Quantifying the Protein Levels of All Nuclear Hormone Receptors by Mass Spectrometry
Notice bibliographique
Résumé
Abstract Nuclear Receptors (NRs) are a family of ligand-activated transcription factors that control the expression of genes involved in a wide range of physiological processes. An atlas detailing the expression of all NRs at the mRNA level was completed in 2006 using quantitative PCR [Bookout et al. Cell 2006]. The comparative measurement of NRs at the protein level, however, has been hindered by the poor quality of commercially available antibodies, as well as the absence of a high throughput method for quantitation. To address this need, we are developing a mass spectrometry-based targeted proteomic assay to quantify the absolute amounts of NR protein in a panel of mouse tissues. NRs were overexpressed in HEK293 cells by transient transfection and protein was isolated. The cell lysates were digested with a combination of trypsin and Lys-C following the Multi-Enzyme Digestion Filter Aided Sample Preparation protocol. The peptides were desalted using an in-house made C18 tip, separated on an EASY-Spray C18 column (75 um x 50 cm, 3Å), and analyzed on a Thermo QExactive HF in Top20 data-dependent acquisition mode. Protein identifications were made using MaxQuant software, and the identifications were mined for members of the NR family. The NR peptides detected were searched against an in silico generated list of optimal NR peptides (filtered for uniqueness, length, absence of post translational modifications, and conservation between human and mouse). The matching peptides were validated by parallel reaction monitoring (PRM) and purchased as synthetic isotopes with a heavy terminal arginine or lysine. Peptide linearity, and lower limits of detection (LLOD) were estimated by spiking digests from a C57Bl/6 mouse liver lysate with increasing amounts of the labeled peptides and analyzing by PRM. Peptides that displayed non-linear behavior were excluded for quantitation. The LLOD were between 100 amol and 1.5 fmol on column. A test panel of tissues (cerebrum, hippocampus, cerebellum, liver, spleen, brown/white adipose, and kidney) showed that we could detect endogenous expression of NRs. To date, we have purchased and validated peptides for 44 of the 49 receptors. We used this assay to quantify the changes in NR protein expression in mouse livers in response to 16 hours of fasting. We found significant changes in the nuclear expression of CAR (3.1-fold increase), RXRβ (1.8-fold increase), SHP (3.9-fold decrease) and RARβ (2.0-fold decrease) in the fasted vs. fed state. Increased CAR activity with fasting was further supported by label-free quantitative proteomics on the same lysates which revealed 210 differentially expressed proteins (2-fold change, p<0.05), with 61 (29%) identified as known CAR target genes. Once complete, this assay will provide researchers with a robust quantitative tool to investigate changes in NR protein expression that will be widely applicable to endocrine research.
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Comment cette classification a été obtenuedéplier
Prédiction distillée sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Apprise à partir de 10 348 étiquettes directes de Codex et de 10 348 étiquettes directes de Gemma. Le mode candidate est l'union des têtes enseignantes seuillées; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont ni des étiquettes humaines ni des étiquettes directes de modèles de pointe.
Scores Codex et Gemma par catégorie
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,001 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,000 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule tête enseignante, pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».