Name: profiling thiol redox proteome using isotope tagging mass spectrometry
Uploaded: 2012-03-24T14:00:00
Duration: 12 min 7 s
Description: Watch this Scientific Journal Video about Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry at JoVE.com

המחברים מבקשים להודות ד&quot;ר גרג מרטין (אוניברסיטת קורנל) וקבוצתו למתן את המתח DC3000, זרעי עגבניות, וייעוץ. הם רוצים גם להודות לד&quot;ר Zhonglin מו לעזרה עם פרוטוקול DAB ואגף Proteomics במרכז הבינתחומי UF למחקר ביוטכנולוגיה לסיוע בפיתוח השיטה. פרוטוקול להפקת חלבונים שונה מ Hurkman ו טנקה 16. פרוטוקול על cysTMT תיוג, על שלבים 4 עד 6 הותאם על בסיס המוצר המקורי תרמו פישר סיינטיפיק פירס ידנית 17. עבודה זו מומנה על ידי הקרן הלאומית למדע (MCB 0818051 ל S חן).

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פרוטוקול זה מספק מידע על ביצוע מכתים DAB כמו גם cysTMT שכותרתו כימות ציסטאין חיזור. נהלים אלה מועילים לבחון ייצור של ROS, כמו גם השפעה על ויסות חלבון כאשר Solanum lycopersicum הוא מחוסן עם Pseudomonas syringae. השיטות שהוצגו בפרוטוקול זה מספק דרך לבחון ROS בדגימות עלים שלמים בצורה שגורמת את הכמות המינימלית של פגיעה רקמת העלה. הליך תיוג מספק דרך לבחון חלבונים מוסדר חמזור פוטנציאלי על ידי ניצול שיטת תיוג ציסטאין. זה מועיל כאשר בוחנים בשלב מוקדם של תגובת הלחץ. שיטות כגון תג איזוטופ מקודדת זיקה (ICAT) ו cysTMT ניתן להשתמש בבדיקת פוטנציאליים חלבונים מוסדרים חמזור של דגימות ביולוגיות. ICAT מאפשר תיוג והשוואה של שני מדגמים 12. שתי השיטות לתייג cysteines בחינם והוא יכול לשמש חלבון quantification 10,12. עם זאת, השיטה cysTMT מאפשר ירידה וריאציה ניסיוני וכן ריבוב 10. מספר התגים זמינים מאפשר לחוקרים כוללים משכפל או דוגמאות רבות בתכנון הניסוי שלהם. שיש דוגמאות יותר מספק פוטנציאל למספר גבוה יותר של חלבונים המזוהים. החיסרון העיקרי של השיטה cysTMT היא שזה פוגע באיכות הכוללת של זיהוי החלבון בגלל הצעדים העשרה סלקטיבית cysTMT שכותרתו למוצרי פפטידים (6.5-6.6). מספר פפטידים לזיהוי החלבון תלוי בעיקר במספר שאריות ציסטאין ברצף החלבון. בעיה זו ניתן להתגבר על ידי הגשת חלק מדגם tryptic לפני העשרה לזיהוי ספקטרומטריית מסה חלבון. בשל אופיו של עיצוב ניסיוני, כמו גם מנגנון תיוג כי שיטת cysTMT מנצל, צעדים מסוימים הן קריטיות. בעת ביצוע חלבון precipitation ו גלולה כביסות (3.9) חשוב לשמור על דגימות קרה כיסתה את הקרח כדי לצמצם פירוק חלבונים. במהלך תיוג cysTMT, הסרת מגיב צמצום (4.6) חשוב כי דגימות עשוי לעבור תיוג הפוך. תיוג הפוך אפשרי אם הפחתת מגיב נשאר המדגם. אם דגימות מופחתים לאחר תיוג, תג cysTMT ניתן להסיר. פעם התוויות נוספות דגימות, רמת ה-pH יש לבדוק (4.7) כדי שיהיה תיוג יעילות אופטימלית. בנוסף, ניתוח נתונים תלוי מה נדרש החוקר ואת המטרה הסופית באמצעות פרוטוקול. זה תלוי גם תוכנות בשימוש כתוכנה לכל אחד מהם יש אלגוריתמים שונים. ניסוי זה מנצל הפתוגן כמו elicitor לייצור משופר של מינים חמצון תגובתי ב עגבניות, עם זאת, אחרים תגובות מוסדרים חמזור ניתן למדוד בהתאם. זה עיצוב ניסיוני ניתנת להתאמה צמח המערכות האחרות של בעלי חיים. </p&gt;

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגי </td></tr><tr><td> MetroMix 500 </td><td> BWI חברות </td><td> TX-500 </td></tr><tr><td> 3,3 &quot;, Diaminobenzidine </td><td> Sigma-Aldrich </td><td> D8001 </td></tr><tr><td> ReadyPrep הפקת רצופות ערכת מגיב 3 </td><td> ביו רד </td><td> 163-2104 </td></tr><tr><td> CB-X חלבון assay </td><td> Geno טכנולוגיה </td><td> 786-12x </td></tr><tr><td> cysTMT ריאגנטים </td><td> Thermo Scientific מחקר פירס אבקות חלבון </td><td> 90071 </td></tr><tr><td> Laemmli מאגר לדוגמה </td><td> ביו רד </td><td> 161-0737 </td></tr><tr><td> Bio-Safe Comassie (G-250 הכתם) </td><td> ביו רד </td><td> 161-0786 </td></tr><tr><td> Microcon טור 3KD </td><td> Millipore </td><td> 42403 </td></tr><tr><td> משותקת נגד TMT שרף </td><td> Thermo Scientific מחקר פירס אבקות חלבון </td><td> 90076 </td></tr><tr><td> צנטריפוגה עמודה </td><td> Thermo Scientific מחקר פירס אבקות חלבון </td><td> 89896 </td></tr><tr><td> Proteopep II C18 עמודה </td><td> חדש המטרה </td><td> PFC7515-PP2-10 </td></tr><tr><td> NanoLC-1D HPLC </td><td> א.ב. Sciex </td><td> 90389 </td></tr><tr><td> LTQ Orbitrap XL </td><td> Thermo Scientific </td><td> 0020137580 </td></tr><tr><td> SpeedVac </td><td> Labconco </td><td> 7812013 </td></tr><tr><td> Discoverer Proteome 1.2 התוכנה </td><td> Thermo Scientific מחקר פירס אבקות חלבון </td><td></td></tr><tr><td> טריפסין </td><td> Promega </td><td> V5111 </td></tr><tr><td> Oakridge Tube צנטריפוגה </td><td> Thermo Scientific Nalgeנה החברה </td><td> 3139-0050 </td></tr><tr><td> Microcentrifuge צינור (2ml) </td><td> ארה&quot;ב מדעי </td><td> 1620-2700 </td></tr><tr><td> 12% מיני הפושט צורה ולובש צורה TGX Precast ג&#39;ל </td><td> ביו רד </td><td> 456-1043 </td></tr><tr><td> גבי טופס &gt; Bio-Safe Coomassie StainBottom של טופס </td><td> ביו רד </td><td> 161-0786 </td></tr><tr><td> TMT ערכת ההעשרה </td><td> Thermo Scientific מחקר פירס אבקות חלבון </td><td> 90077 </td></tr></tbody></table>

profiling thiol redox proteome using isotope tagging mass spectrometry

Pseudomonas syringae PV. עגבניות DC3000 זן לא רק גורם למחלות גרגר החיידקים Solanum lycopersicum אלא גם על מינים Brassica, כמו גם על thaliana ארבידופסיס, צמח צייתן גנטית 1,2 המארח. הצטברות של מינים החמצן מגיב (ROS) ב cotyledons מחוסן עם DC3000 מצביע על תפקידו של ROS במוות ויסות תא נמקי בעת מחלה חיידקית גרגר של עגבניות 3. מי חמצן, מרכיב של ROS, מופק לאחר חיסון של שיחי עגבניות עם Pseudomonas 3. מי חמצן ניתן לאתר באמצעות histochemical כתם diaminobenzidine 3&#39;-3 &quot;(DAB) 4. מכתים DAB מגיב עם מי חמצן כדי לייצר חום כתם 4 רקמת העלה. ROS יש תפקיד הרגולציה של הסביבה חמזור הסלולר, אשר יכול לשנות את הסטטוס חיזור של חלבונים מסוימים 5. ציסטאין היא חומצה אמינית חשובה רגישחמזור שינויים. תחת חמצון קל, חמצון הפיך של קבוצות ציסטאין sulfhydryl משמש חיישנים חמזור ו מתמרים האות המווסתים מגוון תהליכים פיזיולוגיים 6,7. טנדם המוניים תג (TMT) ריאגנטים לאפשר זיהוי משתף מרובב quantitation של חלבונים בדגימות שונות באמצעות טנדם ספקטרומטריית מסה 8,9. את תגובתי ציסטאין-TMT (cysTMT) ריאגנטים לאפשר תיוג סלקטיבי quantitation היחסי של ציסטאין המכיל פפטידים ממרחק של עד שש דגימות ביולוגיות. כל תג cysTMT isobaric יש אותה מסה הורה הנומינלי מורכב קבוצה sulfhydryl-reactive, הזרוע MS-נייטרלי spacer ואת הכתב MS / MS 10. לאחר תיוג, הדגימות היו נתונים העיכול פרוטאז. פפטידים ציסטאין, שכותרתו היו מועשר באמצעות שרף המכיל נוגדנים נגד TMT. במהלך ניתוח MS / MS, סדרה של יוני הכתב (כלומר, 126-131 דה) מופיעים באזור מסה נמוכה, מתן מידע על quantitation יחסית.זרימת עבודה יעילה להפחתת המורכבות המדגם, שיפור טווח דינמי ולומדים שינויים ציסטאין. כאן אנו מציגים ניתוח proteomic חיזור של עגבניות ה-pst DC3000 מטופלים (Rio Grande) עוזבת באמצעות טכנולוגיית cysTMT. שיטה זו תפוקה גבוהה יש פוטנציאל להיות מיושם על לימוד אחרים חמזור מוסדר תהליכים פיזיולוגיים.

Watch this Scientific Journal Video about Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry at JoVE.com

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

רמת תגובתי מינים החמצן הוא גבוה כאשר תאים נתקלים בתנאי עקה. כאן אנו מציגים דוגמה מכתים diaminobenzidine &#39;3&#39;-3, כמו גם תיוג cysTMT ו ספקטרומטריית מסה לפרופיל proteome חיזור ב<em&gt; Pseudomonas syringae</em&gt; טיפול עלים עגבניות.

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 ...

profiling-thiol-redox-proteome-using-isotope-tagging-mass-spectrometry

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Biology

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 ...

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 ...

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 ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (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 chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

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

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by 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 ...

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 ...

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 ...

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 ...

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ability to proliferate or they can differentiate and fuse into multinucleated myotubes, which maturate further to form myofibers. Skeletal muscle terminal differentiation is orchestrated by the coordinated action of various transcription factors, in particular the members of the Muscle Regulatory Factors or MRFs (MyoD, Myogenin, Myf5, and MRF4), also called the myogenic bHLH transcription factors family. These factors cooperate with chromatin-remodeling complexes within elaborate transcriptional regulatory network to achieve skeletal myogenesis. In this, MyoD is considered the master myogenic transcription factor in triggering muscle terminal differentiation. This notion is strengthened by the ability of MyoD to convert non-muscle cells into skeletal muscle cells. Here we describe an approach used to identify MyoD protein partners in an exhaustive manner in order to elucidate the different factors involved in skeletal muscle terminal differentiation. The long-term aim is to understand the epigenetic mechanisms involved in the regulation of skeletal muscle genes, i.e., MyoD targets. MyoD partners are identified by using Tandem Affinity Purification (TAP-Tag) from a heterologous system coupled to mass spectrometry (MS) characterization, followed by validation of the role of relevant partners during skeletal muscle terminal differentiation. Aberrant forms of myogenic factors, or their aberrant regulation, are associated with a number of muscle disorders: congenital myasthenia, myotonic dystrophy, rhabdomyosarcoma and defects in muscle regeneration. As such, myogenic factors provide a pool of potential therapeutic targets in muscle disorders, both with regard to mechanisms that cause disease itself and regenerative mechanisms that can improve disease treatment. Thus, the detailed understanding of the intermolecular interactions and the genetic programs controlled by the myogenic factors is essential for the rational design of efficient therapies.

MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.

זיהוי של Myod Interactome שימוש טיהור טנדם זיקה מצמידים ספקטרומטריית מסה

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ...

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM).

התאוששות נייד באמצעות פרופילים תוכן גבוהה מיקרוסקופית

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...