עבודה זו נתמכה על ידי ארגון Human תוכנית Frontier Science (פרס HFSP 2007, RGP0058/2007-C). אנו מודים פרופ &#39;ריצ&#39;רד רוברטס ליזום את הפרויקט לדיונים פוריים.

<ol>
	<li>Katayama, H., Oda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+proteomics+for+drug+discovery+based+on+compound-immobilized+affinity+chromatography.">Chemical proteomics for drug discovery based on compound-immobilized affinity chromatography.</a> J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 855, 21-27 (2007).</li><li>Barglow, K. T., Cravatt, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-based+protein+profiling+for+the+functional+annotation+of+enzymes.">Activity-based protein profiling for the functional annotation of enzymes.</a> Nat. Methods. 4, 822-827 (2007).</li><li>Koster, H., Little, D. P., Luan, P., Muller, R., Siddiqi, S. M., Marappan, S., Yip, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capture+compound+mass+spectrometry:+a+technology+for+the+investigation+of+small+molecule+protein+interactions.">Capture compound mass spectrometry: a technology for the investigation of small molecule protein interactions.</a> Assay Drug Dev. Technol. 5, 381-390 (2007).</li><li>Dalhoff, C., Hueben, M., Lenz, T., Poot, P., Nordhoff, E., Koster, H., Weinhold, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+S-Adenosyl-L-homocysteine+Capture+Compounds+for+Selective+Photoinduced+Isolation+of+Methyltransferases.">Synthesis of S-Adenosyl-L-homocysteine Capture Compounds for Selective Photoinduced Isolation of Methyltransferases.</a> ChemBioChem. 11, 256-265 (2010).</li><li>Lu, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S-Adenosylmethionine.">S-Adenosylmethionine.</a> Int. J. Biochem. Cell. Biol. 32, 391-3952 (2000).</li><li>Cantoni, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+methylation:+selected+aspects.">Biological methylation: selected aspects.</a> Annu. Rev. Biochem. 44, 435-451 (1975).</li><li>Jeltsch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+Watson+and+Crick:+DNA+methylation+and+molecular+enzymology+of+DNA+methyltransferases.">Beyond Watson and Crick: DNA methylation and molecular enzymology of DNA methyltransferases.</a> ChemBioChem. 3, 274-293 (2002).</li><li>Chow, C. S., Lamichhane, T. N., Mahto, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+nucleotide+repertoire+of+the+ribosome+with+post-transcriptional+modifications.">Expanding the nucleotide repertoire of the ribosome with post-transcriptional modifications.</a> ACS Chem. Biol. 2, 610-619 (2007).</li><li>Grillo, M. A., Colombatto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S-Adenosylmethionine+and+protein+methylation.">S-Adenosylmethionine and protein methylation.</a> Amino Acids. 28, 357-362 (2005).</li><li>Fujioka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+small+molecule+methyltransferases:+their+structural+and+functional+features.">Mammalian small molecule methyltransferases: their structural and functional features.</a> Int. J. Biochem. 24, 1917-1924 (1992).</li><li>Borchardt, R. T., Salvatore, F., Borek, E., Zappia, V., Williams-Ashman, H. G., Schlenk, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Biochemistry of S-Adenosylmethionine. , 151-171 (1977).</li><li>Hoffman, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatographic+analysis+of+the+chiral+and+covalent+instability+of+S-adenosyl-l-methionine.">Chromatographic analysis of the chiral and covalent instability of S-adenosyl-l-methionine.</a> Biochemistry. 25, 4444-4449 (1986).</li><li>Keller, A., Nesvizhskii, A. I., Kolker, E., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Empirical+statistical+model+to+estimate+the+accuracy+of+peptide+identifications+made+by+MS/MS+and+database+search.">Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.</a> Anal. Chem. 74, 5383-5392 (2002).</li><li>Nesvizhskii, A. I., Keller, A., Kolker, E., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+statistical+model+for+identifying+proteins+by+tandem+mass+spectrometry.">A statistical model for identifying proteins by tandem mass spectrometry.</a> Anal. Chem. 75, 4646-4658 (2003).</li></ol>

ThomasLenz, OliviaGrabner, MirkoGlinski, MathiasDreger ו HubertKosterare המועסקים על ידי caprotec bioanalytics GmbH, Inc המייצרת כמה חומרים כימיים המשמשים במאמר זה.

אמצעי הזהירות הבאים והערות עשוי להיות שימושי כאשר בעקבות פרוטוקול תיאר: א) היתרון העיקרי של CCS טמון ביצירת קשר קוולנטי בין CC לבין MTase, כמו זה היתרי התנאים הבאים כביסה מחמירים. Crosslink קוולנטיים מושגת על ידי photoreaction מופעלות על ידי אור אולטרה סגול (310 ננומטר מקסימום.). אור המנורה רגיל מכיל רק חלק קטן של UV, לעומת זאת, להגן על אדון-CC מחשיפה עוד תקורה או אור השמש אפילו עד הקרנה מבוקרת מקורר caproBox. ב) דגימות ביולוגיות שממנו MTases הם להיות מבודדים עשויים להכיל חלבונים נוטים denaturation, ולכן זו חובה לשמור על הדגימות קריר להימנע מקציפים בכל עת. ג) caproBox מתקרר דגימות כדי 0-4 מעלות צלזיוס, המנורות פולטות אור UV, אך גם פולטים חום. לכן יש צורך בקצרה צנטריפוגות מבחנות לפני ההקרנה, ולכן חלבונים דבקות כמוסות או קירות הבקבוקון לא יכול להיווצר משקעים זרעים. ד) מחדש את ההשעיה של חרוזים מגנטיים (למשל הפתרונות לשטוף) לא ניתן יד, זמן קצר חלה אולטרסאונד על ידי הצבת דגימות לאמבטיה אולטרסאונד. ה) טרי להכין את הפתרון 0.2% ACN TFA/60%. מצאנו כי אחרת את החלבונים שנתפסו לא יכול להיות ביקע מן החרוזים. ו) בסופו של חלבונים שנתפסו מתבצעת על ידי LC-MS/MS. ספקטרומטריית מסה היא שיטה רגישה מאוד. זה הכרחי להשתמש ריאגנטים בלעדי LC-MS כיתה השלבים הסופיים (2.11 צעד ועוד). הימנע זיהום של ניסויים על ידי מקורות חלבון חיצוני, קרטין כגון אבק או שמקורם מן הנסיין. במיוחד במהלך השלבים העיכול הסופי, מומלץ לשים לב שטח עבודה נקי, ללבוש כפפות וחלוק מעבדה ואולי רשת שיער או באופן אידיאלי לבצע את השלבים הסופיים מתחת לספסל נקי. הכן את 50 mM אמוניום ביקרבונט המאגר משמש לעיכול tryptic במים MS LC כיתה, דרך מסנן 0.22 מיקרומטר, חנות לסנן, aliquot ב -20 ° C, ולהשתמש בכל aliquot רק פעם אחת, כדי למנוע זיהומים. הפתרון טריפסין (סידור כיתה, רוש, להכין 0.5 מיקרוגרם / פתרון μl ידי הוספת 1 mM HCl כדי lyophilized טריפסין) ניתן לאחסן 4 ° C למשך מספר שבועות. ז) כדי לקבל ספקטרום המוני אמין חיוני יש לרסס יציב בניתוח ESI-MS/MS. ח) עבור מערכות LC-MS/MS שונה מזו השתמשו במחקר הנוכחי, מדידת פרמטרים אלגוריתמים לזיהוי פפטידים חייב להיות מותאם באופן אינדיבידואלי. את השינויים הבאים הם אפשריים ביחס פרוטוקול המתואר:) לחילופין כדי לשחרר את החלבונים מן החרוזים ידי TFA 60% ACN/0.2% (שלב 2.11), חלבונים ניתן ישירות מתעכל tryptically בתוך ההשעיה חרוז (כרכים זהה בשלב 5.1) או, SDS-PAGE, חלבונים ניתן שפורסמו על ידי השעיית וחימום החרוזים שנאסף לאחר שלב 2.9-95 מעלות צלזיוס למשך 10 דקות במאגר מדגם SDS (שניהם, את ההשעיה כולו או רק את supernatant ניתן לטעון לכיס ג&#39;ל). מצאנו כי החרוזים לאט לשחרר כמויות קטנות של הפולימר לתוך בתמיסה מימית לעכל tryptic במהלך לעכל על חרוז tryptic גם לאחר מספר צעדים לשטוף מימית. זיהום פולימר מפריע זיהוי הפפטיד MS וניתן לשטוף את החרוזים באמצעות ACN 80% (לפחות שלוש פעמים). לאחר השלבים לשטוף 80% ACN, החרוזים יש לשטוף מיד עם מים לפני לעכל על חרוז tryptic. ב) כתמים המערבי באמצעות streptavidin-horseraddish peroxidase ואת המצע ECL יכול לשמש גם כדי להמחיש crosslinking מוצלח של CC ביוטין המכיל את החלבונים. לכן, גם הג&#39;ל המתקבל לאחר שלב 3.2 ניתן מחק או, כי הרגישות היא על פי 10 גבוה יותר מאשר צביעת כסף של ג&#39;לים, 10 דגימות μl אחרי צעד 2.7 עשוי גם להיות מנותח על ידי כתם המערבי. Mind כי במקרה האחרון חלבונים biotinylated endogeneously יהיה גם זוהה מלבד החלבונים biotinylated באופן מלאכותי על ידי אדון-CC. ג) מצאנו כי זה תלוי במערכת מיוחדת (lysate, התייחס חלבונים היעד, סלקטיביות ותפקוד תגובתיות של CC הוא), האם &quot;off-חרוז&quot; תצורה, שם את התגובה crosslinking מתרחש בין CC חופשית חלבון בתמיסה 4 או המתואר כיום &quot;על חרוז&quot; תצורה (איור 1B) מבצעת טוב יותר. באופן כללי, השיטה צריכה גם להיות תואם עם כל מדינה-of-the איזוטופ יציב אמנות החלבון או הפפטיד תיוג הטכנולוגיה, או הערכה של המדגם ללכוד על ידי ג&#39;ל אלקטרופורזה 2D. ב התצורה &quot;off-חרוז&quot;, אפשר גם ללכוד חלבונים בתוך תאים שלמים (תוצאות לא פורסם). יתר על כן, התרופה או אתר cofactor מחייב של חלבון יכול להיות שתוארו על ידי קביעת עמדה מקרוב על ידי crosslinking של CC בתוך רצף החלבון על ידי רצף הפפטיד MS. מצב המחייב של מולקולה קטנה לחלבון ניתן לחקור באמצעות ג שונהמצורף עמדות hemical על הפונקציה סלקטיביות, והמקשר באורכים שונים. כפי שמוצג במחקר הנוכחי, גם שותפים חלבון מחייב של חלבונים פנה ידי הפונקציה סלקטיביות (RplK כמו המצע של PrmA) או לא ידוע חלבון קטן מולקולה אינטראקציות (אדון על GlnA) ניתן לזהות. סיכם, תכונה נוספת של CCS, תגובתיות צילום crosslinking, מאפשר בידוד וזיהוי של חלבונים בשפע נמוך או משפחות חלבון פונקציונלי מתערובות חלבון מורכב עם רגישות גבוהה ומספקת למדענים כלי נוסף ללימוד מולקולה קטנה - אינטראקציות חלבון .

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
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<td>SAH caproKit&trade;</td>
<td>&nbsp;</td>
<td>caprotec bioanalytics GmbH</td>
<td>1-1010-050 (50 reactions) 1-1010-010 (10 reactions)</td>
<td>Includes the SAH-CC, SAH competitor, streptavidin coated magnetic beads, capture buffer, and wash buffer</td>
</tr>
<tr>
<td>caproBox&trade;</td>
<td>&nbsp;</td>
<td>caprotec bioanalytics GmbH</td>
<td>1-5010-003 (110 V) 1-5010-004 (230 V)</td>
<td>For reproducible photo-activation while cooling the samples</td>
</tr>
<tr>
<td>caproMag&trade;</td>
<td>&nbsp;</td>
<td>caprotec bioanalytics GmbH</td>
<td>1-5100-001</td>
<td>For easy handling of magnetic particles without pipetting</td>
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profiling of methyltransferases and other s-adenosyl-l-homocysteine-binding proteins by capture compound mass spectrometry (ccms)

יש מגוון של גישות כדי להפחית את המורכבות של proteome על בסיס פונקציונלי של מולקולות חלבון אינטראקציות קטנות כגון זיקה כרומטוגרפיה 1 או חלבון על בסיס פעילות פרופיל 2. תרכובות לכידת Trifunctional (CCS, איור 1 א) 3 הם בסיס לגישה גנרי, שבו שיווי המשקל הראשונית מונחה אינטראקציה בין בדיקה מולקולה קטנה (הפונקציה סלקטיביות, כאן S-adenosyl-L-הומוציסטאין, אדון, איור 1 א) חלבונים היעד הוא קבוע ובלתי הפיך על crosslinking-photo בין פונקציית צילום activable עצמאית תגובתיות (כאן phenylazide) של CC לבין פני השטח של חלבונים היעד. הפונקציה מיון (כאן ביוטין) משמש כדי לבודד את CC - conjugates חלבון מתערובות ביולוגיות מורכבות בעזרת בשלב מוצק (כאן חרוזים streptavidin מגנטי). שתי תצורות הניסויים הם אפשריים: &quot;מחוץ חרוז&quot; 4 או המתואר כיום &quot;על חרוז&quot; תצורה (איור 1B). הפונקציה הסלקטיביות עשויה להיות כמעט כל מולקולה קטנה של עניין (מצעים, מעכבי, מולקולות התרופה). S-Adenosyl-L-מתיונין (SAM, איור 1 א) היא ככל הנראה, שני ATP, cofactor הנפוץ ביותר בטבע 5, 6. הוא משמש בתור התורם העיקרי קבוצת מתיל בכל היצורים החיים עם התגובה הכימית להיות catalyzed ידי SAM תלויי methyltransferases (MTases), אשר methylate 7-DNA, RNA 8, 9 חלבונים או מולקולות קטנות 10. לאור תפקיד מכריע של תגובות פיזיולוגיות מתילציה בתרחישים שונים (ויסות הגנים, אפיגנטיקה, מטבוליזם), פרופילים של MTases ניתן לצפות להיות חשיבות דומה proteomics פונקציונלי כמו פרופיל של קינאזות. כלים אנליטיים עבור פרופילים שלהם, לעומת זאת, לא היה זמין. אנו הציגה לאחרונה CC עם אדון כקבוצה סלקטיביות למלא את הפער הטכנולוגי (איור 1A). אדון, תוצר של SAM לאחר העברת מתיל, הוא מעכב ידוע בכלל MTase המוצר 11. מסיבה זו וגם בגלל הטבעית cofactor SAM נמצא בשימוש על ידי אנזימים נוסף העברת חלקים אחרים של cofactor או ליזום התגובות הרדיקלי, כמו גם בגלל חוסר היציבות הכימית שלה 12, אדון הוא פונקציה סלקטיביות אידיאלי CC למקד MTases. כאן אנו מדווחים על השירות של אדון-CC ו CCMS ידי MTases פרופילים אחרים אדון מחייב חלבונים מן DH5α את המתח של Escherichia coli (E. coli), אחד הטובים ביותר, מאופיין פרוקריוטים, אשר שימש המודל המועדף האורגניזם מחקרים ביוכימיים, ביולוגיים ביוטכנולוגיים אינספור. תמונה המופעל crosslinking משפר את התשואה ואת הרגישות של הניסוי, ואת הספציפיות ניתן לבדוק בקלות עבור בניסויים התחרות באמצעות עודף של אדון חינם.

Watch this Scientific Journal Video about Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS at JoVE.com

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

תרכובות לכידת הם מולקולות קטנות trifunctional כדי להפחית את המורכבות של proteome על ידי תפקודית אינטראקציה הפיך מולקולות חלבון קטן ואחריו crosslinking-צילום טיהור. כאן אנו משתמשים מתחם לכידת עם<em&gt; S</em&gt;-Adenosyl-<sub&gt; L</sub&gt;-הומוציסטאין מחייב כפונקציה סלקטיביות לבודד methyltransferases מ<em&gt; Escherichia coli</em&gt; התא כולו lysate לזהות אותם על ידי MS.

פרופיל של Methyltransferases ועל אחרים S-Adenosyl- L-הומוציסטאין מחייב חלבונים על ידי ספקטרומטריית מתחם המיסה לכידה (CCMS)

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

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

15.5K Views.  caprotec bioanalytics GmbH. תרכובות לכידת הם מולקולות קטנות trifunctional כדי להפחית את המורכבות של proteome על ידי תפקודית אינטראקציה הפיך מולקולות חלבון קטן ואחריו crosslinking-צילום טיהור. כאן אנו משתמשים מתחם לכידת עם S-Adenosyl- L-הומוציסטאין מחייב כפונקציה סלקטיביות לבודד methyltransferases מ

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

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 ...

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

נוגדן פרופיל על ידי מערכות immunoprecipitation בלוציפראז (שפתיים)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

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 ...

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

אוליגו פרופיל המוניים (OLIMP) של סוכרים תאית

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

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

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

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

בלחץ אטמוספרי מולקולרית והדמיה של רקמות ביולוגיות באמצעות Biofilms LAESI המוניים ספקטרומטריית

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

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

ניתוח ישיר של תאים בודדים באמצעות ספקטרומטריית מסה בלחץ אטמוספרי

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

כימות של חלבונים באמצעות העשרה Immunoaffinity פפטיד יחד עם ספקטרומטריית מסה

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

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

פרופיל Proteome חיזור תיאול באמצעות תיוג איזוטופ ספקטרומטריית מסה

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

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 ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

תיוג רצף ספציפי של חומצות גרעין וחלבונים עם Methyltransferases ואנלוגים כקו-פקטורים

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...