This research was supported by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases.

The authors have nothing to disclose.

כימות חלבון מוחלט חיוני למגוון מאוד רחב של יישומים ביו-רפואיים כגון אימות סמן ביולוגי ודוגמנות מסלול העברת אותות. לאחרונה, פרוטאומיקה ממוקדת באמצעות LC-SRM נהנתה משיפורים רבים בטכנולוגיות כולל הכנת פפטיד סטנדרטית, HPLC, QQQ-MS, וניתוח נתונים LC-SRM. כתוצאה מכך, היא הפכה חלופה ר...

ניסויי proteomic המשתמשים בספקטרומטר מסה (MS) יכולים להיות מתוכננים לשימוש או שאינם ממוקד (רובה) או שיטות ממוקדות. פרוטאומיקה גילוי בדרך כלל מסתמכת על הרובה מלמטה למעלה MS, או על ידי שימוש במצב רכישת הנתונים תלויים מסורתי, או על ידי שימוש באחת מטכניקות הנתונים עצמאיים שפותחו לאחרונה (למשל, MS E, רצועה) 1,2. פרוטאומיקה רובה היא כלי רב עוצמה לזיהוי פפטיד תפוקה גבוהה וכימות יחסי, אבל זה בדרך כלל אינו מתאים לכימות מוחלט או למיקוד קבוצות קטנות, מוגדרים (~ עשרות) של חלבונים. שיטת MS משמשת לרוב לפרוטאומיקה ממוקדת נבחרה תגובת ניטור (SRM) בגלל הרגישות גבוהה, המהירות, והטווח הדינמי שלה 3-5. חלופות לSRM כוללות ניטור תגובה מקביל, שמנצל ברזולוציה גבוהה, סריקה מלאה MS 6. SRM מבוצע בדרך כלל באמצעות ננו-זרימת reverכרומטוגרפיה SED-שלב ביצועים גבוהים נוזלי (ננו-RP-LC) מכשיר מצמידים את יינון ננו electrospray יון מקור (ננו &#39;חדשות ארכיאולוגיות) מחובר לספקטרומטר מסת quadrupole משולש (QQQ-MS). בניסוי טיפוסי, חלבוני מדגם proteolytically מתעכלים, ופפטידים וכתוצאה מכך chromatographically מופרדים, desorbed, ומיוננים. היונים מבשר וכתוצאה מכך הם מ &#39;/ z מסונן על ידי quadrupole הראשון (Q1) ומקוטע בquadrupole השני (Q2) על ידי התנגשותם עם גז התנגשות. יוני שבר כתוצאה הם מ &#39;/ בquadrupole השלישי (Q3) ולכמת ידי dynode-מסונן z. כל זוג מבשר ויון בר נקרא מעבר, וכל מעבר הוא פיקוח לתקופה קצובה בזמן (הזמן להתעכב, בדרך כלל 2-50 אלפיות שנייה). במהלך LC-SRM, מחזורי QQQ-MS באמצעות רשימה מוגדרת מראש של מעברים (מחזור העבודה היא בדרך כלל ≤3 שניות), וchromatogram של כל מעבר מיוצר. strateg האלטרנטיביies לכימות חלבון בדרך כלל להשתמש immunoassays כגון כתמי נקודה, כתמים מערביים, ELISAs, microarrays נוגדן, microarrays חלבון שלב הפוך, immunoassays microfluidic, ELISAs הדיגיטלי, וimmunoassays מבוסס microsphere 7. Immunoassays הטוב ביותר יכול להיות משמעותי יותר רגיש מאשר LC-SRM, ותפוקת מדגם של immunoassays יכולה להיות גבוהה באופן משמעותי מזה של LC-SRM 5. עם זאת, immunoassays פיתוח יכול להיות יקר ו / או זמן רב, וכתוצאה מכך יכולים להיות מבחני פגיעים לתגובה צולבת ו / או הפרעות, עולים בקנה אחד עם תא / תמוגה רקמה / שיטות הומוגניות, ו / או לא נוח לריבוב 5,8. חלק מבעיות אלה ניתן לטפל על ידי צימוד טכניקות antibody- ומבוסס MS. לדוגמא, ניתן להעשיר חלבוני היעד באמצעות immunoprecipitation לפני proteolysis וLC-SRM 9-12. לחלופין, טכניקת SISCAPA מעסיקה immunoprecipitation לאחר proteolysis בleve פפטידl 13,14. בנוסף לimmunoenrichment אסטרטגיות, immunodepletion של חלבוני שפע גבוהים יכול להיות מועסק על מנת להגביר את רגישות LC-SRM על ידי צמצום הפרעות על ידי coeluting analytes 15,16. כימות חלבון מבוסס MS יכול להיות מחולק לכימות יחסי ומוחלט, וגם לתיוג ויציב ללא תווית איזוטופ (למשל, תיוג חילוף חומרים, תיוג כימי, וחלבון שכותרתו כבד ופפטיד סטנדרטים פנימיים). טכניקות ללא תווית יכולות להיות שימושיות עבור כימות חלבון יחסית, אך אינן מתאימים לכימות מדויק מוחלט. לשם השוואה, בטכניקות תיוג צמצמו שגיאה הקשורות להכנת מדגם ושונות MS, ולעתים קרובות משמשות לכימות חלבון ביחס 17. לדוגמא, proteome איזוטופ היציבה שכותרת סטנדרטים (SILAP) מוכנים באמצעות שורת תאי אדם בתרבית אפשרה כימות יחסי של סמנים ביולוגיים פוטנציאליים באמצעות LC-SRM של סרום אנושי 18. כימות מדויק חלבון מוחלט על ידי MS דורש כי סטנדרטים פנימיים חלבון מטוהר, לכמת, isotopically כותרת או הפפטיד להיות ממוסמרים-לדגימות ביולוגיות לפני טרשת נפוצה. השילוב של סטנדרטים הפנימיים שכותרתו איזוטופ הכבד לעבודת LC-SRM מאפשר כימות מוחלטות שהוכחה להיות לשחזור והעברה בין מעבדות 16,19 מאוד. הסטנדרטים הפנימיים שכותרתו איזוטופ היציב לכימות חלבון מוחלט על ידי MS כוללים תקני פפטיד מוכנים באמצעות סינתזה מוצקה שלב 20, חלבונים מורכבים מתקני פפטיד בשרשור פרוטאז-cleavable 21, וסטנדרטים חלבון באורך מלא 22. יעד שינוי קוולנטיים חלבון והכנת מדגם חלקי (כלומר, תמוגה שלמה מדגם ולתפעול, וsolubilization חלבון שלם, denaturation, אלקילציה, וproteolysis) יכול לערער כימות מדויק. עמ &#39;הפנימיהסטנדרטים rotein הם הפחות סביר להיות מושפע מרוב הבעיות הפוטנציאליות אלה, אבל הם בדרך כלל הקשים ביותר להכנה. אלטרנטיבה היא לנתח כל חלבון מטרת שימוש בתקני פפטיד פנימיים רבים אשר נועדו כוללת שאריות ילידי איגוף amino- וcarboxy המסוף. לא משנה איזה סוג של התקן הפנימי מועסק, זה צריך להיות ממוסמר-לדגימות ביולוגיות כבתחילת שלב במהלך הכנת מדגם ככל האפשר. כמו כן, טכניקות מדגם מרובה הכנה (למשל, תנאי denaturation שונים) צריכים להיבדק. השימוש בטכניקות מרובות המאונכות ניסיוניות (אימות צולבת ניסיונית) הוא אסטרטגיה מעשית להתגברות על רוב כימות פוטנציאל אתגרי 23-25. כימות LC-SRM של חלבונים הוא טכניקה גמישה מאוד שכבר בשימוש במגוון רחב של יישומים. יש לציין, זה כבר נעשה שימוש כדי ללמוד סמנים ביולוגיים פפטיד וחלבונים בתוךדגימות קליניות כגון סרום, ביופסיות ליבה, וaspirates מחט הדק 5. LC-SRM שימש גם כדי למדוד את ההרכב של קומפלקסי חלבונים 5,26, כדי לזהות עצביים בוטולינום 27, לכמת דינמיקת זרחון החלבון בתוך מסלולי איתות 5, ולכמת שינויים בקונפורמציה חלבון 28. המעבדה שלנו היא באמצעות LC-SRM לכמת את חלבוני האיתות שלתווך chemotaxis מקרופאג כדי לתמוך בפיתוח של סימולציות מסלול chemotaxis. התכנית הכוללת של הפרוטוקול (איור 1) מתחילה בדירוג פפטידים היעד המהוסס. בהמשך לכך, סטנדרטים פפטיד חיצוניים גולמיים מסונתזים ומשמשים לפיתוח מבחני LC-SRM לניתוחים איכותיים של דגימות ביולוגיות. אם פפטיד היעד נגזר מדגם הביולוגי מזוהה, הסטנדרטים פפטיד מטוהרים שכותרתו כבדה פנימיים מוכנים לLC-SRM כמותיים. פרוטוקול זה יכול לשמש כדי ACצורה מדויקת לכמת חלבונים ממגוון רחב של דגימות ביולוגיות, ולתמוך בחקירות במגוון רחב של יעדי חלבון.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Acetonitrile (ACN), LC-MS grade</td>
			<td>Fisher</td>
			<td>A955-1</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA (bicinchoninic acid) protein assay kit</td>
			<td>Fisher</td>
			<td>23235</td>
			<td></td>
		</tr>
		<tr>
			<td>Beads for bead beating, zirconia-silica, 0.1mm</td>
			<td>BioSpec Products</td>
			<td>11079101z</td>
			<td></td>
		</tr>
		<tr>
			<td>Bestatin hydrochloride</td>
			<td>Sigma</td>
			<td>B8385-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture DMEM (with glucose, without L-glutamine)</td>
			<td>Lonza</td>
			<td>12-614F</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture EDTA, 500mM, pH8</td>
			<td>Gibco</td>
			<td>15575</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture fetal bovine serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S11550</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture L-glutamine</td>
			<td>Sigma</td>
			<td>G8540-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture phosphate buffered saline (PBS) pH 7.4</td>
			<td>Gibco</td>
			<td>10010-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture Trypan Blue viability stain, 0.4% w/v</td>
			<td>Lonza</td>
			<td>17-942E</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellometer Auto T4 cell counter</td>
			<td>Nexcelom Bioscience</td>
			<td>Cellometer Auto T4</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellometer Auto T4 disposable counting chambers</td>
			<td>Nexcelom Bioscience</td>
			<td>CHT4-SD100-014</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Sigma</td>
			<td>D5545-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid, LC-MS grade, ampules</td>
			<td>Fisher</td>
			<td>A117-10X1AMP</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer, Neubauer-improved, 0.1mm deep</td>
			<td>Marienfeld-Superior</td>
			<td>0640030</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES, 1M, pH 7.2</td>
			<td>Mediatech</td>
			<td>25-060-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid, 37% w/w</td>
			<td>VWR</td>
			<td>BDH3028-2.5LG</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>Sigma</td>
			<td>I1149-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Based Micropipette Puller</td>
			<td>Sutter Instrument Co.</td>
			<td>P-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>LC Magic C18AQ, 5&#181;m, 200&#8491;, loose media</td>
			<td>Michrom Bioresources</td>
			<td>PM5/61200/00</td>
			<td></td>
		</tr>
		<tr>
			<td>LC Halo ES-C18, 2.7&#181;m, 160&#8491;, loose media</td>
			<td>Michrom Bioresources</td>
			<td>PM3/93100/00</td>
			<td></td>
		</tr>
		<tr>
			<td>LC coated silica capillary, 50&#181;m id</td>
			<td>Polymicro Technologies</td>
			<td>1068150017</td>
			<td></td>
		</tr>
		<tr>
			<td>LC vial, autosampler, 12x32mm polypropylene</td>
			<td>SUN SRI</td>
			<td>200-268</td>
			<td></td>
		</tr>
		<tr>
			<td>LC vial screw cap, autosampler, pre-slit PTFE/silicone</td>
			<td>SUN SRI</td>
			<td>500-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Luciferase, from Photinus pyralis</td>
			<td>Sigma</td>
			<td>L9506-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepstatin A</td>
			<td>EMD Millipore</td>
			<td>516481-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>pH strips colorpHast (pH 0.0-6.0)</td>
			<td>EMD Chemicals</td>
			<td>9586-1</td>
			<td></td>
		</tr>
		<tr>
			<td>PhosStop phosphatase inhibitor cocktail</td>
			<td>Roche</td>
			<td>04906837001</td>
			<td></td>
		</tr>
		<tr>
			<td>RapiGest SF</td>
			<td>Waters</td>
			<td>186001861</td>
			<td></td>
		</tr>
		<tr>
			<td>Sep-Pak SPE, C18 1ml 100mg cartridge</td>
			<td>Waters</td>
			<td>WAT023590</td>
			<td></td>
		</tr>
		<tr>
			<td>Sep-Pak SPE, extraction manifold, 20 position</td>
			<td>Waters</td>
			<td>WAT200609</td>
			<td></td>
		</tr>
		<tr>
			<td>Sep-Pak SPE, flat-surfaced rubber bulb</td>
			<td>Fisher</td>
			<td>03-448-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Fisher</td>
			<td>S318-500</td>
			<td></td>
		</tr>
		<tr>
			<td>SpeedVac vacuum concentrator</td>
			<td>Fisher</td>
			<td>SPD111V</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid (TFA), LC-MS grade</td>
			<td>Fisher</td>
			<td>A116-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin, sequencing grade, modified</td>
			<td>Promega</td>
			<td>V5113</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube decapper for Micronic tubes</td>
			<td>USA Scientific</td>
			<td>1765-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubes, 2ml microcentrifuge, o-ring screw-cap, sterile</td>
			<td>Sarstedt</td>
			<td>72.694.006</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma</td>
			<td>U0631-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>Water, LC-MS grade</td>
			<td>Fisher</td>
			<td>W6-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

הפיתוח של מודלים חישוביים חזוי של מסלולי העברת אותות הוא אחת ממטרות היסוד של ביולוגיה מערכות 53. למרבה הצער, גם למסלולי איתות שנחקרו רב ויש לו משמעות קלינית גבוהה, זה עדיין בדרך כלל לא ניתן לחזות התנהגות כמותית מסלול בתגובה להפרעות (למשל, זה נכון לMAPK ERK המסלו?...

Watch this Scientific Journal Video about Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification at JoVE.com

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

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 ...

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16.9K Views.  National Institutes of Health. 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.

Video: Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

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 ...

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 ...

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

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N2) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (&Omega;). 
Recently, IM spectrometry was coupled with mass spectrometry and a traveling-wave (T-wave) Synapt ion mobility mass spectrometer (IM-MS) was released. Integrating mass spectrometry with ion mobility enables an extra dimension of sample separation and definition, yielding a three-dimensional spectrum (mass to charge, intensity, and drift time). This separation technique allows the spectral overlap to decrease, and enables resolution of heterogeneous complexes with very similar mass, or mass-to-charge ratios, but different drift times. Moreover, the drift time measurements provide an important layer of structural information, as &Omega; is related to the overall shape and topology of the ion. The correlation between the measured drift time values and &Omega; is calculated using a calibration curve generated from calibrant proteins with defined cross-sections1.
The power of the IM-MS approach lies in its ability to define the subunit packing and overall shape of protein assemblies at micromolar concentrations, and near-physiological conditions1. Several recent IM studies of both individual proteins2,3 and non-covalent protein complexes4-9, successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available)10. We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical &Omega; values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field.

Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.

גל T-Ion Mobility-ספקטרומטריית מסה: הפרוצדורות בסיסי לניתוח חלבונים מורכבים

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. ...

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 ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

זיהוי של קומפלקסי חלבונים ב<em&gt; חיידקי Escherichia</em&gt; באמצעות טיהור זיקת פפטיד סדרתית בשילוב עם טנדם המוני ספקטרומטריית

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

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

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

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

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

פרוטוקול לזיהוי של אינטראקציות בין חלבונים על הסמך<sup&gt; 15</sup&gt; תיוג N מטבולית, immunoprecipitation, כמותית המונית ספקטרומטריית וזיקת ​​אפנון

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

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

העשרה תאית מטריקס חלבונים מרקמות ועיכול לפפטידים לניתוח ספקטרומטריית המסה

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

Combining Chemical Cross-linking and Mass Spectrometry of Intact Protein Complexes to Study the Architecture of Multi-subunit Protein Assemblies

Proteins interact with their ligands to form active and dynamic assemblies which carry out various cellular functions. Elucidating these interactions is therefore fundamental for the understanding of cellular processes. However, many protein complexes are dynamic assemblies and are not accessible by conventional structural techniques. Mass spectrometry contributes to the structural investigation of these assemblies, and particularly the combination of various mass spectrometric techniques delivers valuable insights into their structural arrangement.
In this article, we describe the application and combination of two complementary mass spectrometric techniques, namely chemical cross-linking coupled with mass spectrometry and native mass spectrometry. Chemical cross-linking involves the covalent linkage of amino acids in close proximity by using chemical reagents. After digestion with proteases, cross-linked di-peptides are identified by mass spectrometry and protein interactions sites are uncovered. Native mass spectrometry on the other hand is the analysis of intact protein assemblies in the gas phase of a mass spectrometer. It reveals protein stoichiometries as well as protein and ligand interactions. Both techniques therefore deliver complementary information on the structure of protein-ligand assemblies and their combination proved powerful in previous studies.

The architecture of protein complexes is essential for their function. Combining various mass spectrometric techniques proved powerful to study their assembly. We provide protocols for chemical cross-linking and native mass spectrometry and show how these complementary techniques help to elucidate the architecture of multi-subunit protein assemblies.

שילוב cross-linking כימי ספקטרומטר מסה של מתחמי חלבון שלם ללמוד את הארכיטקטורה של יחידת משנה רב חלבון להרכבות

Proteins interact with their ligands to form active and dynamic assemblies which carry out various cellular functions. Elucidating these interactions is ...

Absolute Quantification of Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse Transcription PCR

RT-qPCR is one of the most common methods to assess individual target miRNAs. MiRNAs levels are generally measured relative to a reference sample. This approach is appropriate for examining physiological changes in target gene expression levels. However, absolute quantification using better statistical analysis is preferable for a comprehensive assessment of gene expression levels. Absolute quantification is still not in common use. This report describes a protocol for measuring the absolute levels of plasma miRNA, using RT-qPCR with or without pre-amplification.
A fixed volume (200 &#181;L) of EDTA-plasma was prepared from the blood collected from the femoral vein of conscious cynomolgus monkeys (n = 50). Total RNA was extracted using commercially available system. Plasma miRNAs were quantified by probe-based RT-qPCR assays which contains miRNA-specific forward/reverse PCR primer and probe. Standard curves for absolute quantification were generated using commercially available synthetic RNA oligonucleotides. A synthetic cel-miR-238 was used as an external control for normalization and quality assessment. The miRNAs that showed quantification cycle (Cq) values above 35 were pre-amplified prior to the qPCR step.
Among the 8 miRNAs examined, miR-122, miR-133a, and miR-192 were detectable without pre-amplification, whereas miR-1, miR-206, and miR-499a required pre-amplification because of their low expression levels. MiR-208a and miR-208b were not detectable even after pre-amplification. Sample processing efficiency was evaluated by the Cq values of the spiked cel-miR-238. In this assay method, technical variation was estimated to be less than 3-fold and the lower limit of quantification (LLOQ) was 102 copy/&#181;L, for most of the examined miRNAs.
This protocol provides a better estimate of the quantity of plasma miRNAs, and allows quality assessment of corresponding data from different studies. Considering the low number of miRNAs in body fluids, pre-amplification is useful to enhance detection of poorly expressed miRNAs.

This report describes a protocol for measuring the absolute levels of plasma miRNA, using quantitative real-time reverse transcription PCR with or without pre-amplification. This protocol affords better understanding of the quantity of plasma miRNAs and allows qualitative assessment of corresponding data from different studies or laboratories.

כימות מוחלטת של פלזמה רמות MicroRNA בקופים Cynomolgus, באמצעות שעתוק במהופך בזמן אמת PCR כמותי

RT-qPCR is one of the most common methods to assess individual target miRNAs. MiRNAs levels are generally measured relative to a reference sample. This ...