This work has been supported by the DFG grant JE 252/7.

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No conflicts of interest declared.

التوليف SPOT كما هو موضح هنا هو وسيلة قوية لرسم خريطة مواقع التفاعل البروتين البروتين والتحقيق في التعرف على الركيزة من الببتيد تعديل الانزيمات. ومع ذلك، SPOT التوليف لا يزال لديه بعض العيوب، لأنه على الرغم يتم الإبلاغ عن الببتيدات توليفها من خلال طريقة SPOT أن يكون أكثر م...

في العقدين الأخيرين، أظهرت عدة تقارير على أهمية التعديلات بعد متعدية (PTM) في التنمية الخلوية والعديد من الأمراض مثل السرطان، ولكن في الآونة الأخيرة البروتين يسين مثيلة قد ظهرت كعامل حيوي PTM آخر. في حين تم العثور على هيستون في البداية مثيلة ليسين ليكون علامة لونين الأساسية، وأظهر العمل في وقت لاحق أيضا يسين مثيلة من عدة بروتينات غير بسيطة 1-4. يتم تحفيز نقل متتابعة من مجموعات الميثيل من S-adenosyl-L-ميثيونين إلى مجموعة-ε الأمينية من بقايا يسين من قبل عائلة من الإنزيمات يسمى البروتين ليسين ميثيل (PKMTs) الذي يحتوي على أكثر من 60 البروتينات في الجينوم البشري. وقد اكتشفت في البداية PKMTs كما هيستون تعديل الانزيمات التي ميثيل بقايا يسين محددة ولكن أظهرت تقارير لاحقة أنها يمكن أيضا ميثيل البروتينات غير بسيطة 5. حتى الآن، تم تحديد ما يقرب من 5،000 مواقع مثيلة ليسين على البروتينات المختلفة 6 </sتصل&gt;، ولكن غالبا ما لا يتم التعرف على الانزيمات المسؤولة عن هذه التعديلات. وأحد أسباب ذلك هو أن خصوصية أكثر PKMTs لم يدرس على نطاق واسع. ولذلك، فإنه يحدث متكرر أن ركائز جديدة للPKMTs يتم اكتشافها. عدم وجود معرفة مفصلة من خصوصية الركيزة من PKMTs يعيق فهم وظيفتها البيولوجية ودورها. لدراسة خصوصية من PKMT بالتفصيل، يجب أن تقاس معدلات مثيلة من العديد من ركائز الببتيد التي تختلف في واحد أو عدد قليل من الأحماض الأمينية ومقارنة، والتي تتم بشكل مثالي باستخدام صفائف الببتيد. استنادا إلى ملامح خصوصية الناتجة عن ذلك، يمكن تحديد ركائز المحتملة للPKMTs التي يمكن مزيد من الدراسة. وتستخدم على نطاق واسع صفائف الببتيد أدوات لتحليل الكيمياء الحيوية من الأجسام المضادة، الببتيد الانزيمات تعديل ورسم خرائط لمواقع التفاعل البروتين البروتين (الضد مستضد، مستقبلات يجند) 7-9. وهناك حاجة إلى عدة مئات من الببتيدات لSUCح التطبيقات. وطرق المختلفة المتاحة لتخليق الببتيد، الببتيد التوليف بينها على الراتنج يستخدم عادة للغاية، ولكن له حدود في الإنتاجية وأنها مكلفة نسبيا. تم حل هذه القضايا مع إدخال طريقة تركيب SPOT فرانك وزملاؤه 10. يسمح للطريقة التوليف SPOT تجميع لعدة مئات من الببتيدات بالتوازي وفي المتوسط ​​أنها غير مكلفة مقارنة توليف الراتنج. الببتيدات توليفها على غشاء السليولوز يمكن استخدام إما مباشرة لمختلف التطبيقات أو الببتيدات يمكن المشقوق من الغشاء واستخدامها كأداة الببتيدات المجانية للفي المقايسات حل أو لإعداد ميكروأرس الببتيد 10-13. SPOT التوليف هو البديل من الببتيد مرحلة التوليف الصلبة، والذي يستخدم غشاء السليلوز كدعم قوي وتوظف القياسية Fmoc الكيمياء 10-13. وبالتالي، فإن تركيب سلاسل الببتيد يبدأ في نهاية-C محطة ويمضي نحونهاية-N محطة على النقيض من التوليف البيولوجي في ريبوسوم. وبين functionalized أغشية السليلوز لإلحاق أول الأحماض الأمينية تنشيط (الشكل 1). وتستند هذه الطريقة SPOT على تسليم متتابعة من الأحماض الأمينية تفعيلها في بقطرة من المذيب إلى مناطق محددة على الغشاء باستخدام نظام pipetting لالآلي. والاستغناء عن قطرة من السائل على غشاء مسامي حيث أنها تشكل بقعة دائرية الرطب، الذي يعمل في وقت لاحق كما مفاعل مفتوحا لتفاعلات كيميائية في التوليف الببتيد. يتم تحديد حجم بقعة على حجم الاستغناء والقدرة الاستيعابية للغشاء، يتم ترتيب مضاعفات مثل هذه البقع كما المصفوفات. حجم التوليف يرتبط حجم البقعة وقدرة التحميل من الغشاء. المسافة بين البقع وكثافة صفائف تدار من قبل متفاوتة الأحجام الفور. غشاء السليلوز لديها العديد من المزايا على النحو المرحلة الصلبة في التوليف الببتيد، أنها غير مكلفة، متسامح إلى كيميائياعتلال الأعصاب الحركية المستخدمة في تركيب الببتيد، مستقرة في المحاليل المائية وسهلة في التعامل معها. وبالإضافة إلى ذلك، طبيعته المحبة للماء يجعلها مناسبة لعدة أنظمة الفحص البيولوجية. يمكن أن يتم تركيب SPOT يدويا أو آليا (ل1000s من الببتيدات) اعتمادا على العدد المطلوب من الببتيدات. ويستخدم مركب SPOT مؤتمتة بالكامل من Intavis (كولن، ألمانيا) لطلباتنا. ويسمح تركيب الببتيدات في كميات مختلفة وطول مختلف. وقد تم تجميع الببتيدات الخطية بانتظام مع 15 إلى 20 الأمينية طول حامض، في الببتيدات إضافة ما يصل إلى 42 من الأحماض الأمينية يمكن أيضا أن تكون خطوة حكيمة التوليف 14،15 إعدادها. ومع ذلك، زيادة عدد الأحماض الأمينية يؤدي إلى تخفيضات في العوائد اقتران الشاملة، مما يؤثر على نوعية من الببتيدات. ونظرا لكمية قليلة من الببتيدات في بقعة، والمنتجات وغالبا ما تكون صعبة لتنقية ونوعية الببتيدات الفردية لا يمكن تقييمها بسهولة. ولذلك، فإن النتائج التي تم الحصول عليها من SPOT peptلا بد من تأكيد صفائف بيئة تطوير متكاملة سواء مع الببتيدات توليفها من قبل الأساليب القياسية في نطاق أوسع، والتي يمكن تنقيتها وتحليلها وفقا للمعايير في التوليف الببتيد أو عن طريق تجميع البروتينات التي تحتوي على سلاسل الببتيد المطلوب. ومع ذلك، وجدنا التوليف SPOT أن تكون موثوق بها للغاية ويؤدي عموما أن يكون استنساخه. لا يقتصر التوليف SPOT لproteinogenic الأحماض الأمينية، والعديد من الأحماض الأمينية تعديل المتاحة تجاريا كما يمكن استخدامها لتخليق، مما يسمح الببتيدات إلى أن يتم تعديل قبل وبعد الانقسام النهائي للمجموعة حماية الجانب السلسلة، وعلاوة على ذلك، فإنه يسمح أيضا إدماج فسفرته، مميثل أو الأسيتيل الأحماض الأمينية 11. مكتبات الببتيد ثبتوا توليفها من خلال طريقة SPOT يمكن استخدامها مباشرة للعديد من المقايسات البيولوجية والبيوكيميائية. نحن العاملين صفائف الببتيد تضم 300-400 الببتيدات للتحقيق في خصوصية ركيزة من PKMTs. لmodifi الأنزيميالموجبة، يتم تحضين صفائف الببتيد مع PKMT منها وصفت [ميثيل 3 H] -AdoMet في العازلة المناسبة. ويتم تحليل الحامض الركيزة منها باتباع نقل الأنزيمية للمجموعات الميثيل المسمى بالإشعاع من AdoMet إلى الركيزة الببتيد عبر تصوير الإشعاع الذاتي (الشكل 3). بواسطة هذا الإجراء، صفائف الببتيد تسمح دراسة مثيلة من ركائز الببتيد مختلفة في نفس الوقت. أحد المزايا المهمة لهذا الأسلوب هو أن جميع الببتيدات وميثليته في المنافسة، هذا إلى أنه خلال المرحلة خطية من حركية مثيلة، ومثيلة النسبي لكل الببتيد يتناسب مع معدل ثابت الحفاز مقسوما على ثابت التفكك (ك القط / K D) من انزيم لالببتيد الركيزة منها. ولذلك، يرتبط مقدار النشاط الإشعاعي إدراجها في كل بقعة مباشرة مع النشاط الأنزيمي نحو الببتيد معين. باستخدامنتائج تجربة مثيلة مجموعة الببتيد، والوضع خصوصية PKMT يمكن تعريف وبناء على هذه الرواية ركائز يمكن أن يستند. صفائف الببتيد تسمح التحقق من صحة كفاءة السريع وتكلفة مثيلة من ركائز جديدة على مستوى الببتيد. لهذا، يتم إعداد المصفوفات التي تحتوي على ركائز جديدة توقع مع الببتيدات المعدلة تحتوي على علاء بدلا من ليز في المواقع المستهدفة وكذلك الببتيدات الإيجابية والسلبية السيطرة. وأخيرا، فإن ركائز الرواية يمكن أن تكون على استعداد كما البروتينات مع المسوخ، والتي يتم تبديل اليس هدف لعلاء ويمكن التأكد من مثيلة في مستوى البروتين. اعتمادا على نتائج، وهذا هو ثم تليها الدراسات البيولوجية معالجة الأدوار المحتملة للمثيلة من ركائز البروتين وصفها حديثا.

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			<td height="21" style="height:21px;width:325px;">Name of Material/ Equipment</td>
			<td style="width:112px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:179px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol abs Gradient Grade HPLC</td>
			<td>Honeywell</td>
			<td>10299901</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N,N-Dimethylformamide Peptide Synthesis</td>
			<td>Biosolve</td>
			<td style="width:119px;">4193302</td>
			<td>Flammable, Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Piperidine &#8805; 99 % for Peptide Synthesis</td>
			<td>Roth</td>
			<td>A122.1</td>
			<td>Flammable, Toxic, corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Oxyma Pure (Ethyl (hydroxyimino)cyanoacetate )</td>
			<td>Novabiochem</td>
			<td>8510860100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">N,N&#8217;-Diisopropylcarbodiimide purum &#8805; 98% GC</td>
			<td>Fluka</td>
			<td>38370</td>
			<td>Flammable, Toxic, corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetic anhydride</td>
			<td>Roth</td>
			<td>CP28.1</td>
			<td>Flammable, Toxic, corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triisopropylsilane 99%,</td>
			<td>Aldrich</td>
			<td>233781</td>
			<td>Flammable, Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dichlormethane &#8805;99,9%</td>
			<td>Roth</td>
			<td>P089.1</td>
			<td>Carcinogenic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-Methyl-2-Pyrrolidone</td>
			<td>Roth</td>
			<td>4306.2</td>
			<td>Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Derivatized cellulose Membrane</td>
			<td style="width:112px;">Intavis AG K&#246;ln</td>
			<td style="width:119px;">32.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trifluoroacetic acid</td>
			<td>Roth</td>
			<td>P088.2</td>
			<td>Toxic, corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bromphenolblue</td>
			<td>AppliChem</td>
			<td>A3640.0010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium Hydrogen Carbonate</td>
			<td>Roth</td>
			<td>T871.2</td>
			<td>Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Dodecyl Sulfate Pellets</td>
			<td>Roth</td>
			<td>CN30.3</td>
			<td>Toxic, Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">MultiPep Synthesizer</td>
			<td style="width:112px;">Intavis AG K&#246;ln</td>
			<td style="width:119px;">n.a.</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">HyperfilmTM high performance film</td>
			<td style="width:112px;">GE Healthcare</td>
			<td style="width:119px;">28906837</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:325px;">Phoretix software</td>
			<td style="width:112px;">TotalLab</td>
			<td style="width:119px;">n.a.</td>
			<td style="width:179px;"></td>
		</tr>
	</tbody>
</table>

specificity analysis of protein lysine methyltransferases using spot peptide arrays

يسين مثيلة هو التعديل بعد الترجمة الناشئة وتم التعرف على العديد من هيستون وغير هيستون-البروتينات، حيث أنه يلعب دورا حاسما في تطوير خلية والعديد من الأمراض. وقد تم تحديد مواقع 5،000 مثيلة ليسين تقريبا على البروتينات المختلفة، وهي تحدد من قبل عدد قليل العشرات من ميثيل البروتين ليسين. وهذا يشير إلى أن كل PKMT methylates بروتينات متعددة، ولكن حتى الآن تم التعرف على واحد أو اثنين فقط ركائز للعديد من هذه الإنزيمات. تحليلات لنهج هذه المشكلة، أدخلنا مجموعة الببتيد تستند الركيزة خصوصية PKMTs. صفائف الببتيد هي أدوات قوية لتوصيف خصوصية PKMTs بسبب مثيلة من عدة ركائز مع سلاسل مختلفة يمكن اختبارها على مجموعة واحدة. نحن توليفها صفائف الببتيد على غشاء السليلوز باستخدام المزج Intavis SPOT وتحليلها خصوصية مختلف PKMTs. وبناء على هذه النتائج، بالنسبة للعديد من هذه الإنزيمات، رواية الصورةيمكن تحديد ubstrates. على سبيل المثال، لNSD1 من خلال توظيف صفائف الببتيد، أظهرنا أنه methylates K44 من H4 بدلا من H4K20 ذكرت وبالإضافة H1.5K168 هو الركيزة يفضل بدرجة كبيرة خلال H3K36 المعروفة سابقا. وبالتالي، صفائف الببتيد هي أدوات قوية لتوصيف كيميائيا وPKMTs.

كانت تستخدم صفائف الببتيد بنجاح لتوصيف كيميائيا خصوصية PKMTs، وتم تحديد عدة هيستون رواية وغير هيستون-ركائز من PKMT هذا النهج 5،16-19. تحديد الطيف الركيزة الصحيح لPKMT (أو أي انزيم) هو خطوة ضرورية نحو فهم آليتها الجزيئية والخلوية وظائف. <p class="jove_content" style=";text-align:right;direction:r...

Watch this Scientific Journal Video about Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays at JoVE.com

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

Peptide arrays synthesized by the SPOT method can be used to analyze the substrate specificity of Protein lysine methyltransferases (PKMTs) and to define the substrate spectrum of PKMTs to understand their biological role. This protocol describes how to synthesize peptide arrays, methylate them with PKMTs, and analyze the results.

تحليل خصوصية البروتين ليسين ميثيل عن طريق SPOT الببتيد صفائف

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays ...

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Research

JoVE Journal

Biology

13.8K Views.  Stuttgart University. Peptide arrays synthesized by the SPOT method can be used to analyze the substrate specificity of Protein lysine methyltransferases (PKMTs) and to define the substrate spectrum of PKMTs to understand their biological role. This protocol describes how to synthesize peptide arrays, methylate them with PKMTs, and analyze the results.

Video: Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

تلطيخ من البروتينات في الهلام مع Coomassie G - 250 دون عضوي حمض الخليك والمذيبات

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

باستخدام نظام التقطير GELFREE 8100 لتقطير الوزن الجزيئي وبناء مع مرحلة الانتعاش السائل

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

الطفرات والتحليل الوظيفي للقنوات ايون أعربت Heterologously في خلايا الثدييات

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

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.

الكمي من البروتينات عن طريق التخصيب الببتيد Immunoaffinity اقترن الطيف الكتلي

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

Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small molecule studies and drug development 4. Many epigenetic regulators have only recently been discovered and are still in the process of being classified. Among these enzymes are lysine demethylases which remove methyl groups from lysines on histones and other proteins. Due to the novel nature of this class of enzymes, few assays have been developed to study their activity. This has been a road block to both the classification and high throughput study of histone demethylases. Currently, very few demethylase assays exist. Those that do exist tend to be qualitative in nature and cannot simultaneously discern between the different lysine methylation states (un-, mono-, di- and tri-). Mass spectrometry is commonly used to determine demethylase activity but current mass spectrometric assays do not address whether differentially methylated peptides ionize differently. Differential ionization of methylated peptides makes comparing methylation states difficult and certainly not quantitative (Figure 1A). Thus available assays are not optimized for the comprehensive analysis of demethylase activity.
Here we describe a method called MassSQUIRM (mass spectrometric quantitation using isotopic reductive methylation) that is based on reductive methylation of amine groups with deuterated formaldehyde to force all lysines to be di-methylated, thus making them essentially the same chemical species and therefore ionize the same (Figure 1B). The only chemical difference following the reductive methylation is hydrogen and deuterium, which does not affect MALDI ionization efficiencies. The MassSQUIRM assay is specific for demethylase reaction products with un-, mono- or di-methylated lysines. The assay is also applicable to lysine methyltransferases giving the same reaction products. Here, we use a combination of reductive methylation chemistry and MALDI mass spectrometry to measure the activity of LSD1, a lysine demethylase capable of removing di- and mono-methyl groups, on a synthetic peptide substrate 5. This assay is simple and easily amenable to any lab with access to a MALDI mass spectrometer in lab or through a proteomics facility. The assay has ~8-fold dynamic range and is readily scalable to plate format 5.

We present a method for using MALDI mass spectrometry and reductive methylation chemistry to quantify changes in lysine methylation.

تطبيق MassSQUIRM لقياسات كمية من نشاط Demethylase ليسين

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small ...

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.

تحديد بروتين في مجمعات<em&gt; كولاي</em&gt; استخدام المتعاقبة تنقية تقارب الببتيد في تركيبة مع جنبا إلى جنب الطيف الكتلي

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

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

A تجليد الفحص إنتاجية عالية MHC II لتحليل الكمي لالببتيد الحواتم

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

تحديد مواقع التفاعل البروتين البروتين عن طريق الببتيد صفائف

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

الأخضر الفلورسنت القائم على البروتين التعبير فحص غشاء البروتينات في<em&gt; الإشريكية القولونية</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating interactions between DNA and its binding proteins. Here, we report a method that uses fluorescent microscopy for visualizing large DNA molecules tethered on the surface. First, glass coverslips are biotinylated and passivated by coating with biotinylated polyethylene glycol, which specifically binds biotinylated DNA via avidin protein linkers and significantly reduces undesirable binding from non-specific interactions of proteins or DNA molecules on the surface. Second, the DNA molecules are biotinylated by two different methods depending on their terminals. The blunt ended DNA is tagged with biotinylated dUTP at its 3' hydroxyl terminus, by terminal transferase, while the sticky ended DNA is hybridized with biotinylated complimentary oligonucleotides by DNA ligase. Finally, a microfluidic shear flow makes single DNA molecules stretch to their full contour lengths after being stained with fluorescent protein-DNA binding peptide (FP-DBP).

We present an approach for visualizing fluorescent protein DNA binding peptide (FP-DBP)-stained large DNA molecules tethered on the polyethylene glycol (PEG) and avidin-coated glass surface and stretched with microfluidic shear flows.

التصور-المربوطة السطحية الكبيرة جزيئات الحمض النووي مع الحمض النووي البروتين الفلوري ملزم الببتيد

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating ...