המחברים מודים לד&quot;ר קנט סילי, מנהל המרכז לגילוי תרופות וחדשנות (CDDI) מתקן פרוטאומיקה ליבה באוניברסיטה של ​​דרום פלורידה לשימוש במתקן זה. עבודה זו נתמכה על ידי מענק NIH / NIDCD R01 DC004295 לBHAS

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המחברים מצהירים שום אינטרסים מתחרים.

<html> צעדי המפתח למקסם זיהוי חלבון מהאפיתל חושית השבלול הם: 1) שימוש בendoproteinases מרובה לעיכול, 2) שימוש בטכניקות הפרדה מרובות, ו3) ניצול של ספקטרומטר מסה ברזולוציה גבוהה. היישום של אנזימים מרובים מגדיל את מספר פפטידים ומשפר את כיסוי רצף חלבון, ומכאן שיפור במספר חלבונים המזוה...

פרוטאומיקה היא המחקר של מערכות ביולוגיות מורכבות על ידי ניתוח ביטוי חלבון, תפקוד, שינויים, ואינטראקציות 1. כמה שיטות כבר נוצלו לניתוח proteome של האוזן הפנימית, כולל נוגדן microarray 2, ג&#39;ל אלקטרופורזה דו ממדי 3-5, וDIGE 6. עם זאת, רק מספר מוגבל של חלבונים זוהה ואופיינו 2,7-10, בהשוואה לגנים מעל 10,000 ותגים הביעו רצף (ESTs) המזוהים באוזן הפנימית 11,12, הטרשת הנפוצה היא הטכניקה המקיפה ביותר והנפוץ לשימוש בפרוטאומיקה לאפיון חלבונים. ניתוח של דגימות proteomic מורכבות, כגון השבלול, יכול להיות מאתגר. עם זאת, שילוב של טכניקות הפרדה מרובות עם MS מאפשר זיהוי של מספר גדול יותר של פפטידים וחלבונים, בשל טווח ריכוז דינמי מוגבר וקיבולת שיא 13. chromatogra רב ממדיPHY מפחית תערובות חלבון מורכבות מאוד על ידי המאפשר שימוש במנגנוני ספיחה שונים. ישנן שתי גישות נפוצות MS ניתוח proteome, רובה ציד ופרוטאומיקה מלמטה למעלה. בפרוטאומיקה רובה הציד, תערובת של חלבונים שלמים מתעכלת enzymatically והמופרד באמצעות כרומטוגרפיה רב ממדית עם כרומטוגרפיה קטיון מטבע חזקה (SCX) ואחריו כרומטוגרפיה הפוכה-שלב נוזלי (RPLC) 14,15. פפטידים המופרדים חשופים לטנדם MS ומסד נתוני חיפוש 15. יתרון עיקרי של שיטה זו הוא כי אלפי חלבונים יכולים להיות מזוהה בניתוח יחיד והטכניקה היא מתאימה יותר לחלבונים בממברנה. בגישה מלמטה למעלה, תערובת החלבונים מופרדת, בדרך כלל על ידי אחד או אלקטרופורזה דו ממדים, ולהקות חלבון או כתמים הבודדים לגזור ומתעכלות עם אנזים כגון טריפסין, בדרך כלל כתוצאה מפפטידים מרובים. עם זאת, עוד יותר אחרוניםly פיתח גישת electrophoretic, המשמש בפרוטאומיקה מלמטה למעלה, הוא GELFrEE. טכניקה זו fractionates דגימות חלבון בנוזל שלב והופכת אותם פחות מורכב לפני הניתוח. טכניקה זו היא לשחזור, מציעה התאוששות חלבון גבוהה, ומפחיתה את החלוקה של חלבונים בשפע גבוהים בדגימות חלבון מורכבות 16. פפטידים, וכתוצאה מחלבונים הופרדו, נותחו על ידי MS, באמצעות טביעת אצבע פפטיד המונית או טנדם MS (MS / MS), כדי ליצור רצף תגים עבור מסד הנתונים חיפוש 17-19. חלק מן היתרונות העיקריים של שימוש בגישה מלמטה למעלה הוא היכולת להשיג הפרדות ברזולוציה גבוהה וכיסוי חלבון מקיף. פרוטאומיקה מלמטה למעלה היא הטכניקה הנפוצה ביותר בפרוטאומיקה 20, ולכן, כמה כלים לביואינפורמטיקה זמינים. בנוסף, ניתן להפריד חלבונים בתערובת מורכבת לפני העיכול, ולכן יש סיכוי גדול יותר של הזדהות. אחד האתגרים הגדוליםבשימוש באוזן הפנימית לניתוח proteomic הוא הגודל שלה קטן, נגישות מוגבלת, וגיוון סוג התא 21. בנוסף, חלבונים מרכזיים המבדילה את הפונקציונליות שלו, כגון תעלות יונים, מובילים וקולטנים, הם חלבונים בממברנה, אשר יכול להיות קשה לבודד 22. לכן, הכנה בעזרת מסנן מדגם (FASP) היא יתרון עבור ניתוחי proteomic של רקמות כי הם מוגבלים להפקת חלבון ושדורשת חומרי ניקוי לsolubilize קרומים 23. סינון זה מאפשר ניתוח MS של הממברנה וחלבונים מסיסים וליכולת לבודד פפטידים ממזהמים במשקל מולקולריים נמוכים 23,24. הפרוטוקול הנוכחי מתאר גישות proteomic נפוצות שמשולבות והותאמו לניתוח חלבונים הן מסיסים וקרום וכדי למקסם את מספר תעודות הזהות של חלבון מהאפיתל החושית השבלול. נתאר באמצעות פרוטאומיקה רובה עם FASP רב לעכליון, כרומטוגרפיה החלפת יונים, ברזולוציה גבוהה בטרשת נפוצה, וניתוח נתונים. בנוסף, נתאר פרוטאומיקה מלמטה למעלה עם GELFrEE, FASP רב לעיכול, ברזולוציה גבוהה בטרשת נפוצה, וניתוח נתונים.

<table border="0" cellpadding="0" cellspacing="0" width="749">
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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:264px;">8% Tris-acetate cartridge&#160;</td>
			<td style="width:159px;">Protein Discovery</td>
			<td style="width:133px;">42103</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>179124</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetonitrile</td>
			<td>Honeywell</td>
			<td>015-1L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AEBSF</td>
			<td>Calbiochem</td>
			<td>101500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium formate</td>
			<td>Fisher Scientific</td>
			<td>AC16861</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Aprotinin</td>
			<td>Calbiochem</td>
			<td>616370</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ASB-14&#160;</td>
			<td>Calbiochem</td>
			<td>182750-5GM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin</td>
			<td>BioRad</td>
			<td>500-0112</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">C18 column&#160;</td>
			<td>New Objective</td>
			<td>A25112</td>
			<td>75 &#956;m &#215; 10 cm&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DC Protein Assay</td>
			<td>BioRad</td>
			<td>500-0116</td>
			<td>Microplate Assay Protocol</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E9884</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Endoproteinase Lys-C</td>
			<td>Sigma-Aldrich</td>
			<td>P3428</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FASP Protein Digestion Kit</td>
			<td>Protein Discovery</td>
			<td>44250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formic acid&#160;</td>
			<td>Fluka</td>
			<td>94318</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GELFrEE Fractionation System</td>
			<td>Protein Discovery</td>
			<td>42001</td>
			<td>GELFrEE 8100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leupeptin</td>
			<td>Calbiochem</td>
			<td>108975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MacroSpin Column</td>
			<td>The Nest Group</td>
			<td>SMM SS18V</td>
			<td>Silica C18</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microcystin</td>
			<td>Calbiochem</td>
			<td>475815</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pepstatin</td>
			<td>Sigma-Aldrich</td>
			<td>P5318</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polysulfoethyl A Column</td>
			<td>The Nest Group</td>
			<td>202SE0503</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma-Aldrich</td>
			<td>L3771</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sonic Dismembrator&#160;</td>
			<td>Thermo Fisher</td>
			<td>15-338-53</td>
			<td>Model 100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>T6567</td>
			<td>Proteomics Grade</td>
		</tr>
	</tbody>
</table>

bottom-up and shotgun proteomics to identify a comprehensive cochlear proteome

פרוטאומיקה היא גישה נפוץ שיכול לספק תובנות לתוך מערכות ביולוגיות מורכבות. האפיתל החושית השבלול מכילה קולטנים שtransduce האנרגיה מכאנית של קול לאנרגית אלקטרו כימי מעובד על ידי מערכת עצבים ההיקפית ומרכזית. כמה טכניקות proteomic פותחו כדי ללמוד את שבלול האוזן פנימית, כגון אלקטרופורזה דו ממדי ג&#39;ל ההבדל (2D-DIGE), microarray נוגדן, וספקטרומטריית מסה (MS). הטרשת הנפוצה היא הכלי המקיף והמגוונים ביותר בפרוטאומיקה בשיתוף עם שיטות הפרדה יכולה לספק proteome מעמיקה של דגימות ביולוגיות. יש שיטות הפרדה בשילוב עם MS היכולת להעשיר דגימות חלבון, לזהות במשקל מולקולרי נמוך וחלבונים הידרופובי, ולזהות חלבונים בשפע נמוכים על ידי צמצום הטווח הדינמי proteome. ניתן ליישם אסטרטגיות עיכול שונות לכל lysate או לlysate חלבון המופרד כדי לשפר את פפטיד וחלבוניםכיסוי רצף. ניצול של טכניקות שונות הפרדה, כוללים חילופי חזקים קטיון (SCX), התהפך השלב (RP), ואלקטרופורזה-eluted ג&#39;ל הנוזלי מלכוד שבריר (GELFrEE) ניתן ליישם כדי להפחית את מורכבות מדגם לפני ניתוח MS לזיהוי חלבון.

כדי להשיג את proteome המקיפה ביותר של האפיתל החושית השבלול, נתיחת רקמות מהירה נדרשת לפני מיצוי חלבון והכנת מדגם. ניתן להשתמש בשתי שיטות proteomic, פרוטאומיקה רובה ומלמטה למעלה. כדי להכין את הדגימות לפרוטאומיקה רובה הציד, הליך עיכול FASP שימש כפי שמודגם באיור 1. שיטת FASP ?...

Watch this Scientific Journal Video about Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome at JoVE.com

Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome

ניתוח Proteome של האפיתל החושית השבלול יכול להיות מאתגר בשל גודלו הקטן ובגלל חלבוני קרום קשים לבודד ולזהות. הממברנה וחלבונים מסיסים שניהם יכולים להיות מזוהות על ידי שילוב של שיטות הכנה של מספר רב של טכניקות והפרדה יחד עם ספקטרומטריית מסה ברזולוציה גבוהה.

מלמטה למעלה ופרוטאומיקה Shotgun לזהות מקיף שבלול Proteome

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that ...

bottom-up-shotgun-proteomics-to-identify-comprehensive-cochlear

Research

JoVE Journal

Biology

18.6K Views.  University of South Florida.  ניתוח Proteome של האפיתל החושית השבלול יכול להיות מאתגר בשל גודלו הקטן ובגלל חלבוני קרום קשים לבודד ולזהות. הממברנה וחלבונים מסיסים שניהם יכולים להיות מזוהות על ידי שילוב של שיטות הכנה של מספר רב של טכניקות והפרדה יחד עם ספקטרומטריית מסה ברזו?...

Video: Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

בידוד ניתוח בתאי גזע hematopoietic מן השליה

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

Proteomics כדי לזהות חלבונים אינטראקציה עם ליגנד P2X2-מגודרת ערוצי קטיון

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

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 ...

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

חלבון טיהור אורתוגונלית בהנחיית תג זיקה קטנים Bispecific

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi הקרנת לזהות פנוטיפים Postembryonic ב ג elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

הבידוד של הריבוזום פוליפפטידים המתהווה כרוכים<em&gt; במבחנה</em&gt; כדי לזהות אתרים השהה של תרגום לאורך ה-mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

ייצור של<em&gt; Xenopus tropicalis</em&gt; ביצה מחלץ לזהות RNAs microtubule הקשורים

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

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 ...

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2 incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.

Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for &#62;6 months.

אינו אנזימטית, תרבות רקמות סרום ללא נגעי שד טרום פולשני לדור ספונטני של Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Xenopus laevis כמודל לזיהוי תרגום ירידת ערך

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...