מחקר זה פורסם במקור בפרוטאומיקה הנייד מול. סולדי מ &#39;וBonaldi ט proteomic חקירת תחומים פונקציונליים הכרומטין חושף Synergisms הרומן בין ברור heterochromatin הרכיבים MCP. 2013; 12: 64-80. © האגודה האמריקנית לביוכימיה וביולוגיה מולקולרית. אנו מודים לרוברטה Noberini (האיטלקי מכון טכנולוגי ולIEO, איטליה) לקריאה ביקורתית של כתב היד. עבודת TB נתמכת על ידי מענקים מArmenise-הרווארד תכנית ג&#39;ובאני קרן לפיתוח קריירה, האיטלקית האגודה לחקר הסרטן ומשרד בריאות האיטלקית. עבודת MS נתמכה על ידי מענק FIRC.

<ol>
	<li>Kornberg, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+structure:+a+repeating+unit+of+histones+and+DNA.">Chromatin structure: a repeating unit of histones and DNA.</a> Science. 184, 868-871 (1974).</li><li>Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F., Richmond, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+nucleosome+core+particle+at+2.8+A+resolution.">Crystal structure of the nucleosome core particle at 2.8 A resolution.</a> Nature. 389, 251-260 (1997).</li><li>Kouzarides, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+modifications+and+their+function.">Chromatin modifications and their function.</a> Cell. 128, 693-705 (2007).</li><li>Bannister, A. J., Kouzarides, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+chromatin+by+histone+modifications.">Regulation of chromatin by histone modifications.</a> Cell Res. 21, 381-395 (2011).</li><li>Taverna, S. D., Li, H., Ruthenburg, A. J., Allis, C. D., Patel, 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=How+chromatin-binding+modules+interpret+histone+modifications:+lessons+from+professional+pocket+pickers.">How chromatin-binding modules interpret histone modifications: lessons from professional pocket pickers.</a> Nat Struct Mol Biol. 14, 1025-1040 (2007).</li><li>Jenuwein, T., Allis, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+the+histone+code.">Translating the histone code.</a> Science. 293, 1074-1080 (2001).</li><li>Spotswood, H. T., Turner, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+increasingly+complex+code.">An increasingly complex code.</a> J Clin Invest. 110, 577-582 (2002).</li><li>Soldi, M., Cuomo, A., Bremang, M., Bonaldi, T. <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-based+proteomics+for+the+analysis+of+chromatin+structure+and+dynamics.">Mass spectrometry-based proteomics for the analysis of chromatin structure and dynamics.</a> Int J Mol Sci. 14, 5402-5431 (2013).</li><li>Sidoli, S., Cheng, L., Jensen, O. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+in+chromatin+biology+and+epigenetics:+Elucidation+of+post-translational+modifications+of+histone+proteins+by+mass+spectrometry.">Proteomics in chromatin biology and epigenetics: Elucidation of post-translational modifications of histone proteins by mass spectrometry.</a> J Proteomics. 75, 3419-3433 (2012).</li><li>Britton, L. M., Gonzales-Cope, M., Zee, B. M., Garcia, 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=Breaking+the+histone+code+with+quantitative+mass+spectrometry.">Breaking the histone code with quantitative mass spectrometry.</a> Expert Rev Proteomics. 8, 631-643 (2011).</li><li>Taverna, S. 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=Long-distance+combinatorial+linkage+between+methylation+and+acetylation+on+histone+H3+N+termini.">Long-distance combinatorial linkage between methylation and acetylation on histone H3 N termini.</a> Proc Natl Acad Sci U S A. 104, 2086-2091 (2007).</li><li>Garcia, B. A., Shabanowitz, J., Hunt, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+histones+and+their+post-translational+modifications+by+mass+spectrometry.">Characterization of histones and their post-translational modifications by mass spectrometry.</a> Curr Opin Chem Biol. 11, 66-73 (2007).</li><li>Villar-Garea, A., Imhof, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+analysis+of+histone+modifications.">The analysis of histone modifications.</a> Biochim Biophys Acta. 1764, 1932-1939 (2006).</li><li>Plazas-Mayorca, M. 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=One-pot+shotgun+quantitative+mass+spectrometry+characterization+of+histones.">One-pot shotgun quantitative mass spectrometry characterization of histones.</a> J Proteome Res. 8, 5367-5374 (2009).</li><li>Ohta, S., 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+protein+composition+of+mitotic+chromosomes+determined+using+multiclassifier+combinatorial+proteomics.">The protein composition of mitotic chromosomes determined using multiclassifier combinatorial proteomics.</a> Cell. 142, 810-821 (2010).</li><li>Vermeulen, 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=Selective+anchoring+of+TFIID+to+nucleosomes+by+trimethylation+of+histone+H3+lysine+4.">Selective anchoring of TFIID to nucleosomes by trimethylation of histone H3 lysine 4.</a> Cell. 131, 58-69 (2007).</li><li>Vermeulen, 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=Quantitative+interaction+proteomics+and+genome-wide+profiling+of+epigenetic+histone+marks+and+their+readers.">Quantitative interaction proteomics and genome-wide profiling of epigenetic histone marks and their readers.</a> Cell. 142, 967-980 (2010).</li><li>Bartke, T., 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=Nucleosome-interacting+proteins+regulated+by+DNA+and+histone+methylation.">Nucleosome-interacting proteins regulated by DNA and histone methylation.</a> Cell. 143, 470-484 (2010).</li><li>Dejardin, J., Kingston, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+proteins+associated+with+specific+genomic+Loci.">Purification of proteins associated with specific genomic Loci.</a> Cell. 136, 175-186 (2009).</li><li>Lambert, J. P., Mitchell, L., Rudner, A., Baetz, K., Figeys, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+proteomics+approach+for+the+discovery+of+chromatin-associated+protein+networks.">A novel proteomics approach for the discovery of chromatin-associated protein networks.</a> Mol Cell Proteomics. 8, 870-882 (2009).</li><li>Soldi, M., Bonaldi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+proteomic+investigation+of+chromatin+functional+domains+reveals+novel+synergisms+among+distinct+heterochromatin+components.">The proteomic investigation of chromatin functional domains reveals novel synergisms among distinct heterochromatin components.</a> Mol Cell Proteomics. 12, 764-780 (2013).</li><li>Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V., 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=In-gel+digestion+for+mass+spectrometric+characterization+of+proteins+and+proteomes.">In-gel digestion for mass spectrometric characterization of proteins and proteomes.</a> Nat Protoc. 1, 2856-2860 (2006).</li><li>Shevchenko, A., Wilm, M., Vorm, O., 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=Mass+spectrometric+sequencing+of+proteins+silver-stained+polyacrylamide+gels.">Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.</a> Anal Chem. 68, 850-858 (1996).</li><li>Bonaldi, T., Regula, J. T., Imhof, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+mass+spectrometry+for+the+analysis+of+histone+modifications.">The use of mass spectrometry for the analysis of histone modifications.</a> Methods Enzymol. 377, 111-130 (2004).</li><li>Cuomo, A., Moretti, S., Minucci, S., Bonaldi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SILAC-based+proteomic+analysis+to+dissect+the+&amp;#34;histone+modification+signature&amp;#34;+of+human+breast+cancer+cells.">SILAC-based proteomic analysis to dissect the &amp;#34;histone modification signature&amp;#34; of human breast cancer cells.</a> Amino Acids. 41, 387-399 (2011).</li><li>Rappsilber, J., Mann, M., Ishihama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+micro-purification,+enrichment,+pre-fractionation+and+storage+of+peptides+for+proteomics+using+StageTips.">Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips.</a> Nat Protoc. 2, 1896-1906 (2007).</li><li>Rappsilber, J., Ishihama, Y., 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=Stop+and+go+extraction+tips+for+matrix-assisted+laser+desorption/ionization,+nanoelectrospray,+and+LC/MS+sample+pretreatment+in+proteomics.">Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics.</a> Anal Chem. 75, 663-670 (2003).</li><li>Olsen, J. V., Ong, S. E., 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=Trypsin+cleaves+exclusively+C-terminal+to+arginine+and+lysine+residues.">Trypsin cleaves exclusively C-terminal to arginine and lysine residues.</a> Mol Cell Proteomics. 3, 608-614 (2004).</li><li>Jung, H. R., Pasini, D., Helin, K., Jensen, O. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+mass+spectrometry+of+histones+H3.2+and+H3.3+in+Suz12-deficient+mouse+embryonic+stem+cells+reveals+distinct,+dynamic+post-translational+modifications+at+Lys-27+and+Lys-36.">Quantitative mass spectrometry of histones H3.2 and H3.3 in Suz12-deficient mouse embryonic stem cells reveals distinct, dynamic post-translational modifications at Lys-27 and Lys-36.</a> Mol Cell Proteomics. 9, 838-850 (2010).</li><li>Cox, J., 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=MaxQuant+enables+high+peptide+identification+rates,+individualized+p.p.b.-range+mass+accuracies+and+proteome-wide+protein+quantification.">MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.</a> Nat Biotechnol. 26, 1367-1372 (2008).</li><li>Cox, 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=Andromeda:+a+peptide+search+engine+integrated+into+the+MaxQuant+environment.">Andromeda: a peptide search engine integrated into the MaxQuant environment.</a> J Proteome Res. 10, 1794-1805 (2011).</li><li>Lachner, M., O'Carroll, D., Rea, S., Mechtler, K., Jenuwein, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylation+of+histone+H3+lysine+9+creates+a+binding+site+for+HP1+proteins.">Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins.</a> Nature. 410, 116-120 (2001).</li><li>Bannister, A. 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=Selective+recognition+of+methylated+lysine+9+on+histone+H3+by+the+HP1+chromo+domain.">Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain.</a> Nature. 410, 120-124 (2001).</li><li>Ram, O., 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=Combinatorial+patterning+of+chromatin+regulators+uncovered+by+genome-wide+location+analysis+in+human+cells.">Combinatorial patterning of chromatin regulators uncovered by genome-wide location analysis in human cells.</a> Cell. 147, 1628-1639 (2011).</li><li>Barski, A., 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=High-resolution+profiling+of+histone+methylations+in+the+human+genome.">High-resolution profiling of histone methylations in the human genome.</a> Cell. 129, 823-837 (2007).</li><li>Beck, H. C. <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+in+epigenetic+research.">Mass spectrometry in epigenetic research.</a> Methods Mol Biol. 593, 263-282 (2010).</li><li>Ernst, J., Kellis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+and+characterization+of+chromatin+states+for+systematic+annotation+of+the+human+genome.">Discovery and characterization of chromatin states for systematic annotation of the human genome.</a> Nat Biotechnol. 28, 817-825 (2010).</li><li>Tipton, J. 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=Analysis+of+intact+protein+isoforms+by+mass+spectrometry.">Analysis of intact protein isoforms by mass spectrometry.</a> J Biol Chem. 286, 25451-25458 (2011).</li><li>Young, N. 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=High+throughput+characterization+of+combinatorial+histone+codes.">High throughput characterization of combinatorial histone codes.</a> Mol Cell Proteomics. 8, 2266-2284 (2009).</li><li>Boyne, M. T., Pesavento, J. J., Mizzen, C. A., Kelleher, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precise+characterization+of+human+histones+in+the+H2A+gene+family+by+top+down+mass+spectrometry.">Precise characterization of human histones in the H2A gene family by top down mass spectrometry.</a> J Proteome Res. 5, 248-253 (2006).</li></ol>

אין ניגודי האינטרסים הכריזו.

יש לנו לאחרונה תיארתי ChroP, אסטרטגיה כמותית לאפיון בקנה מידה גדולה של רכיבי החלבון של הכרומטין. ChroP משלב שתי גישות משלימות בשימוש בתחום אפיגנטיים, שבב וטרשת נפוצה, מרוויח מנקודתי החוזק שלהם ולהתגבר על המגבלות שלהם. שבב מצמידים את רצף עמוק (שבב Seq) מאפשר מיפוי הגנום של שינויי היסטון ברזולוציה של כמה nucleosomes 35. למרות יתרון לרגישות שלהם, מבחני מבוססי נוגדנים מוגבלים ביכולת שלהם להבחין בשינויים דומים ובלנתח את ההיבט קומבינטורית של קוד היסטון 36. מצד השני, בעוד MS מספק ניתוח מקיף ובלתי מוטה של hPTMs, ביחידים ובשילובים 9, זה עד כה פנה לניתוח של הכרומטין בתפזורת, עם חוסר הסוגר של מידע על דפוסי PTMs מוקד ספציפי. אנו מעסיקים גרסה שונה של שבב לבודד מבחינה תפקודיתתחומים שונים הכרומטין וספקטרומטריית מסה לאפיין את דפוסי PTM היסטון וחלבונים שאינם histonic במיוחד שיתוף מועשר. באמצעות N-ChroP, הניתוח של hPTMs שיתוף קשורים H3K9me3 חשף העשרה משמעותית של סמנים הקשורים הכרומטין השקט, ודלדול של שינויים הקשורים הכרומטין הפעיל. הסכם של תוצאות אלו עם מחקרים קודמים 37 הוכיח את החוסן של האסטרטגיה. ב X-ChroP של תחומים H3K9me3, החקירה מבוססת SILAC של חלבוני אינטראקציה-הכרומטין אישרה כמה interactors שתואר קודם לכן, ובכך מאמת את השיטה. ChroP מציג שני היבטים מקוריים עיקריים בביחס לאסטרטגיות כבר זמינות כדי לחקור את מרכיב proteomic של הכרומטין: למשל, את האפשרות לחשוף synergisms בין מולקולרי בין השינויים לקשט ההיסטונים ליבה שונים בתוך אותו מונו nucl ללא פגעeosome מטוהר על ידי N-שבב; שני את ההזדמנות להעריך את המידור הספציפי של גרסאות היסטון ותת היסטון מקשר. מאז החקירה של וריאנטים היסטון מוחזקת בחזרה על ידי חוסר של חומרים כימיים באיכות טובה (כלומר, נוגדנים), ChroP מתגלה ככלי הייחודי זמין על מנת להעריך את מיקומם ותפקיד פונקציונלי. מגבלה אחת של ChroP בה הנוכחי להגדיר שקרים בגישת פפטיד ממוקד (&quot;מלמטה למעלה&quot;) המשמשת בטרשת נפוצה, עם ההיסטונים מתעכלים בפפטידים קצרים וכתוצאה מכך לירידת ערך באיתור הקישוריות למרחקים ארוכים בין שינויים. ככזה, נטיה של ChroP עם ניתוח MS &quot;מלמטה למעלה&quot; מאפשרת הערכה חלקית של ההיבט קומבינטורית של קוד היסטון. אנו חוזים כי יישומן של אסטרטגיות חלופיות MS, כגון &quot;-Down למעלה התיכון ו&quot; למיפוי hPTMs על פפטידים ארוכים יותר (&gt; 20 אא) עד בחלבונים שלמים 38-40, יתגברהאיפוק הזה. בסך הכל, N-ו-X-ChroP משלים מאוד, עם האפשרות לנתח את המורכבות של מבנה הכרומטין בתחומים פונקציונלי שונים, עם רזולוציה של מונו לאוליגו nucleosomes. אנו צופים כי ChroP יהיה שימושי גם כדי לאפיין את ההרכב של אזורי הכרומטין מסומנים על ידי נוכחותם של חלבונים גרעיניים שאינם histonic הספציפיים, לדוגמא גורמי שעתוק (TFS). בנוסף, יכול להיות מועסק ChroP במחקרים פונקציונליים למפה הקומפוזיציה הדינמית של הכרומטין בלוקוסים ספציפיים על הפרעות שונות, למשל בעת הפעלת תעתיק הגלובלית. מסיבות אלה, ChroP מתגלה ככלי שימושי נוסף בארסנל של אסטרטגיות אנליטיות זמינות לנתח את נוף proteomic של הכרומטין.

הכרומטין הוא מורכב nucleoprotein דינמי מאוד כי הוא מעורב כתבנית ראשונית לכל התהליכים בתיווך ה-DNA. הנוקלאוזום הוא היחידה הבסיסית החוזרת ונשנית של הכרומטין והוא מורכב מליבת octameric חלבוניים המכילה שתי מולקולות של כל H2A הקנונית היסטון, H2B, H3 ו-H4, שסביבו 147 נ&quot;ב של ה-DNA עטופים 1,2. כל ההיסטונים הליבה בנויים כתחום כדורי ו&quot; זנב &quot;N-מסוף גמיש שבולט מחוץ להנוקלאוזום. אחד המנגנונים העיקריים להסדרת מבנה הכרומטין ודינמיקה מבוסס על שינויים קוולנטיים לאחר translational (PTMs), שבעיקר להתרחש על N-Termini של ההיסטונים 3,4. שינויי היסטון יכולים לתפקד גם על ידי שינוי מבנה הכרומטין סדר גבוה יותר, על ידי שינוי קשרים בין ההיסטונים-DNA או בין nucleosomes, ובכך שליטה על הנגישות של חלבוני ה-DNA מחייבת (מנגנוני cis), או על ידי מתנהג כמו dockinאתרים גרם לחלבונים רגולטוריים, או כיחידות בודדות, או משובץ במתחמי Multimeric. חלבוני ויסות כזה יכול להפעיל את תפקידם בדרכים שונות: על ידי ויסות ישירות ביטוי גנים (כלומר חלבוני TAF), או על ידי שינוי מיקום הנוקלאוזום (כלומר הכרומטין שיפוץ מכלולים) או על ידי שינוי שאריות אחרות היסטון (כלומר חלבונים עם מתיל-transferase או אצטיל פעילות transferase) (מנגנוני טרנס) 5. התצפית כי אשכול דפוסים ברור PTM בלוקוסי הכרומטין ספציפיים הוביל לפירוט של את ההשערה כי שינויים שונים באתרים שונים עשויים synergize ליצור קוד מולקולרי המתווך את המצב התפקודי של ה-DNA הבסיסי. &quot;ההשערה של קוד היסטון&quot; צברה קונסנסוס גדול בשנים, אבל אימות ניסיוני שלה כבר התאפקה על ידי מגבלות טכניות 6,7. פרוטאומיקה מבוססת ספקטרומטריית מסה (MS) התפתחהכלי רב עוצמה למפה דפוסי שינוי היסטון ולאפיין מחייב חלבוני הכרומטין 8. MS מזהה שינוי כΔmass ספציפי בין המסה הניסיונית ותיאורטית של פפטיד. ברמה של ההיסטונים בודדים, MS מספק שיטה משוחדת ומקיפה למפה PTMs, המאפשר זיהוי של שינויים חדשים וinterplays החושפני ביניהם 9-14. בשנים האחרונות, מספר האסטרטגיות פותחו כדי לנתח את הרכב proteomic של הכרומטין, כולל האפיון של כרומוזומים שלמים המיטוטי 15, זיהוי של חלבוני קושרי hPTM המסיסים 16-18 והבידוד והניתוח של אזורי הכרומטין הספציפיים (כלומר הטלומרים) 19,20. עם זאת, החקירה של סינרגיות המוקד ספציפי בין PTMs היסטון, גרסאות, והחלבונים הקשורים הכרומטין היא עדיין לא הושלמה. כאן, אנו מתארים גישה חדשה, בשם ChroP (roteomics chro P matin) 21, שפיתחנו כדי לאפיין ביעילות תחומים הכרומטין תפקודי מובהקים. גישה זו מתאים את עצמו immunoprecipitation הכרומטין (שבב), פרוטוקול מבוסס היטב בשימוש במחקר אפיגנטיים, לניתוח proteomic מבוסס MS יעיל של המדגם המועשר. פיתחנו שני פרוטוקולים שונים, בהתאם לסוג של הכרומטין משמש כקלט והשאלה מופנה על ידי MS; בפרט: 1) שבב של הכרומטין ילידים מבולבל מתעכל עם MNase מועסק לטהר מונו nucleosomes ולנתח את hPTMs שיתוף שיוך (N-ChroP); 2) שבב של הכרומטין crosslinked מקוטע על ידי sonication משמש בשילוב עם אסטרטגית interactomics מבוסס SILAC לאפיין כל הכרומטין שיתוף ההעשרה מחייב חלבונים (X-ChroP). אנו מדגימים כאן שילוב של N-ו-X-ChroP להעשרת ולימוד heterochromatin, באמצעות H3K9me3 כפיתיון לצעדי immunoprecipitation. השימוש בChroP יכול להתארךללמוד או אזורים שונים על הכרומטין, או שינויים בהרכב הכרומטין בתוך האזור אותו במהלך המעבר למצב תפקודי שונה, ובכך סלל את הדרך ליישומים שונים באפיגנטיקה.

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</table>

the chrop approach combines chip and mass spectrometry to dissect locus-specific proteomic landscapes of chromatin

הכרומטין הוא מורכב nucleoprotein מאוד דינמי עשויים דנ&quot;א וחלבונים השולט בתהליכי ה-DNA תלוי שונים. מבנה הכרומטין ותפקוד באזורים מסוימים מוסדר על ידי ההעשרה המקומית של שינויי היסטון לאחר translational (hPTMs) וגרסאות, מחייב חלבוני הכרומטין, ובכלל זה גורמי שעתוק, ומתילציה בדנ&quot;א. אפיון proteomic של הרכב הכרומטין באזורים פונקציונליים שונים כבר הקשו עד כה על ידי חוסר פרוטוקולים יעילים להעשיר תחומים כגון בטוהר והכמות המתאימים לניתוח מעמיק לאחר מכן על ידי ספקטרומטריית מסה (MS). אנו מתארים כאן אסטרטגיה חדשה תוכננה פרוטאומיקה הכרומטין, בשם ChroP (roteomics P matin chro), לפיה immunoprecipitation הכרומטין preparative משמש כדי לבודד אזורי הכרומטין ברורים שתווים, במונחים של hPTMs, וריאנטים הקשורים שותף חלבונים שאינו histonic, הם נותח על ידי טרשת נפוצה. אנו ממחישים הואמחדש הקמת ChroP להעשרה והניתוח של אזורי heterochromatic תעתיק שקטים, שסומנה על ידי נוכחותו של Tri-מתילציה של ליזין 9 בהיסטון H3. התוצאות שהושגו להדגים את הפוטנציאל של ChroP באפיון יסודיות proteome heterochromatin ולהוכיח את זה כאסטרטגיה אנליטיים רבת עוצמה להבנת אופן שבו גורמי החלבון הייחודיים של הכרומטין אינטראקציה וsynergize להקים תצורות מבניות ותפקודיות מוקד ספציפי.

Watch this Scientific Journal Video about The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin at JoVE.com

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

על ידי שילוב של ילידים וcrosslinking immunoprecipitation הכרומטין עם רזולוציה גבוהה ספקטרומטריית מסה, גישת ChroP מאפשרת לנתח את ארכיטקטורת proteomic המורכבת של שינויי היסטון, וריאנטים וחלבונים שאינם histonic synergizing בתחומי הכרומטין תפקודי מובהקים.

גישת ChroP משלבת שבב וספקטרומטריית המסה לנתח נופי proteomic לוקוס ספציפיים של הכרומטין

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...

the-chrop-approach-combines-chip-mass-spectrometry-to-dissect-locus

Universidad San Sebastian

Research

JoVE Journal

Biology

18.1K Views.  European Institute of Oncology.  על ידי שילוב של ילידים וcrosslinking immunoprecipitation הכרומטין עם רזולוציה גבוהה ספקטרומטריית מסה, גישת ChroP מאפשרת לנתח את ארכיטקטורת proteomic המורכבת של שינויי היסטון, וריאנטים וחלבונים שאינם histonic synergizing בתחומי הכרומטין תפקודי מובהקים. 

Video: The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

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 הכנה לדוגמא: שיטת אולטרה שכבה דקה

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

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

ההפרדה Electrophoretic של חלבונים

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

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

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

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

הערכה כמותית של Lipidome שמרים באמצעות Ionization electrospray ספקטרומטריית מסה

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

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

ניתוח מערכות חלבון גדול על ידי ספקטרומטריית מסה מבנית

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

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

הכרומטין immunoprecipitation (שבב) באמצעות תסיסנית רקמה

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

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.

גישה חדשה לניתוח השוואתי של multiprotein מכלולים בהתבסס על<sup&gt; 15</sup&gt; N מטבולית תיוג וכמותי ספקטרומטריית המסה

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

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

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

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

ספקטרומטריית מתחם מיסת לכידה - כלי רב עוצמה לזיהוי הרומן ג-di-GMP חלבונים מפעיל

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

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

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

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

תגובה נבחרת ניטור ספקטרומטריית מסה לכימות חלבון מוחלט

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

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

הגברה, רצף הדור הבא, ואת הגנום מיפוי ה- DNA של אתרי אינטגרצית retroviral

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

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

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

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

יישומים של-הבדיקה הבודדה: הדמיה ספקטרומטריית מסה וניתוח תא יחיד בתנאי הסביבה

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