Work in the Ait-Si-Ali lab was supported by the Association Fran&#231;aise contre les Myopathies T&#233;l&#233;thon (AFM-T&#233;l&#233;thon); Institut National du Cancer (INCa); Agence Nationale de la Recherche (ANR), Fondation Association pour la Recherche sur le Cancer (Fondation ARC); Groupement des Entreprises Fran&#231;aises pour la Lutte contre le Cancer (GEFLUC); CNRS; Universit&#233; Paris Diderot and the ''Who Am I?'' Laboratory of Excellence #ANR-11- LABX-0071 funded by the French Government through its ''Investments for the Future'' program operated by the ANR under grant #ANR-11-IDEX-0005-01. EB was supported by an INCa grant.

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The authors have nothing to disclose.

제시된 방법은 전사 인자, MyoD의 파트너 철저한 식별을 허용한다. 그것은 MyoD 유도 분화, 즉 헬라-S3 세포에 강한 이종 시스템에서 MyoD 파트너를 한 것으로 밝혀졌습니다. 따라서, 정의, 식별 MyoD 파트너는 보편적으로 표현된다. 이러한 일반 및 서열 - 특이 적 전사 요소, 염색질 변형 효소, RNA 프로세싱 단백질 키나제 (표 1 및 2)를 포함한다. 헬라-S3 세포 MyoD 유도 트랜스 분화...

진핵 다세포 생물은 다른 기관 및 조직 구성된다. 각 기능 조직 각 분화 단계에서 결정되는 특정 유전자의 발현 패턴을 갖는다. 세포 분화 유전자 세트 세포 증식에​​ 관여하는 등 사람들의 침묵, 일반적으로, 특정 유전자의 활성화, 발현의 유지를 포함하고. 골격근 분화 또는 근육 생성, 즉 근육 아세포로 중배엽 간세포의 결정으로 시작 따라서 다단계 공정, 그 후 제 모노 - 핵 생성하고 다중 핵, myotubes에서 이러한 아세포의 말단 분화를 유도 . 따라서, 근육 아세포는 계속 증식 할 수있는 세포를, &quot;결정&quot;되어 있지만, 그들은 배아 개발하는 동안 또는 성인 근육 재생에 어느 골격 근육 세포에 전적으로 구별하실 수 있습니다 따라서 골격 근육 계통 위해 노력하고 있습니다. 골격근 단말의 처리가 다를entiation 그러한 E2F 대상 유전자 1과 증식 관련 유전자의 결정적인 사일런 리드 근원 세포 전구체 세포의 세포주기에서 영구 출구에서 시작하여 특정 유전자 프로그램 조율된다. 실제로 단말 분화 과정에서, 근육 모세포 증식 정지 골격근 특정 유전자의 발현 및 (2)에 myotubes 아세포의 융합 선행 중요한 단계이다. 이러한 프로그램은 골격근 손상 후 재생 공정 동안에 분화하는 성체 근육 줄기 세포라고도 위성 세포를 허용한다. 포유 동물의 근육 생성이 근육 조직의 기본 나선 루프 나선의 가족에 결정적으로 의존 (bHLH) 전사 자주 골격 근육의 결정 요인 또는 MRFs (근육 규제 요인) (3)의 가족이라 MyoD, Myf5, MRF4 및 오게 닌을, 요인. 그들 각각의 사양 및 DIF에 필수적인 역할을한다골격 근육 세포의 ferentiation과 특정 식 패턴 (4) 5-7 있습니다. Myf5 및 MyoD의 활성화는 근육 조직 계보에 세포를 커밋 결정적인 단계를 구성하고 오게 닌의 후속 표현은 MCK (근육 크레아틴 키나제) 등의 골격근 특정 유전자의 활성화와 근육 생성을 트리거합니다. 근육 조직 bHLH의 전사 인자는 이전에 침묵 궤적 8에서 근육 유전자의 활성화에 MEF2 가족의 구성원들과 협력한다. 또한 다양한 유전자 조절 영역 (8)에서 이른바 E-E 박스 결합 단백질로 알려진 유비쿼터스 bHLH 단백질, E12 및 E47,와 같은 이종 골격근 유전자 전사를 자극한다. 트위스트 이드 (분화 억제제) 및 기타 요인에 부정적인 8 바인딩 E 단백질에 대한 MyoD와 경쟁하여,이 과정을 조절한다. MyoD 근육 터미널 차별화 9 트리거에서 주요 플레이어로 간주됩니다 </s업&gt; 그것은 많은 완전 비 분화 된 근육 시스템 10-13의 근원 성 결정 / 분화 (트랜스 분화) 프로그램을 유도하는 능력을 가지고 있기 때문이다. 실제로, MyoD의 강제 표현은 서로 다른 세포 유형의 다른 태생 학적 기원 (12)로부터 유래도 사람들의 트랜스 분화를 유도한다. 예를 들어, MyoD 근육과 같은 세포로 간세포, 섬유 아 세포, 멜라닌 세포, neuroblasts 및 지방 세포로 변환 할 수 있습니다. MyoD의 트랜스 differentiative 작업은 원래의 유전 적 프로그램 (특히, 증식 유전자)의 침묵에 수반 비 근육 환경에서 근육 조직 유전 적 프로그램의 비정상적인 활성화 (특히 그 표적 유전자)를 포함한다. 근육 아세포 증식에 MyoD 표현하지만 그들의 발기인 14 ~ 16에 결합하는 경우에도 그 표적 유전자를 활성화 할 수 없습니다입니다. 따라서, MyoD가 지속적으로 미분화 된 근육 아세포에서 발현되는 대한 요구 사항은 매우 ELU 유지시브. MyoD 이전 염색질 리모델링을 활성화하는 14,17 효소의 로딩에 근육 아세포 증식에 억압 염색질 - 수정 효소의 채용으로 인해 그 표적 유전자를 억제 할 수있다. 예를 들어, 근육 아세포 증식에​​ MyoD은 히스톤 H3 라이신 9 H3K9 및 H3K27 KMTs 포함한 전사 공동 리프레 KAP-1, 히스톤 데 아세틸 라제 (HDACs) 및 억압 리신 methyltransferases (KMTs)와 연관되고, 적극적으로 표적 유전자의 발현을 억제한다 로컬 억압 크로 마틴 구조 14,17을 설정하여. 중요한 것은, 최근의 보고서는 MyoD 자체가 직접 H3K9 KMT G9a가 transactivating 활동 (16)의 억제 결과에 의해 메틸화 인 것으로 나타났다. MyoD 의한 비 근육 세포의 이러한 트랜스 분화에 관련된 후성 메커니즘은 대부분 알려져 있지 않다. 특히, 일부 세포주 MyoD 유도 트랜스 분화에 내성이다. 따라서, HeLa 세포에서 MyoD은 하나 또는 비활성심지어 인해 복잡한 SWI / SNF (18) 리모델링 염색질의 BAF60C 서브 유닛의 표현의 부족으로 리프레보다는 전사의 활성화로 작동 할 수 있습니다. 이 모델은 따라서 더 나은 MyoD 유도 유전자 억압의 메커니즘을 특성화하는 선택이 될 수 있습니다. 관련 파트너와 목표 궤적에서 억압 염색질 환경을 유도하기 때문에 터미널 차별화를 미세 조정하는 근육 아세포 증식에 MyoD 종속 억압 메커니즘을 밝히기 위해서 MyoD의 능력을 분석하는 것이 적합하다. 여기에서 우리는 질량 분석기 (MS)의 특성에 결합 탠덤 선호도 정화 (TAP-태그)를 사용하여 MyoD 파트너의 식별을위한 프로토콜을 설명합니다. 안정 플래그-HA-MyoD 발현-S3 헬라 세포의 사용은 분별 핵 추출물에서 MyoD 복합체를 정제하기에 충분한 재료를 얻을 수. 이종 시스템의 MyoD 파트너의 식별은 validati으로 이어졌습니다관련 시스템에.

<table><tbody><tr><td>&lt;strong&gt;Cell lines&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>HeLa-S3</td><td>ATCC</td><td>CCL-2.2</td><td></td></tr><tr><td>C2C12</td><td>ATCC</td><td>CRL-1772</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Spinner</td><td>Corning</td><td>778531</td><td></td></tr><tr><td>Homogenizer:&amp;nbsp; Dounce homogenizer&amp;nbsp;</td><td>Wheaton</td><td>432-1273 and 432-1271</td><td></td></tr><tr><td>Agitating device for spinners</td><td>Bellco</td><td>778531</td><td></td></tr><tr><td>Sonicator</td><td>Diagenode</td><td>UCD 200</td><td></td></tr><tr><td>Hemocytometer</td><td>Marienfeld Superior&amp;nbsp;</td><td>640610</td><td></td></tr><tr><td>Low-binding tubes</td><td>Sorenson</td><td>27210</td><td></td></tr><tr><td>Empty spin column</td><td>Bio-Rad</td><td>7326204</td><td></td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>SDS-polyacrylamide 4-12%&amp;nbsp; gel</td><td>Life technologies</td><td>NP0336BOX</td><td></td></tr><tr><td>4x loading buffer&amp;nbsp;</td><td>Life technologies</td><td>NP0007</td><td></td></tr><tr><td>10x reducing agent</td><td>Life technologies</td><td>NP0004</td><td></td></tr><tr><td>Silver staining kit</td><td>Life technologies</td><td>A8592</td><td></td></tr><tr><td>Centrifugal filter units, 10K</td><td>Millipore</td><td>UFC501024</td><td></td></tr><tr><td>Protein G agarose beads</td><td>Sigma-Aldrich</td><td>P4691</td><td></td></tr><tr><td>Protein A/G&amp;nbsp; Resin</td><td>Thermo Scientific</td><td>53132</td><td></td></tr><tr><td>Flag resin (anti-Flag M2-agarose affinity gel)</td><td>Sigma-Aldrich</td><td>A2220</td><td></td></tr><tr><td>HA resin (monoclonal Anti-HA agarose)</td><td>Sigma-Aldrich</td><td>A2095</td><td></td></tr><tr><td>MNase</td><td>Sigma-Aldrich</td><td>N3755</td><td></td></tr><tr><td>Flag free peptide (DYKDDDDK)</td><td>Ansynth Service BV</td><td>Custom synthesis</td><td>Resuspend up to 4 mg/ml in 50 mM Tris-HCl, pH 7.8</td></tr><tr><td>HA free peptide (YPYDVPDYA)</td><td>Ansynth Service BV</td><td>Custom synthesis</td><td>Resuspend up to 4 mg/ml in 50 mM Tris-HCl, pH 7.8</td></tr><tr><td>Bicinchoninic acid based protein assay kit : BCA kit</td><td>Thermo Scientific</td><td>23225</td><td></td></tr><tr><td>Protease inhibitors</td><td>Sigma-Aldrich</td><td>S8830</td><td></td></tr><tr><td>Luminol-based enhanced chemiluminescence (ECL) HRP substrate</td><td>Life technologies</td><td>34075</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A9647</td><td></td></tr><tr><td>Sheared salmon sperm DNA (Deoxyribonucleic acid sodium salt from salmon testes)</td><td>Sigma-Aldrich</td><td>D1626</td><td></td></tr><tr><td>Spermine tetrahydrochloride</td><td>Sigma-Aldrich</td><td>S1141</td><td></td></tr><tr><td>Spermidine</td><td>Sigma-Aldrich</td><td>S0266</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>MOPS running buffer</td><td>Life technologies</td><td>NP0001</td><td></td></tr><tr><td>DMEM (high glucose)</td><td>Sigma-Aldrich</td><td>D0822</td><td>Pre-warm&amp;nbsp; at 37 &amp;deg;C before use</td></tr><tr><td>Trypsin-EDTA (0.05% phenol red</td><td>Life technologies</td><td>25300-054</td><td></td></tr><tr><td>Serum</td><td>GE healthcare life sciences&amp;nbsp;</td><td>PAA A15-102</td><td>Each lot of serum has to be first tested for your cells.</td></tr><tr><td>Penicillin and Streptomycin&amp;nbsp;</td><td>Life technologies</td><td>15140-122</td><td></td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Life technologies</td><td>15250-061</td><td></td></tr><tr><td>Water (sterile-filtered)</td><td>Sigma-Aldrich</td><td>W3500</td><td></td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>HA from rat (12CA5)</td><td>Roche</td><td>11583816001</td><td></td></tr><tr><td>Flag</td><td>Sigma-Aldrich</td><td>A8592</td><td></td></tr><tr><td>MyoD</td><td>Santa Cruz</td><td>sc-32758</td><td>To use for western blotting</td></tr><tr><td>MyoD</td><td>Santa Cruz</td><td>SC-760</td><td>To use for immunoprecipitation and western blotting</td></tr><tr><td>CBFbeta</td><td>Santa Cruz</td><td>FL-182&amp;nbsp;</td><td></td></tr><tr><td>HP1alpha</td><td>Euromedex</td><td>2HP2G9</td><td></td></tr><tr><td>HP1beta</td><td>Euromedex</td><td>1MOD1A9AS</td><td></td></tr><tr><td>HP1gamma</td><td>Euromedex</td><td>2MOD1GC</td><td></td></tr><tr><td>Suv39h1</td><td>Cell Signaling Technology</td><td>8729</td><td></td></tr><tr><td>BAF47</td><td>BD Biosciences</td><td>612111</td><td></td></tr><tr><td>Myf5</td><td>Santa Cruz</td><td>SC-302</td><td></td></tr><tr><td>Tubulin</td><td>Sigma-Aldrich</td><td>T9026</td><td></td></tr><tr><td>Actin</td><td>Sigma-Aldrich</td><td>A5441</td><td></td></tr><tr><td>IgG Mouse</td><td>Santa Cruz</td><td>SC-2025</td><td></td></tr><tr><td>IgG Rabbit</td><td>Santa Cruz</td><td>SC-2027</td><td></td></tr><tr><td>Goat anti-rat -HRP</td><td>Sigma-Aldrich</td><td>A9037</td><td></td></tr><tr><td>Goat anti-rabbit -HRP</td><td>Sigma-Aldrich</td><td>A6154&amp;nbsp;</td><td></td></tr><tr><td>Goat anti-mouse -HRP</td><td>Sigma-Aldrich</td><td>A4416</td><td></td></tr></tbody></table>

identification of myod interactome using tandem affinity purification coupled to mass spectrometry

골격 근육 터미널 차별화는 근육 아세포에 만능 중배엽 전구 세포의 의지로 시작합니다. 이러한 세포는 여전히 증식 할 수있는 능력을 가지고 있거나 그들은 근섬유를 형성하도록 더 성숙하다 다핵 myotubes로 차별화 퓨즈있다. 골격근 단말 차별화 특히 근육 규제 요인 또는 MRFs의 멤버 (MyoD이 오게 닌은 Myf5 및 MRF4)는 또한 근육 조직 bHLH 전사 인자 계열이라는 다양한 전사 인자의 협조 작용에 의해 조율된다. 이러한 요인은 골격 근육 생성을 달성하기 위해 정교한 전사 조절 네트워크 내에서 염색질 리모델링 복합체와 협력. 이러한면에서, MyoD 근육 터미널 차별화를 트리거의 마스터 근육 조직 전사 인자로 간주됩니다. 이 개념은 골격근 세포에 비 - 근육 세포를 변환 MyoD의 능력에 의해 강화된다. 여기에서 우리는 MyoD를 식별하는 데 사용되는 방법을 설명골격근 단말 분화에 관련된 다른 요인을 해명하기 위해 철저하게 단백질 파트너. 장기 목표 골격근 유전자의 조절, 즉,., MyoD 타겟 관련된 후생 유전 학적 메카니즘을 이해하는 것이다. MyoD 파트너는 골격근 단말 분화 동안 관련된 파트너의 역할을 확인 하였다 질량 분석 (MS)의 특성에 결합 된 이종 시스템에서 직렬 선호도 그래피 (T​​AP 태그)를 이용하여 식별된다. 근육 재생에 선천성 중증, 근긴장 성 이영양증, 횡문근 육종 결함 : 근육 조직 요인, 또는 비정상적인 규제의 비정상적인 형태, 근육 질환의 수와 연관됩니다. 이와 같이, 근육 조직 인자가 질병의 치료를 향상시킬 수있는 질병 자체 회생기구 원인 메커니즘에 대하여 모두 근육 질환의 잠재적 치료 타겟의 풀을 제공한다. 따라서, 상세한 understan근육 조직 요인에 의해 제어되는 분자간 상호 작용과 유전 적 프로그램 땡 효율적인 치료의 합리적인 디자인을 위해 필수적이다.

MyoD 활성의 조절을 이해하기 위해서 질량 분석 (MS)에 의해 다음의 두 MyoD 태그 형태의 immunopurification에 기초한 생화학 적 정제를 사용 MyoD 착체의 완전한 특성화 착수. 플래그-HA-태그 MyoD 플래그-HA 허가를 발현 제어 세포주를 발현 헬라-S3 세포주의 사용 플래그-HA-MyoD 이중 화성 정제를 수행하여 MyoD 복합체를 정제하기에 충분한 재료를 얻을 수있다. <p class="jove_content" fo:kee...

Watch this Scientific Journal Video about Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry at JoVE.com

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

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.

탠덤 선호도 정화를 사용하여 MyoD 상호 작용 체의 식별 질량 분석에 결합

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

identification-myod-interactome-using-tandem-affinity-purification

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

9.8K 조회수.  Centre National de la Recherche Scientifique CNRS - Université Paris Diderot, Sorbonne Paris Cité. 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.

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

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

깊은 프로테옴 Isobaric 라벨, 광범위 한 액체 크로마토그래피, 질량 분석 및 소프트웨어 기반 정량화 하 여 프로 파일링

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

연구

생화학

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

대규모 핵 상호 작용 프로파일링을 위한 라벨 없는 면역 침전 질량 분광 워크플로우

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

TMT Sample Preparation for Proteomics Facility Submission and Subsequent Data Analysis

Proteomic technologies are powerful methodologies that can aid our understanding of mechanisms of action in biological systems by providing a global view of the impact of a disease, treatment, or other condition on the proteome as a whole. This report provides a detailed protocol for the extraction, quantification, precipitation, digestion, labeling, and subsequent data analysis of protein samples. Our optimized TMT labeling protocol requires a lower tag-label concentration and achieves consistently reliable data. We have used this protocol to evaluate protein expression profiles in a variety of mouse tissues (i.e., heart, skeletal muscle, and brain) as well as cells cultured in vitro. In addition, we demonstrate how to evaluate thousands of proteins from the resulting dataset.

We present an optimized tandem mass tag (TMT) labeling protocol that includes detailed information for each of the following steps: protein extraction, quantification, precipitation, digestion, labeling, submission to a proteomics facility, and data analyses.

프로테오믹스 시설 제출 및 후속 데이터 분석을 위한 TMT 샘플 준비

Proteomic technologies are powerful methodologies that can aid our understanding of mechanisms of action in biological systems by providing a global view ...

A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors

Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein phosphatases (PPPs) coordinate over 90% of all cellular serine/threonine dephosphorylation. Deregulation of protein phosphorylation has been implicated in the pathophysiology of various diseases, including cancer and neurodegeneration. Despite their widespread activity, the molecular mechanisms controlling PPPs and those controlled by PPPs are poorly characterized. Here, a proteomic approach termed phosphatase inhibitor beads and mass spectrometry (PIB-MS) is described to identify and quantify PPPs, their posttranslational modifications, and their interactors in as little as 12 h using any cell line or tissue. PIB-MS utilizes a non-selective PPP inhibitor, microcystin-LR (MCLR), immobilized on sepharose beads to capture and enrich endogenous PPPs and their associated proteins (termed the PPPome). This method does not require the exogenous expression of tagged versions of PPPs or the use of specific antibodies. PIB-MS offers an innovative way to study the evolutionarily conserved PPPs and expand our current understanding of dephosphorylation signaling.

Here, we present a protocol for the enrichment of endogenous phosphoprotein phosphatases and their interacting proteins from cells and tissues and their identification and quantification by mass spectrometry-based proteomics.

Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

탠덤 동질 정제를 사용하여 단백질 나누기 파트너의 식별

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

생물학

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.

의 단백질 단지의 식별<em&gt; 대장균</em&gt; 직렬 질량 분석법과 함께 순차적 펩타이드의 동질 정화를 사용하여

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

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

단백질 - 단백질 상호 작용을 확인<em&gt; 초파리</em탠덤 선호도 정화로&gt; 성인 머리 (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into protein function. Despite the importance of multi-PTM enrichment studies, few studies investigate more than one PTM at a time, due partially to the expenses, time, and large protein quantities required to perform multiple global proteomic analysis of PTMs. The &#34;one-pot&#34; affinity enrichment detailed in this protocol overcomes these barriers by permitting the simultaneous identification and quantification of peptides with lysine residues containing acetylation and succinylation PTMs with low amounts of sample input. The protocol involves preparation of protein lysate from mouse livers of SIRT5 knockout mice, performance of trypsin digestion, enrichment for PTMs, and performance of mass spectrometric analysis using a data-independent acquisition (DIA) workflow. Because this workflow allows for the enrichment of two PTMs from the same sample simultaneously, it provides a practical tool to study PTM crosstalk without requiring large amounts of samples, and it greatly reduces the time required for sample preparation, data acquisition, and analysis. The DIA component of the workflow provides comprehensive PTM-specific information. This is particularly important when studying PTM site localization, as DIA provides comprehensive sets of fragment ions that can be computationally deciphered to differentiate between different PTM localization isoforms.

This workflow describes the performance of time- and cost-efficient enrichment of multiple protein post-translational modifications (PTMs) simultaneously for quantitative global proteomic analysis. The protocol utilizes peptide-level PTM enrichment with multiple conjugated antibodies, followed by data-independent acquisition mass spectrometry analysis to gain biological insights into PTM crosstalk.

정량화 및 사이트 현지화를 위한 두 가지 번역 후 수정의 동시 선호도 강화

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into ...

High-throughput and Deep-proteome Profiling by 16-plex Tandem Mass Tag Labeling Coupled with Two-dimensional Chromatography and Mass Spectrometry

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an expanded 16-plex TMT method has been introduced, which further increases the throughput of proteomic studies. In this manuscript, we present an optimized protocol for 16-plex TMT-based deep-proteome profiling, including protein sample preparation, enzymatic digestion, TMT labeling reaction, two-dimensional reverse-phase liquid chromatography (LC/LC) fractionation, tandem mass spectrometry (MS/MS), and computational data processing. The crucial quality control steps and improvements in the process specific for the 16-plex TMT analysis are highlighted. This multiplexed process offers a powerful tool for profiling a variety of complex samples such as cells, tissues, and clinical specimens. More than 10,000 proteins and posttranslational modifications such as phosphorylation, methylation, acetylation, and ubiquitination in highly complex biological samples from up to 16 different samples can be quantified in a single experiment, providing a potent tool for basic and clinical research.

Presented here is an optimized high-throughput protocol developed with 16-plex tandem mass tag reagents, enabling quantitative proteome profiling of biological samples. Extensive basic pH fractionation and high-resolution LC-MS/MS mitigate ratio compression and provide deep proteome coverage.

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an ...

Tandem Mass Spectrometry

In tandem mass spectrometry a biomolecule of interest is isolated from a biological sample, and then fragmented into multiple subunits in order to help elucidate its composition and sequence. This is accomplished by having mass spectrometers in series. The first spectrometer ionizes a sample and filter ions of a specific mass to charge ratio. Filtered ions are then fragmented and passed to a second mass spectrometer where the fragments are analyzed.
This video introduces the principles of tandem mass spectrometry, including mass-to-ratio selection and dissociation methods. Also shown is a general procedure for analyzing a biochemical compound using tandem mass spectrometry with collision-induced dissociation. The applications section covers selection reaction monitoring, determination of protein post-translation modifications, and detection of tacrolimus levels in blood.
<div class="chapter_text" id="script_0">
Tandem mass spectrometry links together multiple stages of mass spectrometry to first isolate a biomolecule, and then determine aspects of its chemical makeup. Biomolecules have large, complex structures, making it difficult to determine their molecular composition. Tandem mass spectrometry selects a molecule of interest that is later fragmented into multiple subunits, which can help elucidate its identification and sequence. This video will show the concepts of tandem mass spectrometry, a general procedure, and some of its uses in biochemistry.
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<div class="chapter_text" id="script_1">
Tandem mass spectrometry begins as a typical mass spec instrument: with an ion source, which converts the sample into ions, and a mass analyzer, which separates the ions based on their mass-to-charge ratio. A common mass analyzer, the quadrupole, only allows ions with a specific ratio through, while the others crash into the rods of the apparatus. The species allowed through, called the precursor ion, is the biomolecule of interest. The ion moves into a collision cell, typically another quadrupole, where energy is applied to fragment the ion in a predictable pattern.
These fragments move into another mass analyzer, such as a time-of-flight, which separates these &#34;product ions&#34;. The product ions are then sent to the detector, as in a normal MS instrument. In the case of an unknown protein, the resulting spectrum contains numerous overlapping fragments, making a definitive complete sequence of the biomolecule difficult to generate. However, the spectral pattern is unique for a given protein. Analysis software compares the spectrum to a database of known peptide sequences, elucidating the unknown protein from the overlapping fragments.
Depending on the sample and desired degree of fragmentation, multiple fragmentation methods are possible. Fragmentation patterns depend on how the energy is transferred, its amount, and how it is distributed through the precursor ion. Energy can be transferred via neutral particles, radiation, or electrons. Using neutral atoms, a process called collision-induced dissociation or CID, primarily cleaves at the peptide bond between the amino acids, ideal for their identification.
Now that the basics of the technique have been covered, let&#39;s look at CID tandem mass spectrometry being used to study a component of bacterial cell envelopes.
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<div class="chapter_text" id="script_2">
As with all mass spectrometric experiments, the first step is to ionize the sample. For biomolecules, this is typically done with matrix assisted laser desorption or electrospray ionization. The precursor ion signal is then optimized by tuning of the ion optics. Once done, the target is isolated and the fragmentation method is chosen, such as CID.
The strength of an applied voltage, which accelerates the precursor ion into the collision cell, affects the degree of fragmentation. This voltage is increased until the precursor is roughly 10% abundance compared to the highest product ion. Multiple spectra are acquired and averaged until a sufficient signal-to-noise ratio is achieved. The number of scans needed is dependent on the signal intensity of the original precursor ion and can range from 3 to 300.
The analyte in this example, lipid A from Escherichia coli K-12, had 19 major fragments after CID. Lipid A&#39;s general structure is well known, allowing software to reconstruct the specific composition from the sample.
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<div class="chapter_text" id="script_3">
Now that we&#39;ve looked the procedure, let&#39;s look at some of the ways tandem mass spectrometry is used in biochemistry.
A common scanning mode in tandem mass spectrometry is selected reaction monitoring, or SRM. In SRM, both mass analyzers are fixed to a selected mass-to-charge ratio, focusing on specific precursor and product ions. Because of SRM&#39;s high degree of sensitivity, the spectra of peptide standards of known concentration can be utilized and compared to that of the unknown samples, allowing proteins of interest to be quantified.
Proteins are commonly modified after translation, typically by the addition of functional groups such as methyl groups, phosphate groups, or sugars, known as glycans. These are important in cell signaling processes, elucidating how cells communicate with one another. Because tandem mass spectrometry fragments the proteins into smaller components, it is possible to determine the location of the PTM to the specific fragment or even an amino acid. Some modifications, such as acetylation and trimethylation, are difficult to differentiate by mass alone, so chromatographic separation is performed before the mass spectrometry.
Many analytes in patient&#39;s blood are found at concentrations below the limit of detection for typical mass spectrometry. Another advantage of SRM is that it discards all but one product ion, increasing the sensitivity and enhancing the lower detection limit by up to 100 fold. In this example, the immunosuppressant drug, tacrolimus, could be detected at levels of 1 ng/mL.
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You&#39;ve just watched JoVE&#39;s video on tandem mass spectrometry. This video described the theory of the instrument, went over a general procedure, and explained some of the ways the technique is currently being utilized. Thanks for watching!
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Tandem Mass Spectrometry: Principle, Instrumentation, Uses

탠덤 질량 분석

In tandem mass spectrometry a biomolecule of interest is isolated from a biological sample, and then fragmented into multiple subunits in order to help ...

탠덤 선호도 정화를 사용하여 MyoD 상호 작용 체의 식별 질량 분석에 결합

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