीत की पहचान के लिए एक प्रोटोकॉल के आधार पर<sup&gt; 15</sup&gt; एन मेटाबोलिक लेबलिंग, immunoprecipitation, मात्रात्मक मास स्पेक्ट्रोमेट्री और आत्मीयता मॉडुलन

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

Microscale Thermophoresis द्वारा बंधन आत्मीयता निर्धारण के प्रोटीन शुद्धीकरण मुक्त विधि

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

Multiprotein परिसरों का तुलनात्मक विश्लेषण के लिए एक नए दृष्टिकोण पर आधारित<sup&gt; 15</sup&gt; एन मेटाबोलिक लेबल और मात्रात्मक मास स्पेक्ट्रोमेट्री

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

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.

निरपेक्ष प्रोटीन की मात्रा के लिए मास स्पेक्ट्रोमेट्री निगरानी चयनित रिएक्शन

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