Os autores agradecem os membros do grupo Sharon para a sua revisão crítica, e por suas contribuições ao manuscrito. Estamos gratos pelo apoio dos Programas Morasha e Bikura, a Israel Science Foundation (Grant n. º s 378/08 e 1823-1807), o Josef Cohn Minerva Center for Research biomembrana, a Família Chais Fellows Program para novos cientistas, o Abraham e Sonia Rochlin Foundation, o Family Trust Wolfson Charitable, a Helen e Milton A. Kimmelman Centro de Estrutura Biomolecular e Montagem; o espólio de Shlomo e Beirzwinsky Sabine; Meil ​​de Botton Aynsley, e Karen Siem, Reino Unido.

<ol>
	<li>Ruotolo, 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=Ion+mobility-mass+spectrometry+analysis+of+large+proteincomplexes.">Ion mobility-mass spectrometry analysis of large proteincomplexes.</a> Nat Protoc. 3 (7), 1139-1152 (2008).</li><li>Scarff, C. A., Thalassinos, K., Hilton, G. R., Scrivens, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Travelling+wave+ion+mobility+mass+spectrometry+studies+of+protein+structure:+biological+significance+and+comparison+with+X-ray+crystallography+and+nuclear+magnetic+resonance+spectroscopy+measurements.">Travelling wave ion mobility mass spectrometry studies of protein structure: biological significance and comparison with X-ray crystallography and nuclear magnetic resonance spectroscopy measurements.</a> Rapid Commun Mass Spectrom. 22 (20), 3297-3304 (2008).</li><li>Smith, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+drift+time+measurements+from+travelling+wave+ion+mobility+spectrometry-mass+spectrometry+studies.">Deciphering drift time measurements from travelling wave ion mobility spectrometry-mass spectrometry studies.</a> Eur J Mass Spectrom (Chichester, Eng). 15 (2), 113-130 (2009).</li><li>Leary, 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=Methodology+for+measuring+conformation+of+solvent-disrupted+protein+subunits+using+T-WAVE+ion+mobility+MS:+an+investigation+into+eukaryotic+initiation+factors.">Methodology for measuring conformation of solvent-disrupted protein subunits using T-WAVE ion mobility MS: an investigation into eukaryotic initiation factors.</a> J Am Soc Mass Spectrom. 20 (9), 1699-1706 (2009).</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=Determination+of+stoichiometry+and+conformational+changes+in+the+first+step+of+the+P22+tail+assembly.">Determination of stoichiometry and conformational changes in the first step of the P22 tail assembly.</a> J Mol Biol. 379 (2), 385-396 (2008).</li><li>Pukala, T. L. <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+multiprotein+assemblies+determined+using+restraints+from+gas-phase+measurements.">Subunit architecture of multiprotein assemblies determined using restraints from gas-phase measurements.</a> Structure. 17 (9), 1235-1243 (2009).</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=Chaperonin+complexes+monitored+by+ion+mobility+mass+spectrometry.">Chaperonin complexes monitored by ion mobility mass spectrometry.</a> J Am Chem Soc. 131 (4), 1452-1459 (2009).</li><li>Ruotolo, 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=Evidence+for+macromolecular+protein+rings+in+the+absence+of+bulk+water.">Evidence for macromolecular protein rings in the absence of bulk water.</a> Science. 310 (5754), 1658-1661 (2005).</li><li>Ruotolo, B. T., 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=Aspects+of+native+proteins+are+retained+in+vacuum.">Aspects of native proteins are retained in vacuum.</a> Curr Opin Chem Biol. 10 (5), 402-408 (2006).</li><li>Giles, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+a+travelling+wave-based+radio-frequency-only+stacked+ring+ion+guide.">Applications of a travelling wave-based radio-frequency-only stacked ring ion guide.</a> Rapid Commun Mass Spectrom. 18 (20), 2401-2414 (2004).</li><li>McKay, A. R., Ruotolo, B. T., Ilag, L. L., 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=Mass+measurements+of+increased+accuracy+resolve+heterogeneous+populations+of+intact+ribosomes.">Mass measurements of increased accuracy resolve heterogeneous populations of intact ribosomes.</a> J Am Chem Soc. 128 (35), 11433-11442 (2006).</li><li>Ruotolo, 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=Ion+mobility-mass+spectrometry+reveals+long-lived,+unfolded+intermediates+in+the+dissociation+of+protein+complexes.">Ion mobility-mass spectrometry reveals long-lived, unfolded intermediates in the dissociation of protein complexes.</a> Angew Chem Int Ed Engl. 46 (42), 8001-8004 (2007).</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>Valentine, S. J., Counterman, A. E., Clemmer, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+database+of+660+peptide+ion+cross+sections:+use+of+intrinsic+size+parameters+for+bona+fide+predictions+of+cross+sections.">A database of 660 peptide ion cross sections: use of intrinsic size parameters for bona fide predictions of cross sections.</a> J Am Soc Mass Spectrom. 10 (11), 1188-1211 (1999).</li><li>Mesleh, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+information+from+ion+mobility+measurements:+effects+of+the+long-range+potential.">Structural information from ion mobility measurements: effects of the long-range potential.</a> J Phys Chem. 100, 16082-16086 (1996).</li><li>Shvartsburg, A. A., Jarrold, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+exact+hard-spheres+scattering+model+for+the+mobilities+of+polyatomic+ions.">An exact hard-spheres scattering model for the mobilities of polyatomic ions.</a> Chem Phys Lett. 261, 86-91 (1996).</li></ol>

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O protocolo aqui descrito permite definir a seção transversal de colisão de proteínas ou complexos de proteínas com uma estrutura de três desconhecidos dimensional, com o objetivo de fornecer informações sobre sua forma geral, embalagem subunidade e topologia. Para este efeito, uma vez valores colisão seção transversal são retratados é necessário converter esses valores para detalhes estruturais. Este processo requer esforços adicionais experimental bem como a análise computacional, que são brevemente discutidos abaixo. Para começar, recomenda-se a análise de proteínas ou complexos de proteínas com estruturas conhecidas. Estas medidas podem proporcionar um controle de qualidade útil da metodologia e permitirá avaliação da acurácia dos parâmetros de aquisição, comparando os valores teóricos e medidos Ω. O teórico áreas das secções transversais podem ser calculadas a partir da estrutura de cristal coordenadas usando o software MOBCAL 15,16, que é um software de código aberto baseado em FORTRAN que permite a edição de código de acordo com as necessidades do operador. Para a execução de tais cálculos é necessário modificar o programa de tal forma que o número de cálculos iterativos realizados por estrutura de entrada é maior e que os arquivos de coordenadas contendo grande número de átomos são aceitos 1. Uma estratégia IM-MS para a definição de arranjos topológicos de subunidades dentro multicomponentes montagens propôs recentemente 4,6. O método envolve o monitoramento de vias dissociação de grupos de proteínas para os componentes menores. Essa dissociação é conseguido através do ajuste das condições controladas fase de solução, que dá origem a uma distribuição de subcomplexes reflexivo da &quot;blocos de construção&quot; das assembléias. A medição simultânea de valores Ω de ambos os complexos intacta a desmontagem e produtos gera restrições estruturais que são então utilizados para calcular os modelos topológicos dos complexos de proteína. O pressuposto básico subjacente a esta metodologia é que o subcomplexes gerada reter seus nativos como confirmações, e na verdade estudos recentes têm demonstrado que a estrutura de solução dos produtos desmontagem é mantida e nenhum rearranjo importante em qualquer solução ou fases de gás têm ocorrido 4,6. O último passo na atribuição de estrutura quaternária de íons em fase gasosa em proteínas complexas é adequado a colisão valores seção transversal de modelos gerados por computador. Abordagens de modelagem são empregadas, a fim de explorar as diferentes possíveis arranjos topológicos de subunidades e seus valores em silico Ω são calculados e comparados com os experimentais. Atualmente, apenas algumas abordagens computacionais são usados, como o método de granulação grossa spheretype que se aproxima o diâmetro de 1,8 subunidades. No geral, este campo ainda está em seus primeiros anos e desenvolvimento é necessário para fazer essa abordagem genérica e aplicável a uma ampla gama de complexos.

t-wave ion mobility-mass spectrometry: basic experimental procedures for protein complex analysis

De mobilidade iônica (IM) é um método que mede o tempo necessário para um íon de viajar através de uma célula pressurizada sob a influência de um campo elétrico fraco. A velocidade pela qual os íons atravessam a região drift depende de seu tamanho: íons grandes vão experimentar um maior número de colisões com o gás de fundo inerte (geralmente N 2) e, assim, viajar mais devagar através do dispositivo de mensagens instantâneas do que os íons que compõem um menor cross-section. Em geral, o tempo que leva para os íons de migrar embora a fase de gás denso separa-los, de acordo com sua colisão transversal (Ω). Recentemente, a espectrometria de IM foi acoplado com espectrometria de massa e uma viagem-wave (onda T) Synapt de mobilidade iônica espectrômetro de massa (IM-MS) foi lançado. Integração de espectrometria de massa com mobilidade de íons permite uma dimensão extra de separação e definição da amostra, produzindo um espectro tridimensional (em massa para o tempo de carga, intensidade e drift). Esta técnica permite a separação de sobreposição espectral a diminuir, e permite a resolução de complexos heterogêneos com massa muito semelhante, ou massa-carga índices, mas os tempos drift diferentes. Além disso, as medidas de tempo deriva fornecer uma importante camada de informações estruturais, como Ω está relacionado com a forma geral e topologia do íon. A correlação entre os valores medidos drift tempo e Ω é calculada usando uma curva de calibração gerada a partir de proteínas de calibração definido com seções transversais 1. O poder da abordagem IM-MS reside na sua capacidade de definir a embalagem subunidade e forma geral das assembleias de proteína em concentrações micromolar, e quase fisiológica condições 1. Vários estudos recentes IM de ambas as proteínas individuais 2,3 e complexos não-covalente de proteínas 4-9, conseguiu demonstrar que a estrutura da proteína quaternária é mantida na fase gasosa, e destacou o potencial dessa abordagem no estudo dos conjuntos de proteínas de geometria desconhecida . Aqui, fornecemos uma descrição detalhada do IMS-MS análise de complexos de proteínas usando o Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrumento (Waters Ltd; o único instrumento IM-MS comerciais disponíveis atualmente) 10. Descrevemos os passos básicos de otimização, a calibração de colisão seções transversais, e métodos de processamento de dados e interpretação. A etapa final do protocolo discute métodos de cálculo dos valores teóricos Ω. No geral, o protocolo não tenta cobrir todos os aspectos do IM-MS caracterização de conjuntos de proteínas, mas sim, seu objetivo é apresentar os aspectos práticos do método para novos pesquisadores no campo.

Watch this Scientific Journal Video about T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis at JoVE.com

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

Ion espectrometria de mobilidade de massa é uma tecnologia emergente fase gasosa que separa íons, com base em sua colisão transversal e massa. O método fornece informações tridimensionais sobre a topologia geral e forma de complexos de proteínas. Aqui, descrevemos um procedimento básico para configuração e otimização de instrumento, calibração de vezes drift, e interpretação dos dados.

T-wave Ion Mobility Spectrometry massa: Basic Procedimentos para Análise Experimental Protein Complex

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. ...

t-wave-ion-mobility-mass-spectrometry-basic-experimental-procedures

Research

JoVE Journal

Biology

24.4K visualizações.  Weizmann Institute of Science. Ion espectrometria de mobilidade de massa é uma tecnologia emergente fase gasosa que separa íons, com base em sua colisão transversal e massa. O método fornece informações tridimensionais sobre a topologia geral e forma de complexos de proteínas. Aqui, descrevemos um procedimento básico para configuração e otimização de instrumento, calibração de vezes drift, e interpretação dos dados.

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

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 preparação da amostra: o método de Camada Ultra Thin

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

Biologia

Cancer Borealis Stomatogastric Nervous System Dissection

The stomatogastric ganglion (STG) is an excellent model for studying cellular and network interactions because it contains a relatively small number of cells (approximately 25 in C. borealis) which are well characterized. The cells in the STG exhibit a broad range of outputs and are responsible for the motor actions of the stomach. The stomach contains the gastric mill which breaks down food with three internal teeth, and the pylorus which filters the food before it reaches the midgut. The STG produces two rhythmic outputs to control the gastric mill and pylorus known as central pattern generators (CPGs). Each cell in the STG can participate in one or both of these rhythms. These CPGs allow for the study of neuromodulation, homeostasis, cellular and network variability, network development, and network recovery.
The dissection of the stomatogastric nervous system (STNS) from the Jonah crab (Cancer borealis) is done in two parts; the gross and fine dissection. In the gross dissection the entire stomach is dissected from the crab. During the fine dissection the STNS is extracted from the stomach using a dissection microscope and micro-dissection tools (see figure 1). The STNS includes the STG, the oesophageal ganglion (OG), and the commissural ganglia (CoG) as well as the nerves that innervate the stomach muscles. Here, we show how to perform a complete dissection of the STNS in preparation for an electrophysiology experiment where the cells in the STG would be recorded from intracellularly and the peripheral nerves would be used for extracellular recordings. The proper technique for finding the desired nerves is shown as well as our technique of desheathing the ganglion to reveal the somata and neuropil.

Cancer Borealis Stomatogastric Nervous System Dissection

The stomatogastric nervous system (STNS) of the Jonah crab (C. borealis) can be used for electrophysiology, immunohistochemistry, and cell culture studies. The STNS extraction is done in two parts: the gross and fine dissection.

Câncer Borealis Dissection Sistema Nervoso Stomatogastric

The stomatogastric ganglion (STG) is an excellent model for studying cellular and network interactions because it contains a relatively small number of ...

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of the stomach is defined by the mouth opening and the posterior end by the bottom of the pylorus. Along the dorsal side of the stomach lies the stomatogastric nervous system (STNS). This nervous system, which contains rhythmic networks that underlie feeding behavior, is an established model system for studying rhythm generating networks and neuromodulation 1,2. While it is possible to study this system in vivo 3, the STNS continues to produce its rhythmic activity when isolated in vitro. In order to study this system in vitro the stomach must be removed from the animal. This video article describes how the stomach can be dissected from the American lobster. In an accompanying video article4 we demonstrate how the STNS can be isolated from the stomach.

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

We describe the gross dissection of the stomach of the American lobster (Homarus americanus).

Dissecação bruta do Estômago da lagosta, Homarus Americanus</em

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of ...

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the richness of biological information it provides. MR does not require any ionizing radiation and can noninvasively provide higher resolution and better signal-to-noise ratio in comparison to other tomographic or spectroscopic modalities. In this protocol, we will focus on small animal MR imaging and MR spectroscopy (MRI/MRS) to noninvasively acquire relaxation weighted 1H images of mouse and to obtain 31P spectra of mouse muscle. This work does not attempt to cover every aspect of small animal MRI/MRS but rather introduces basic procedures of mouse MRI/MRS experiments. The main goal of this work is to inform researchers of the basic procedures for in vivo MR experiments on small animals. The goal is to provide a better understanding of basic experimental procedures to allow researchers new to the MR field to better plan for non-MR components of their studies so that both MR and non-MR procedures are seamlessly integrated.

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

This work describes basic procedures of noninvasive small animal MRI and MRS in vivo.

Não invasivo In Vivo Pequenos Animais MRI e MRS: Basic procedimentos experimentais

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Usando o sistema de fracionamento GELFREE 8100 para Fracionamento baseada no peso molecular com Recovery Fase Líquida

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

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.

Analisar grandes complexos de proteínas por espectrometria de massa estruturais

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

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagênese e Análise Funcional de canais iônicos Heterologously Expresso em células de mamíferos

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

Coleta de sangue para análise bioquímica em Zebrafish Adulto

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Básico<em&gt; Caenorhabditis elegans</em&gt; Métodos: Sincronização e observação

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

A Plataforma de Produção de Proteína Complexo MultiBac no EMBL

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...