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.

<ol>
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The authors have nothing to disclose.

Das vorgestellte Verfahren erlaubt eine erschöpfende Identifizierung von Partnern eines Transkriptionsfaktors, MyoD. Es zeigte sich MyoD Partner in einem heterologen System resistent gegen MyoD-induzierte Differenzierung, nämlich HeLa-S3-Zellen. Somit definitions identifiziert MyoD Partner ubiquitär exprimiert. Diese umfassen allgemein und sequenzspezifischen Transkriptionsfaktoren, Chromatin-modifizierende Enzyme, RNA - Prozessierung Proteine, Kinasen (Tabellen 1 und 2). Da HeLa-S3-...

Eukaryotische mehrzellige Organismen werden aus verschiedenen Organen und Geweben besteht. Jedes funktionale Gewebe hat ein spezifisches Gen-Musterausdruck, der bei jedem Differenzierungsschritt bestimmt wird. Zelldifferenzierung beinhaltet die Aktivierung von spezifischen Genen, Wartung ihrer Expression und, im allgemeinen, die von einem Satz von Genen, beteiligt an der Zellproliferation silencing. Skelettmuskeldifferenzierung oder Myogenese, ist somit ein mehrstufiges Verfahren, das mit der Bestimmung von mesodermalen Stammzellen in Myoblasten beginnt, und dann führt zur terminalen Differenzierung dieser Myoblasten in erste mono nukleiert, und dann mehrkernigen, Myotuben . Somit Myoblasten &quot;bestimmt&quot; Zellen, die noch in der Lage zu proliferieren, aber sie sind auf die Skelettmuskelabstammungslinie gebunden und somit ausschließlich in Skelettmuskelzellen entweder während der embryonalen Entwicklung oder im Erwachsenenmuskelregeneration unterscheiden kann. Der Prozess der Skelettmuskulatur Terminal unterscheidenzierung wird durch eine spezifische genetische Programm orchestriert , die aus dem Zellzyklus der Myoblasten - Vorläuferzellen mit dem Permanent Ausgang beginnt , die mit 1 der Proliferation assoziierten Genen, wie E2F Zielgenen auf eine definitive silencing führt. Tatsächlich während des Prozesses der terminalen Differenzierung, Proliferation Myoblasten Stillstand ist ein entscheidender Schritt, der die Expression von Skelettmuskel - spezifischen Genen und die Fusion von Myoblasten in Myotuben 2 vorausgeht. Ein solches Programm ermöglicht erwachsenen Muskelstammzellen, auch Satellitenzellen genannt, während des Regenerationsprozesses folgende Skelettmuskelverletzung zu unterscheiden. Mammalian myogenesis auf eine Familie von myogenic basischen Helix-Loop-Helix kritisch abhängig ist (bHLH) Transkriptionsfaktoren MyoD, Myf5, MRF4 und Myogenin bezeichnet häufig als die Familie der Skelettmuskulatur Bestimmung Faktoren oder MRF (Muscle Regulatory Factors) 3. Jeder von ihnen spielt eine wesentliche Rolle in der Beschreibung und den difzierung von Skelettmuskelzellen und eine spezifische Expressionsmuster April 05-07. Die Aktivierung von Myf5 und MyoD bildet die bestimmend Schritt, die Zellen in die myogenen Linie, und anschließende Expression der Myogenin löst myogenesis mit der Aktivierung von Skelettmuskel spezifische Gene, wie MCK (Muscle Creatine Kinase) begeht. Myogenen bHLH - Transkriptionsfaktoren wirken mit Mitgliedern der MEF2 - Familie bei der Aktivierung von Muskel Gene von zuvor stillen loci 8. Sie stimulieren auch Skelettmuskel Gentranskription als Heterodimere mit ubiquitären bHLH - Proteinen, E12 und E47, wie E - Proteine ​​bekannt, die in verschiedenen Gen-regulatorischen Regionen 8 sogenannte E-Boxen binden. Twist, Id (Inhibitor der Differenzierung) und andere Faktoren negativ diesen Prozess regulieren, durch die Bindung 8 mit MyoD für E - Proteine ​​konkurrieren. MyoD wird als wichtiger Akteur betrachtet bei der Auslösung von Muskel terminale Differenzierung 9 </sup&gt; da hat es die Fähigkeit , eine myogenic Bestimmung / Differenzierung (trans-Differenzierung) Programm in vielen ausdifferenzierten nicht Muskelsysteme 10-13 zu induzieren. Tatsächlich induziert gezwungen Expression von MyoD die trans-Differenzierung verschiedener Zelltypen , auch diejenigen , die aus einem anderen embryologischen Ursprungs 12. Zum Beispiel MyoD können Hepatozyten, Fibroblasten, Melanozyten, Neuroblasten und Adipozyten in muskelähnliche Zellen umwandeln. Die trans-differentiative Wirkung von MyoD beinhaltet eine abnormale Aktivierung des genetischen Programms myogene (insbesondere seine Zielgene) in einem nicht-muskuläre Umgebung gleichzeitige zum Silencing des ursprünglichen genetischen Programms (insbesondere Proliferationsgene). In proliferierenden Myoblasten wird MyoD ausgedrückt , ist aber nicht imstande, seine Zielgene selbst aktivieren , wenn es um ihre Promotoren bindet 14-16. Daher ist für die Anforderung MyoD kontinuierlich in undifferenzierte Myoblasten exprimiert werden bleibt ganz elusive. MyoD konnte seine Zielgene aufgrund Rekrutierung von repressiven Chromatin-modifizierende Enzyme in proliferierenden Myoblasten vor dem Laden von Chromatin - Remodeling - Enzyme 14,17 Aktivierung unterdrücken . Zum Beispiel Myoblasten in wuchernden, MyoD mit Transkriptions Korepressor KAP-1, Histon-Deacetylasen (HDACs) und repressive Lysin-Methyltransferasen (KMZ) verbunden sind, einschließlich Histon H3 Lysin 9 oder H3K9 und H3K27 Kmts und unterdrücken aktiv seine Zielgene Ausdruck durch eine lokal repressiven Chromatinstruktur 14,17 etablieren. Wichtig ist , zeigte eine kürzlich veröffentlichten Bericht , dass MyoD ist selbst durch die H3K9 KMT G9a was zu einer Hemmung der Aktivität transaktivierend 16 direkt methyliert. Die epigenetische Mechanismen in dieser Transdifferenzierung nicht-Muskelzellen durch MyoD sind weitgehend unbekannt. Bemerkenswerterweise sind einige Zelllinien resistent gegen MyoD-induzierte Transdifferenzierung. So wird in HeLa-Zellen, MyoD ist entweder inaktiv oderkönnte sogar als Repressorfunktion anstatt Aktivator der Transkription aufgrund des Fehlens der Expression der BAF60C Untereinheit des Chromatin - Remodeling - Komplexes SWI / SNF 18. Dieses Modell kann somit der Wahl sein, um besser die Mechanismen der MyoD-induzierte Gen Repression zu charakterisieren. Es ist auch geeignet , um die Fähigkeit von MyoD dem Test an seinem Zielloci mit seinen zugehörigen Partner repressive Chromatin - Umgebung zu induzieren und daher die MyoD abhängigen repressive Mechanismen aufzudecken Myoblasten in proliferierenden terminalen Differenzierung zur Feinabstimmung. Hier beschreiben wir das Protokoll für die Identifizierung von MyoD Partner unter Verwendung von Tandem Affinity Purification (TAP-Tag), die mit der Massenspektrometrie (MS) Charakterisierung. Die Verwendung von HeLa-S3-Zellen stabil Flag-HA-MyoD exprimieren erlaubt genug Material bekommen die MyoD Komplexe aus fraktionierten Kernextrakten zu reinigen. Die Identifizierung von MyoD Partner im heterologen System wurde von validati gefolgtauf in einem relevanten System.

<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

Der Skelettmuskel terminale Differenzierung beginnt mit dem Einsatz von pluripotenten mesodermalen Vorläuferzellen zu Myoblasten. Diese Zellen haben immer noch die Möglichkeit, sich zu vermehren oder sie können unterscheiden und Sicherung in kernige Myotuben, die maturate weiter Muskelfasern zu bilden. Der Skelettmuskel terminalen Differenzierung wird durch die koordinierte Wirkung von verschiedenen Transkriptionsfaktoren orchestriert, insbesondere die Mitglieder der Muskel-regulierende Faktoren oder MRF (MyoD, Myogenin, Myf5 und MRF4), auch genannt die myogenen bHLH Transkriptionsfaktoren Familie. Diese Faktoren wirken mit Chromatin-Remodeling-Komplexe in aufwendigen Transkriptionsregulationsnetzskelett myogenesis zu erreichen. Dabei ist der Master MyoD myogenen Transkriptionsfaktor betrachtet in Muskel terminalen Differenzierung auszulösen. Diese Vorstellung wird durch die Fähigkeit von MyoD verstärkt nicht-Muskelzellen in Skelettmuskelzellen umwandeln. Hier haben wir einen Ansatz beschreiben MyoD zu identifizierenProteinpartner in erschöpfender Weise, um die verschiedenen Faktoren in Skelettmuskel terminalen Differenzierung beteiligt aufzuklären. Das langfristige Ziel ist es, die epigenetische Mechanismen bei der Regulation der Skelettmuskulatur Gene beteiligt zu verstehen, dh., MyoD Ziele. MyoD Partner werden durch die Verwendung Tandem Affinity Purification (TAP-Tag) von einem heterologen System gekoppelt mit der Massenspektrometrie (MS) Charakterisierung, gefolgt von Validierung der Rolle der relevanten Partner während der Skelettmuskulatur terminalen Differenzierung identifiziert. kongenitale Myasthenie, Muskeldystrophie, Rhabdomyosarkom und Defekte in der Regeneration der Muskulatur: Aberrant Formen von myogenen Faktoren oder deren abweichende Regelung, sind mit einer Reihe von Muskelerkrankungen in Verbindung gebracht. Als solche bieten myogenen Faktoren einen Pool von potenziellen therapeutischen Ziele in Muskelerkrankungen, sowohl im Hinblick auf Mechanismen, die Krankheit selbst und regenerative Mechanismen verursachen, die Behandlung von Krankheiten zu verbessern. Somit ist die detaillierte underding der intermolekularen Wechselwirkungen und die genetischen durch die myogene Faktoren gesteuert Programme ist für das rationale Design von effizienten Therapien unerlässlich.

Um die Regulierung der Aktivität MyoD verstehen, unternahmen wir die erschöpfende Charakterisierung der MyoD Komplexe mittels biochemischer Reinigung, basierend auf der Immunreinigung einer Doppel tagged Form MyoD gefolgt von Massenspektrometrie (MS). Die Verwendung von HeLa-S3-Zelllinie exprimiert Flag-HA-markierten MyoD und eine Linie Kontrollzelle, die Flag-HA erlaubt genug Material bekommen die MyoD-Komplex zu reinigen, indem Sie doppelt Affinitätsreinigung von Flag-HA-MyoD durchf...

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.

Identifizierung von MyoD Interaktom Gekoppelt Mit Tandem Affinity Purification zu Mass Spectrometry

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 Aufrufe.  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.

Serie: 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.

Tiefen Proteom Profilierung durch isobare Kennzeichnung, umfangreiche Flüssigchromatographie-Massenspektrometrie und softwaregestützte Quantifizierung

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

Forschung

Biochemie

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.

Etikettenfreier Immunpräzipitations-Massenspektrometrie-Workflow für großangelegtes Nuklear-Interactome-Profiling

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.

TMT-Beispielvorbereitung für proteomics Facility Submission und nachfolgende Datenanalyse

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.

Ein massenspektrometriebasierter Ansatz zur Identifizierung von Phosphoproteinphosphatasen und ihren Interaktoren

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.

Identifizierung von Protein Interaktionspartner mittels 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 ...

Biologie

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.

Identifizierung von Protein-Komplexen in<em&gt; Escherichia coli</em&gt; Mit Sequential Peptide Affinity Purification in Kombination mit Tandem-Massenspektrometrie

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.

Identifizierung von Protein-Protein-Wechselwirkung in<em&gt; Drosophila</em&gt; Adult Heads von Tandem Affinity Purification (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.

Gleichzeitige Affinitätsanreicherung von zwei posttranslationalen Modifikationen für Quantifizierung und Standortlokalisierung

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.
</div>
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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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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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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

Tandem-Massenspektrometrie

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

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