iTRAQ Ribosomal Proteomic Quantification Service
Are you grappling with challenges in deciphering real-time protein production, identifying proteins actively engaged in cellular processes, or understanding how stimuli like drugs or stress impact translation? Our specialized iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) ribosomal proteomics service helps you accurately quantify newly synthesized proteins and map the actively translating ribosome landscape, providing critical insights into cellular dynamics and responses. Our expert team provides end-to-end support, from experimental design consultation to in-depth bioinformatic analysis, ensuring you receive actionable data tailored to your research questions.
Introduction of iTRAQ Ribosomal Proteomics Quantification Service
Ribosomal proteomics, or translatomics, focuses on identifying and quantifying proteins that are actively being synthesized by ribosomes at a specific point in time. This provides a direct measure of gene expression at the translational level. iTRAQ is a powerful chemical labeling technique used in quantitative mass spectrometry. By applying iTRAQ to isolated ribosomal fractions, researchers can accurately compare the levels of newly synthesized proteins across multiple samples (e.g., different conditions, and time points), offering crucial insights into translational regulation, cellular responses, and disease mechanisms.
Fig.1 Identification and analysis of proteins that differentially accumulated between NS and PS.1
Workflow
Our iTRAQ ribosomal proteomics quantification service is a meticulously designed process to ensure the highest quality data and insights.
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Ribosome Isolation & Enrichment
Cells or tissues are gently lysed, and ribosomes are isolated and enriched, often through sucrose gradient centrifugation or specialized column-based methods, to separate them from non-ribosomal proteins and free RNA. Quality control checks may be performed. -
Protein Extraction from Ribosomal Fractions
Proteins are extracted from the isolated ribosomal fractions using robust protocols designed to solubilize ribosomal proteins and associated factors efficiently.
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Protein Digestion & iTRAQ Labeling
Proteins are denatured, reduced, alkylated, and then enzymatically digested into peptides. Each sample's peptides are then labeled with a unique isobaric iTRAQ reagent. Labeled samples are then combined.
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LC-MS/MS Analysis
The combined, labeled peptide mixture is separated by nano-liquid chromatography and analyzed by LC-MS/MS. The MS fragments the peptides and measures the abundance of the iTRAQ reporter ions, providing quantitative information.
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Data Processing & Bioinformatic AnalysisRaw MS data is processed using specialized software to identify peptides and corresponding proteins, and to quantify the relative abundance of each protein across the different samples based on the iTRAQ reporter ion signals. Statistical analysis is performed to identify significantly changing ribosomal proteins.
Final Deliverables
- A Comprehensive Data Report
This includes a detailed summary of the experimental procedure, quality control metrics, a list of all identified and quantified ribosomal proteins with their relative abundance ratios across samples, and statistical analysis highlighting significantly regulated proteins.
- Bioinformatics Analysis Results
Including Gene Ontology (GO) enrichment analysis, pathway mapping, and visualization plots to help interpret the biological significance of the findings.
- Raw and Processed Data Files
Full access to the raw mass spectrometry data files and processed data tables for your records and further independent analysis.
Why Choose Creative Biolabs?
- Specialized Expertise
Our team has extensive experience specifically in ribosomal profiling and quantitative proteomics, ensuring your samples are handled optimally and data is interpreted accurately.
- Advanced Platform
We utilize state-of-the-art mass spectrometry platforms coupled with optimized iTRAQ labeling protocols to maximize sensitivity, coverage, and quantitative accuracy for ribosome-associated proteins.
- Comprehensive Data Analysis
We go beyond simple protein lists, providing in-depth bioinformatic analysis, including pathway and functional enrichment, to help you extract maximum biological meaning from your data.
- Collaborative Approach
We work closely with you from experimental design to data interpretation, ensuring the project is tailored to your specific research aims and that you fully understand the results.
- Commitment to Quality
Stringent QC measures are implemented at every step, from sample receipt to final report generation, guaranteeing reliable and reproducible outcomes.
Applications
- Disease Mechanism Research
This field focuses on identifying dysregulated protein synthesis pathways in cancers, neurodegenerative disorders, cardiovascular diseases, and metabolic syndromes. By mapping aberrant translational programs, researchers aim to uncover how misregulated protein translation drives disease initiation, progression, and drug resistance.
- Drug Discovery and Development
Drug discovery leverages translational profiling to identify novel therapeutic targets and validate mechanisms of action. By pinpointing ribosomal proteins or translation factors uniquely altered in disease states, researchers can design inhibitors to disrupt oncogenic translation. Translatome analysis further reveals how drug candidates directly modulate protein synthesis, enabling efficacy and safety assessments.
- Biomarker Discovery
Biomarker discovery exploits actively synthesized proteins as indicators of physiological states or disease progression. Using techniques like ribosome profiling or mass spectrometry, researchers identify proteins whose translation rates correlate with treatment response or early disease detection.
- Cellular Stress Response Studies
Cells adapt to environmental stressors by reprogramming protein synthesis. Studies reveal that stress-induced phosphorylation of eIF2α suppresses global translation while prioritizing stress-response proteins. Understanding these translational control mechanisms, such as the integrated stress response (ISR), aids in developing therapies for stress-related diseases.
- Agricultural Biotechnology
Understanding translational responses in crops under stress enables the engineering of stress-tolerant varieties. For example, modulating ribosome biogenesis or translation initiation factors in plants enhances drought resistance without yield penalties. Translational profiling also identifies proteins whose synthesis improves nutritional value or post-harvest shelf life. These strategies address global food security challenges by optimizing crop resilience and quality.
FAQs
Q: What is the primary advantage of iTRAQ ribosomal proteomics over standard total proteomics?
A: While total proteomics measures the overall abundance of proteins, iTRAQ ribosomal proteomics specifically quantifies proteins that are actively being synthesized by ribosomes. This provides a more dynamic and direct view of gene expression regulation at the translational level, which can be crucial for understanding rapid cellular responses or the immediate impact of treatments.
Q: What types of biological questions can be addressed with this service?
A: This service is ideal for investigating how various stimuli affect protein synthesis, identifying proteins whose translation is dysregulated, discovering biomarkers for active cellular processes, or understanding the mechanisms of translational control. If you're looking to see how your system is actively responding by making new proteins, this is a powerful tool.
Q: Are there any specific precautions for sample preparation to ensure good results?
A: Yes, preserving the integrity of ribosomes and the associated mRNA-protein complexes is critical. This usually involves rapid sample harvesting, snap-freezing, and often the use of translation inhibitors like cycloheximide during collection if you want to capture a specific snapshot of active translation.
Ready to explore how our iTRAQ ribosomal proteomics quantification service can elevate your research? Contact our team for more information.
Reference
- Zheng, Xuewei, et al. "iTRAQ-based quantitative proteomic analysis reveals cold responsive proteins involved in leaf senescence in upland cotton (Gossypium hirsutum L.)." International Journal of Molecular Sciences 18.9 (2017): 1984. Distributed under Open Access license CC BY 4.0, without modification.
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