Este trabalho foi apoiado pelo Conselho de Pesquisa em Ciências Naturais e Engenharia do Canadá, pela Fonds de Recherche du Quebec Nature et Technologie, pela Canadian Foundation for Innovation e pela McGill University.

<ol>
	<li>Karplus, M., McCammon, 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=Molecular+dynamics+simulations+of+biomolecules.">Molecular dynamics simulations of biomolecules.</a> Nature Structural &amp; Molecular Biology. 9 (9), 646-652 (2002).</li><li>Campbell, E., et al. <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+protein+dynamics+in+the+evolution+of+new+enzyme+function.">The role of protein dynamics in the evolution of new enzyme function.</a> Nature Chemical Biology. 12 (11), 944-950 (2016).</li><li>Tokuriki, N., 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=Protein+dynamism+and+evolvability.">Protein dynamism and evolvability.</a> Science. 324 (5924), 203-207 (2009).</li><li>Wales, T. E., Engen, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+exchange+mass+spectrometry+for+the+analysis+of+protein+dynamics.">Hydrogen exchange mass spectrometry for the analysis of protein dynamics.</a> Mass Spectrometry Reviews. 25 (1), 158-170 (2006).</li><li>Konermann, L., Pan, J., Liu, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+exchange+mass+spectrometry+for+studying+protein+structure+and+dynamics.">Hydrogen exchange mass spectrometry for studying protein structure and dynamics.</a> Chemical Society Reviews. 40 (3), 1224 (2011).</li><li>Katta, V., Chait, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+changes+in+proteins+probed+by+hydrogen-exchange+electrospray-ionization+mass+spectrometry.">Conformational changes in proteins probed by hydrogen-exchange electrospray-ionization mass spectrometry.</a> Rapid Communications in Mass Spectrometry. 5 (4), 214 (1991).</li><li>Zhang, Z., Smith, D. L. <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+amide+hydrogen+exchange+by+mass+spectrometry:+a+new+tool+for+protein+structure+elucidation.">Determination of amide hydrogen exchange by mass spectrometry: a new tool for protein structure elucidation.</a> Protein Science. 2 (4), 522 (1993).</li><li>Smith, D. L., Deng, Y., Zhang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+non-covalent+structure+of+proteins+by+amide+hydrogen+exchange+and+mass+spectrometry.">Probing the non-covalent structure of proteins by amide hydrogen exchange and mass spectrometry.</a> Journal of Mass Spectrometry. 32 (2), 135-146 (1997).</li><li>Bai, Y., Milne, J. S., Mayne, L., Englander, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+structure+effects+on+peptide+group+hydrogen+exchange.">Primary structure effects on peptide group hydrogen exchange.</a> Proteins. 17 (1), 75-86 (1993).</li><li>Xiao, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revealing+the+architecture+of+protein+complexes+by+an+orthogonal+approach+combining+HDXMS,+CXMS,+and+disulfide+trapping.">Revealing the architecture of protein complexes by an orthogonal approach combining HDXMS, CXMS, and disulfide trapping.</a> Nature Protocols. 13 (6), 1403-1428 (2018).</li><li>Smith, D. L., Deng, Y., Zhang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+non-covalent+structure+of+proteins+by+amide+hydrogen+exchange+and+mass+spectrometry.">Probing the non-covalent structure of proteins by amide hydrogen exchange and mass spectrometry.</a> Journal of Mass Spectrometry. 32 (2), 135-146 (1997).</li><li>Masson, G. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+performing,+interpreting+and+reporting+hydrogen+deuterium+exchange+mass+spectrometry+(HDX-MS)+experiments.">Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments.</a> Nature Methods. 16 (7), 595-602 (2019).</li><li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Efficient+Site-Specific+Method+for+Irreversible+Covalent+Labeling+of+Proteins+with+a+Fluorophore.">An Efficient Site-Specific Method for Irreversible Covalent Labeling of Proteins with a Fluorophore.</a> Scientific Reports. 5, 16883 (2015).</li><li>Marion, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overcoming+the+overlap+problem+in+the+assignment+of+proton+NMR+spectra+of+larger+proteins+by+use+of+three-dimensional+heteronuclear+proton-nitrogen-15+Hartmann-Hahn-multiple+quantum+coherence+and+nuclear+Overhauser-multiple+quantum+coherence+spectroscopy:+application+to+interleukin+1.beta.">Overcoming the overlap problem in the assignment of proton NMR spectra of larger proteins by use of three-dimensional heteronuclear proton-nitrogen-15 Hartmann-Hahn-multiple quantum coherence and nuclear Overhauser-multiple quantum coherence spectroscopy: application to interleukin 1.beta.</a> Biochemistry. 28 (15), 6150-6156 (1989).</li><li>Balasubramaniam, D., Komives, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen-exchange+mass+spectrometry+for+the+study+of+intrinsic+disorder+in+proteins.">Hydrogen-exchange mass spectrometry for the study of intrinsic disorder in proteins.</a> Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. 1834 (6), 1202-1209 (2013).</li><li>Goswami, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+window+expansion+for+HDX+analysis+of+an+intrinsically+disordered+protein.">Time window expansion for HDX analysis of an intrinsically disordered protein.</a> Journal of the American Society for Mass Spectrometry. 24 (10), 1584-1592 (2013).</li><li>Keppel, T. R., Howard, B. A., Weis, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+Unstructured+Regions+and+Synergistic+Folding+in+Intrinsically+Disordered+Proteins+with+Amide+H/D+Exchange+Mass+Spectrometry.">Mapping Unstructured Regions and Synergistic Folding in Intrinsically Disordered Proteins with Amide H/D Exchange Mass Spectrometry.</a> Biochemistry. 50 (40), 8722-8732 (2011).</li><li>Mitchell, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Characterization+and+Conformational+Analysis+of+the+Herpesvirus+saimiri+Tip-C484+Protein.">Functional Characterization and Conformational Analysis of the Herpesvirus saimiri Tip-C484 Protein.</a> Journal of Molecular Biology. 366 (4), 1282-1293 (2007).</li><li>Pan, J., Han, J., Borchers, C. H., Konermann, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen/deuterium+exchange+mass+spectrometry+with+top-down+electron+capture+dissociation+for+characterizing+structural+transitions+of+a+17+kDa+protein.">Hydrogen/deuterium exchange mass spectrometry with top-down electron capture dissociation for characterizing structural transitions of a 17 kDa protein.</a> Journal of the American Chemical Society. 131 (35), 12801-12808 (2009).</li><li>Habibi, Y., Uggowitzer, K. A., Issak, H., Thibodeaux, C. J. <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+the+Dynamic+Structural+Properties+of+a+Lanthipeptide+Synthetase+using+Hydrogen-Deuterium+Exchange+Mass+Spectrometry.">Insights into the Dynamic Structural Properties of a Lanthipeptide Synthetase using Hydrogen-Deuterium Exchange Mass Spectrometry.</a> Journal of the American Chemical Society. 141 (37), 14661-14672 (2019).</li><li>Hebling, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+analysis+of+membrane+proteins+in+phospholipid+bilayer+nanodiscs+by+hydrogen+exchange+mass+spectrometry.">Conformational analysis of membrane proteins in phospholipid bilayer nanodiscs by hydrogen exchange mass spectrometry.</a> Analytical Chemistry. 82 (13), 5415-5419 (2010).</li><li>Duc, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+application+of+bicelles+for+conformational+analysis+of+G+protein-coupled+receptors+by+hydrogen/deuterium+exchange+mass+spectrometry.">Effective application of bicelles for conformational analysis of G protein-coupled receptors by hydrogen/deuterium exchange mass spectrometry.</a> Journal of the American Society for Mass Spectrometry. 26 (5), 808-817 (2015).</li><li>Canul-Tec, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+allosteric+inhibition+of+excitatory+amino+acid+transporter+1.">Structure and allosteric inhibition of excitatory amino acid transporter 1.</a> Nature. 544 (7651), 446-451 (2017).</li><li>Reading, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interrogating+Membrane+Protein+Conformational+Dynamics+within+Native+Lipid+Compositions.">Interrogating Membrane Protein Conformational Dynamics within Native Lipid Compositions.</a> Angewandte Chemie International Edition. 56 (49), 15654-15657 (2017).</li><li>Hentze, N., Mayer, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+protein+dynamics+using+hydrogen+exchange+mass+spectrometry.">Analyzing protein dynamics using hydrogen exchange mass spectrometry.</a> Journal of Visualized Experiments. (81), e50839 (2013).</li><li>Lento, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-resolved+ElectroSpray+Ionization+Hydrogen-deuterium+Exchange+Mass+Spectrometry+for+Studying+Protein+Structure+and+Dynamics.">Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics.</a> Journal of Visualized Experiments. (122), e55464 (2017).</li><li>Schowen, K. B., Schowen, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solvent+isotope+effects+on+enzyme+systems.">Solvent isotope effects on enzyme systems.</a> Methods in Enzymology. 87, 551-606 (1982).</li><li>Lau, A. M. C., Ahdash, Z., Martens, C., Politis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deuteros:+software+for+rapid+analysis+and+visualization+of+data+from+differential+hydrogen+deuterium+exchange-mass+spectrometry.">Deuteros: software for rapid analysis and visualization of data from differential hydrogen deuterium exchange-mass spectrometry.</a> Bioinformatics. 35 (7), 3171-3173 (2019).</li><li>Houde, D., Berkowitz, S. A., Engen, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+utility+of+hydrogen/deuterium+exchange+mass+spectrometry+in+biopharmaceutical+comparability+studies.">The utility of hydrogen/deuterium exchange mass spectrometry in biopharmaceutical comparability studies.</a> Journal of Pharmaceutical Sciences. 100 (6), 2071 (2011).</li><li>Burkitt, W., O'Connor, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+the+repeatability+and+reproducibility+of+hydrogen/deuterium+exchange+mass+spectrometry+measurements.">Assessment of the repeatability and reproducibility of hydrogen/deuterium exchange mass spectrometry measurements.</a> Rapid Communications in Mass Spectrometry. 22 (23), 3893-3901 (2008).</li><li>Rob, T., Gill, P. K., Golemi-Kotra, D., Wilson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+electrospray+ms-coupled+microfluidic+device+for+sub-second+hydrogen/deuterium+exchange+pulse-labelling+reveals+allosteric+effects+in+enzyme+inhibition.">An electrospray ms-coupled microfluidic device for sub-second hydrogen/deuterium exchange pulse-labelling reveals allosteric effects in enzyme inhibition.</a> Lab on a Chip. 13, 2528-2532 (2013).</li></ol>

Não temos nada para revelar.

O fluxo de trabalho HDX-MS apresentado neste protocolo fornece uma plataforma notavelmente robusta para mapear a distribuição espacial de elementos estruturalmente dinâmicos em proteínas e para investigar como essas dinâmicas mudam em resposta à perturbação (ligação de ligante, mutagênese enzimática, etc.). O HDX-MS possui várias vantagens distintas sobre outras abordagens de biologia estrutural que são comumente usadas para investigar a dinâmica conformacional. Mais notavelmente, apenas pequenas quantidad...

Proteínas são moléculas estruturalmente dinâmicas que amostram diferentes conformações em escalas de tempo que vão desde vibração de ligação em escala femtosegundo até rearranjos de domínios proteicos inteiros que podem ocorrer ao longo de muitos segundos1. Estas flutuações conformais são frequentemente aspectos críticos da função enzima/proteína. Por exemplo, alterações conformais induzidas pela ligação de ligasão são muitas vezes criticamente importantes para a modulação da função enzimática, seja organizando resíduos ativos do local necessários para a catálise, definindo locais de ligação de substrato em mecanismos cinéticos sequenciais, protegendo intermediários reativos do ambiente ou modulando a função enzimática através de redes alostêmicas. Estudos recentes também mostraram que a dinâmica conformacional pode ser conservada ao longo da evolução e que perturbações a movimentos moleculares conservados podem ser correlacionadas com mudanças na especificidade do substrato e no surgimento de novas funções enzimárias2,3.
Nos últimos anos, a espectrometria de massa de troca de hidrogênio-deutério (HDX-MS) tem emergido rapidamente como uma técnica poderosa para sondar como as paisagens conformais da proteína respondem a perturbações como ligadura ou mutagênese4,5,6,7. Em um experimento típico hdx-ms(Figura 1), uma proteína de interesse é colocada em tampão preparado em D2O, que desencadeia a substituição de prótons trocaveis de solventes por deuteria. A taxa de troca da moiety amida dos títulos de peptídeo depende fortemente do pH, da sequência local de aminoácidos, e do ambiente estrutural local da amida8. Amidas que estão engajadas em interações de ligação de hidrogênio (como as presentes em α-helices e β-folhas) trocam mais lentamente do que em meios em regiões não estruturadas da proteína que são expostas a solventes a granel. Assim, a extensão da captação do deutério é um reflexo da estrutura da enzima. Enzimas que são conformamente dinâmicas, ou que passam por transições estruturais sobre ligação ligante, seriam esperadas para produzir uma resposta HDX mensurável.
A base mecanicista para a taxa de câmbio lenta de uma amida estruturada é mostrada na Figura 25,8,9. Para se submeter ao HDX, a região estruturada deve primeiro amostrar transitoriamente uma conformação desdobrada, de tal forma que as moléculas de solvente que catalisam a troca de HDX através de um mecanismo químico ácido/base específico, tenham acesso ao amide tromável. Em última análise, as magnitudes relativas da taxa de câmbio química (kchem) e as taxas de dobra e reagem(kopen e kclose) determinam a taxa HDX medida no experimento5,8. A partir deste modelo cinético simples, é claro que a extensão da absorção do deutério refletirá a dinâmica conformacional subjacente (definida por kopen e kclose). A maioria dos experimentos HDX-MS são realizados em um fluxo de trabalho de baixo para cima onde, após a reação de troca, a proteína de interesse é digerida em peptídeos e a absorção do deutério por cada peptídeo é medida como um aumento na massa7. Dessa forma, o HDX-MS permite que perturbações para a dinâmica conformacional enzimária sejam mapeadas na escala espacial local dos peptídeos, permitindo que o pesquisador avalie como a perturbação altera a dinâmica em diferentes regiões da enzima de interesse.
As vantagens da abordagem HDX-MS para a dinâmica estrutural proteica elucidação são inúmeras. Em primeiro lugar, o método pode ser realizado com pequenas quantidades de proteína nativa ou em complexos proteicos em sistemas com estrutura quaternária10. Não é mesmo necessário que a preparação da enzima usada no ensaio seja altamente purificada11,12, desde que o fluxo de trabalho HDX-MS de baixo para cima forneça um número suficiente de peptídeos confiantemente identificados que cobrem a sequência proteica de interesse. Além disso, o HDX-MS pode fornecer informações sobre a dinâmica conformacional em condições próximas de nativos sem a necessidade de rotulagem de proteínas específicas do local, como seria usado em estudos de fluorescência de moléculas únicas13, e não há limite de tamanho para o complexo proteico ou proteico que possa ser investigado (o que torna desafiadoras abordagens como ressonância magnética nuclear [NMR] espectroscopia)7,14. Finalmente, métodos HDX-MS resolvidos pelo tempo podem ser empregados para estudar proteínas intrinsecamente desordenadas, que são difíceis de estudar com cristalografia de raios-X15,16,17,18. A principal limitação do HDX-MS é que os dados são de baixa resolução estrutural. Os dados HDX-MS são úteis para apontar para onde a dinâmica conformacional está mudando e para revelar alterações conformais acopláveis, mas muitas vezes não fornecem muita visão do mecanismo molecular preciso que conduz a mudança observada. Avanços recentes na combinação de métodos de dissociação de captura de elétrons com dados de proteína HDX-MS mostraram-se promissores para mapear locais de troca para resíduos de aminoácidosúnicos 19,mas estudos bioquímicos e estruturais de acompanhamento ainda são frequentemente necessários para fornecer clareza aos modelos estruturais encaminhados pelos dados hdx-MS.
Abaixo, um protocolo detalhado para o desenvolvimento de um ensaio HDX-MS é apresentado20. Os protocolos de preparação da amostra apresentados abaixo devem ser geralmente aplicáveis a qualquer proteína que exaque boa solubilidade em tampões aquosos. Métodos de preparação de amostras mais especializados e fluxos de trabalho HDX-MS estão disponíveis para proteínas do que precisam ser avaliados na presença de detergentes ou fosfolipídios21,22,23,24. As configurações instrumentais para a coleta de dados HDX-MS são descritas para um espectrômetro de massa quadrúpole de alta resolução acoplado ao sistema de cromatografia líquida. Dados de complexidade e resolução semelhantes poderiam ser coletados em qualquer um dos vários sistemas de espectrometria de massa líquida (LC-MS) disponíveis comercialmente. Aspectos-chave do processamento de dados usando um pacote de software comercialmente disponível também são fornecidos. Também apresentamos diretrizes para coleta e análise de dados que são consistentes com as recomendações feitas pela comunidade HDX-MS mais ampla12. O protocolo descrito é usado para estudar as propriedades estruturais dinâmicas do HalM2, uma sintetização lanthipeptide que catalisa a maturação multistep de um produto natural de peptídeo antimicrobiano20. Ilustramos como o HDX-MS pode ser usado para revelar sites de ligação de substratos e propriedades alotésicas que iludiram a caracterização anterior. Vários outros protocolos sobre proteína HDX-MS foram publicados nos últimos anos25,26. Juntamente com o presente trabalho, essas contribuições anteriores devem proporcionar ao leitor alguma flexibilidade no design experimental.

<table>
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>[glu-1]-fibrinopeptide B (Glu-Fib)</td>
			<td>BioBasic</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mL Amicon Ultracel 10k centrifugal filtration device (Millipore)</td>
			<td>Milipore Sigma</td>
			<td>UFC501096</td>
			<td></td>
		</tr>
		<tr>
			<td>acetonitrile</td>
			<td>Fisher</td>
			<td>A955-1</td>
			<td></td>
		</tr>
		<tr>
			<td>AMP-PNP</td>
			<td>SIGMA</td>
			<td>A2647-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>SIGMA</td>
			<td>a2383-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>D2O</td>
			<td>ALDRICH</td>
			<td>435767-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>formic acid</td>
			<td>Thermo Fisher</td>
			<td>28905</td>
			<td></td>
		</tr>
		<tr>
			<td>guanidine-HCl</td>
			<td>VWR</td>
			<td>97063-764</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher</td>
			<td>BP310-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>SiGMA-Aldrich</td>
			<td>63068-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>BioBasic</td>
			<td>PB0440</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium phosphate</td>
			<td>BioBasic</td>
			<td>PB0445</td>
			<td></td>
		</tr>
		<tr>
			<td>TCEP Hydrochloride</td>
			<td>TRC Canada</td>
			<td>T012500</td>
			<td>peptide was synthesized upon request</td>
		</tr>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Deuteros</td>
			<td>Andy M C Lau, et al</td>
			<td>version 1.08</td>
			<td></td>
		</tr>
		<tr>
			<td>DynamX</td>
			<td>Waters</td>
			<td>version 3.0</td>
			<td></td>
		</tr>
		<tr>
			<td>MassLynx</td>
			<td>Waters</td>
			<td>version 4.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein Lynx 
			Global Server (PLGS)</td>
			<td>Waters</td>
			<td>version 3.0.3</td>
			<td></td>
		</tr>
		<tr>
			<td>PyMOL</td>
			<td>Schr&#246;dinger</td>
			<td>version 2.2.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Instrument and equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ACQUITY UPLC BEH C18 analytical Column</td>
			<td>Waters</td>
			<td>186002346</td>
			<td></td>
		</tr>
		<tr>
			<td>ACQUITY UPLC BEH C8 VanGuard Pre-column</td>
			<td>Waters</td>
			<td>186003978</td>
			<td></td>
		</tr>
		<tr>
			<td>ACQUITY UPLC M-Class HDX System</td>
			<td>Waters</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HDX Manager</td>
			<td>Waters</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>microtip pH electrode</td>
			<td>Thermo Fisher</td>
			<td>13-620-291</td>
			<td></td>
		</tr>
		<tr>
			<td>Waters Enzymate BEH column or Pepsin solumn</td>
			<td>Waters</td>
			<td>186007233</td>
			<td></td>
		</tr>
		<tr>
			<td>Waters Synapt G2-Si</td>
			<td>Waters</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a hydrogen-deuterium exchange mass spectrometry (hdx-ms) platform for investigating peptide biosynthetic enzymes

Espectrometria de massa de troca de hidrogênio-deutério (HDX-MS) é um método poderoso para a caracterização biofísica de alterações conformacionais enzimáticos e interações entre enzimas enzimistas. Entre seus muitos benefícios, o HDX-MS consome apenas pequenas quantidades de material, pode ser realizado em condições próximas de nativos sem a necessidade de rotulagem enzimada/substrato, e pode fornecer informações espacialmente resolvidas sobre a dinâmica conformacional enzimático– mesmo para grandes enzimas e complexos multiproteínas. O método é iniciado pela diluição da enzima de interesse em tampão preparado em D2O. Isso desencadeia a troca de protium em peptídeos (N-H) com deutério (N-D). Nos pontos de tempo de troca desejados, as alíquotas de reação são saciadas, a enzima é proteolisada em peptídeos, os peptídeos são separados por cromatografia líquida de ultra-desempenho (UPLC), e a mudança na massa de cada peptídeo (devido à troca de hidrogênio por deutério) é registrada pela MS. A quantidade de absorção de deutério por cada peptídeo depende fortemente do ambiente local de ligação de hidrogênio desse peptídeo. Peptídeos presentes em regiões muito dinâmicas do deutério de troca de enzimas muito rapidamente, enquanto peptídeos derivados de regiões bem ordenadas passam por troca muito mais lentamente. Desta forma, a taxa HDX relata a dinâmica conformacional da enzima local. Perturbações aos níveis de absorção de deutério na presença de diferentes ligantes podem então ser usadas para mapear sites de ligação de ligantes, identificar redes alusicais e entender o papel da dinâmica conformacional na função enzimática. Aqui, ilustramos como usamos o HDX-MS para entender melhor a biossíntese de um tipo de peptídeos naturais chamados lanthipeptides. Lanthipeptídeos são peptídeos geneticamente codificados que são modificados pós-translacionalmente por enzimas grandes, multifuncionais e conformacionalmente dinâmicas que são difíceis de estudar com abordagens tradicionais de biologia estrutural. O HDX-MS fornece uma plataforma ideal e adaptável para investigar as propriedades mecanicistas desses tipos de enzimas.

É necessário avaliar a qualidade da digestão proteolítica e a reprodutibilidade do fluxo de trabalho para cada conjunto de injeções amostrais. Assim, antes de realizar ensaios HDX-MS, é essencial estabelecer condições efetivas para a proteólise da proteína de interesse, para a separação de peptídeos utilizando cromatografia líquida de fase inversa e mobilidade de íons de fase de gás, e para a detecção de peptídeos utilizando EM. Para isso, as amostras de referência para a proteína de interesse (cole...

Watch this Scientific Journal Video about A Hydrogen-Deuterium Exchange Mass Spectrometry HDX-MS Platform for Investigating Peptide Biosynthetic Enzymes at JoVE.com

A Hydrogen-Deuterium Exchange Mass Spectrometry HDX-MS Platform for Investigating Peptide Biosynthetic Enzymes

As sintetizações lanthipeptide catalisam reações multistep durante a biossíntese de produtos naturais de peptídeos. Aqui, descrevemos um fluxo de trabalho contínuo, de baixo para cima, de espectrometria de massa de troca de hidrogênio -deutério (HDX-MS) que pode ser empregado para estudar a dinâmica conformacional das sintetizações de lanthipeptídeo, bem como outras enzimas semelhantes envolvidas na biossíntese de produtos naturais peptídeos.

Uma plataforma de espectrometria de massa de troca de hidrogênio (HDX-MS) para investigar enzimas biossintéticas de peptídeos

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for the biophysical characterization of enzyme conformational changes and ...

a-hydrogen-deuterium-exchange-mass-spectrometry-hdx-ms-platform-for

Research

JoVE Journal

Biochemistry

A Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) Platform for Investigating Peptide Biosynthetic Enzymes

7.8K visualizações.  McGill University. As sintetizações lanthipeptide catalisam reações multistep durante a biossíntese de produtos naturais de peptídeos. Aqui, descrevemos um fluxo de trabalho contínuo, de baixo para cima, de espectrometria de massa de troca de hidrogênio -deutério (HDX-MS) que pode ser empregado para estudar a dinâmica conformacional das sintetizações de lanthipeptídeo, bem como outras enzimas semelhantes envolvidas na biossíntese de produtos naturais peptídeos.

Vídeo: A Hydrogen-Deuterium Exchange Mass Spectrometry HDX-MS Platform for Investigating Peptide Biosynthetic Enzymes

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

resolvida no tempo com ionização por electrospray Hidrogénio-deutério troca Espectrometria de Massa para estudar a estrutura e dinâmica das proteínas

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Bioquímica

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Resolver purificada por afinidade complexos de proteínas por Azul Native PAGE e Proteína Correlação Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

Isolamento de gotículas de gordura para análise quantitativa Espectrometria de Massa

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

Preparação da amostra para identificação de espectrometria de massa-baseada das regiões de ligação de RNA

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

Preparação ideal de formol fixada amostras para peptídeo baseado Matrix assistida por espectrometria de massa de ionização/dessorção Laser Imaging fluxos de trabalho

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host ...

Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of reactive oxygen species, and more. These fluctuations can lead to a widespread protein unfolding, aggregation, and cell death. Therefore, cells have evolved a dynamic and stress-specific network of molecular chaperones, which maintain a "healthy" proteome during stress conditions. ATP-independent chaperones constitute one major class of molecular chaperones, which serve as first-line defense molecules, protecting against protein aggregation in a stress-dependent manner. One feature these chaperones have in common is their ability to utilize structural plasticity for their stress-specific activation, recognition, and release of the misfolded client. 
In this paper, we focus on the functional and structural analysis of one such intrinsically disordered chaperone, the bacterial redox-regulated Hsp33, which protects proteins against aggregation during oxidative stress. Here, we present a toolbox of diverse techniques for studying redox-regulated chaperone activity, as well as for mapping conformational changes of the chaperone, underlying its activity. Specifically, we describe a workflow which includes the preparation of fully reduced and fully oxidized proteins, followed by an analysis of the chaperone anti-aggregation activity in vitro using light-scattering, focusing on the degree of the anti-aggregation activity and its kinetics. To overcome frequent outliers accumulated during aggregation assays, we describe the usage of Kfits, a novel graphical tool which allows easy processing of kinetic measurements. This tool can be easily applied to other types of kinetic measurements for removing outliers and fitting kinetic parameters. To correlate the function with the protein structure, we describe the setup and workflow of a structural mass spectrometry technique, hydrogen-deuterium exchange mass spectrometry, that allows the mapping of conformational changes on the chaperone and substrate during different stages of Hsp33 activity. The same methodology can be applied to other protein-protein and protein-ligand interactions.

Defining Hsp33&#039;s Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry

One of the most challenging stress conditions that organisms encounter during their lifetime involves the accumulation of oxidants. During oxidative stress, cells heavily rely on molecular chaperones. Here, we present methods used to investigate the redox-regulated anti-aggregation activity, as well as to monitor structural changes governing the chaperone function using HDX-MS.

Definição da atividade de chaperona regulada por redox de Hsp33 e mapeamento de alterações conformacionais em Hsp33 usando espectrometria de massa de troca de hidrogênio-deutério

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

Detecção de locais de ubiquitinação de proteínas por enriquecimento de peptídeos e espectrometria de massa

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

Perfil e identificação de nitropeptídeos ilustrados pela angiotensina II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

A Plasma Sample Preparation for Mass Spectrometry using an Automated Workstation

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves numerous incubation and liquid transfer steps in order to achieve denaturation, reduction, alkylation, and cleavage. Adapting this workflow onto an automated workstation can increase efficiency and reduce coefficients of variance, thereby providing more reliable data for statistical comparisons between sample types. We previously described an automated proteomic sample preparation workflow1. Here, we report the development of a more efficient and better controlled workflow with the following advantages: 1) The number of liquid transfer steps is reduced from nine to six by combining reagents; 2) Pipetting time is reduced by selective tip pipetting using a 96-position pipetting head with multiple channels; 3) Potential throughput is increased by the availability of up to 45 deck positions; 4) Complete enclosure of the system provides improved temperature and environmental control and reduces the potential for contamination of samples or reagents; and 5) The addition of stable isotope labeled peptides, as well as &#946;-galactosidase protein, to each sample makes monitoring and quality control possible throughout the entire process. These hardware and process improvements provide good reproducibility and improve intra-assay and inter-assay precision (CV of less than 20%) for LC-MS based protein and peptide quantification. The entire workflow for digesting 96 samples in a 96-well plate can be completed in approximately 5 hours.

Here, we present an automated plasma protein digestion method for mass spectrometry-based quantitative proteomic analysis. In this protocol, the liquid transferring and incubation steps for protein denaturation, reduction, alkylation, and trypsin digestion reactions are streamlined and automated. It takes approximately five hours to prepare a 96-well plate with desired precision.

Uma preparação de amostra de plasma para espectrometria de massa usando uma estação de trabalho automatizada

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves ...

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ratios (m/z), which are useful to deduce molecular weights and structures. While it is essentially a destructive analytical method, recent advancements in the ambient ionization technique have enabled us to acquire data while leaving tissue in a relatively intact state in terms of integrity. Probe electrospray ionization (PESI) is a so-called direct method because it does not require complex and time-consuming pretreatment of samples. A fine needle serves as a sample picker, as well as an ionization emitter. Based on the very sharp and fine property of the probe tip, destruction of the samples is minimal, allowing us to acquire the real-time molecular information from living things in situ. Herein, we introduce three applications of PESI-MS technique that will be useful for biomedical research and development. One involves the application to solid tissue, which is the basic application of this technique for the medical diagnosis. As this technique requires only 10 mg of the sample, it may be very useful in the routine clinical settings. The second application is for in vitro medical diagnostics where human blood serum is measured. The ability to measure fluid samples is also valuable in various biological experiments where a sufficient volume of sample for conventional analytical techniques cannot be provided. The third application leans toward the direct application of probe needles in living animals, where we can obtain real-time dynamics of metabolites or drugs in specific organs. In each application, we can infer the molecules that have been detected by MS or use artificial intelligence to obtain a medical diagnosis.

This article introduces sample preparation methods for a unique real-time analytical method based on the ambient mass spectrometry. This method lets us perform real-time analysis of the biological molecules in vivo without any special pretreatments.

Preparação de amostras para espectrometria de ionização de eletrospray de sonda

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...