Introduction of Phosphorylation Modification Proteomic Analysis
Protein phosphorylation is a fundamental and ubiquitous post-translational modification crucial for regulating virtually all aspects of cellular life, from growth and metabolism to immune response and gene expression. This reversible process, orchestrated by a dynamic interplay of protein kinases and phosphatases, acts as a molecular switch, dictating protein activity, localization, and interactions. Aberrant phosphorylation is a common hallmark of numerous diseases, including cancer, diabetes, neurodegenerative disorders like Alzheimer's, and infectious diseases, making its comprehensive analysis paramount for understanding disease mechanisms and identifying therapeutic interventions. Phosphoproteomics, leveraging advanced mass spectrometry, provides an unparalleled window into this complex regulatory landscape.
Providing a specialized phosphorylation modification proteomic quantitative analysis service is not merely a convenience; it is an absolute necessity for modern biological and biomedical research, addressing critical gaps that other technologies cannot fill:
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Unlocking cellular regulatory mechanisms: Phosphorylation is the most pervasive and critical regulatory post-translational modification, acting as the cell's primary signaling language. A dedicated service is necessary to systematically map and quantify these modifications, providing an unparalleled insight into how cells control virtually every biological process.
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Beyond transcriptomics and genomics: While genomics and transcriptomics reveal what can happen (genes present, mRNAs expressed), they do not directly reflect the functional state of proteins, which are the primary workhorses of the cell. Proteins undergo PTMs, like phosphorylation, Acetylation, and Ubiquitination, that dramatically alter their activity, localization, and interactions without changing their mRNA levels. Proteomic Quantitative Analysis provides this crucial, direct layer of functional information, revealing what is happening at the protein level in real-time.
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Identifying actionable drug targets: Many diseases are characterized by aberrant phosphorylation. A specialized service can precisely identify dysregulated phosphorylation events, leading to the discovery of novel and highly specific drug targets.
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Biomarker discovery: Altered phosphorylation patterns can serve as powerful and dynamic biomarkers for disease diagnosis, prognosis, and monitoring treatment response. A quantitative service is critical for the high-confidence identification and rigorous validation of these phosphorylation-based biomarkers.
What Is the Principle of Phosphorylation Modification Proteomic Quantitative Analysis?
The core principle behind our phosphorylation modification proteomic quantitative analysis service is based on the unique chemical properties of the phosphate group. When a phosphate group is added to serine (S), threonine (T), or tyrosine (Y) residues, it introduces an additional mass of approximately 79.9663 Da to the peptide. Mass spectrometry (MS) instruments are highly sensitive detectors of mass and can identify these precise mass shifts, thereby indicating the presence of phosphorylation. Quantitative analysis extends this by measuring the relative or absolute abundance of these modified peptides across different biological samples. This is achieved through various strategies:
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Label-free quantification (LFQ): This method compares the intensity of MS signals (e.g., extracted ion chromatogram peak areas, spectral counts) of phosphopeptides directly between different samples. It relies on the reproducibility of LC-MS runs and sophisticated software algorithms for alignment and quantification. While cost-effective and flexible for many samples, it can be more sensitive to technical variability.
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Stable isotope labeling: This approach incorporates stable, non-radioactive isotopes into proteins or peptides. These "heavy" and "light" versions are chemically identical but differ in mass, allowing them to be distinguished by the mass spectrometer. Labeled samples are typically mixed before MS analysis, enabling highly accurate relative quantification based on the ratio of heavy to light signals within the same MS run, thereby minimizing inter-run variability.
How to Provide a Phosphorylation Modification Proteomic Quantitative Analysis Service?
Providing a high-quality phosphorylation modification proteomic quantitative analysis service requires a meticulous, multi-stage approach, integrating advanced laboratory techniques with sophisticated bioinformatics. Here's a detailed breakdown of Creative Biolabs' specific processes and steps, designed for robust and reliable results:
01Experimental Design
Our scientific team, with over years of specialized experience in Glycoproteomics, engages in in-depth discussions with you. We work to understand your specific biological question, the nature of your samples, experimental conditions, and your desired level of quantitative detail. Based on this, we advise on the most appropriate quantitative strategy (e.g., SILAC, label-free, Data-Independent Acquisition (DIA)) and recommend optimal sample replication to ensure statistical power and robust conclusions.
02Sample Preparation
Upon receipt, samples are immediately processed under stringent conditions. This includes rapid homogenization for tissues and efficient cell lysis. Crucially, a comprehensive cocktail of broad-spectrum protease and phosphatase inhibitors is added immediately to halt all enzymatic activity, thereby preserving the in vivo phosphorylation profile and preventing artifactual modifications post-lysis. Protein concentration is accurately determined (e.g., BCA assay), and initial protein integrity and purity are assessed to ensure sample suitability for the entire workflow.
03Protein Digestion and Quantitative Labeling
Proteins are first denatured (e.g., with urea/thiourea), reduced, and alkylated (e.g., iodoacetamide) to prepare them for enzymatic digestion. They are then precisely digested into peptides using highly specific proteases. If a chemical labeling strategy is chosen, peptides from different samples are then chemically labeled with their respective isobaric tags. Labeled peptides are then pooled in precise ratios before subsequent enrichment steps.
04Phosphopeptide Enrichment
This is a critical and highly optimized step. We employ multi-dimensional enrichment strategies (e.g., cation exchange chromatography, titanium dioxide (TiO2) affinity chromatography) to maximize coverage and specificity.
05Quantitative MS Acquisition and Analysis
The enriched phosphopeptides are separated by high-performance liquid chromatography for optimal resolution and then introduced into state-of-the-art mass spectrometers. Creative Biolabs utilizes advanced hybrid Orbitrap platforms for their high resolution, high mass accuracy, and sensitivity. We employ various cutting-edge acquisition modes, including Data-Dependent Acquisition (DDA), DIA, Targeted Multiple Reaction Monitoring (MRM), Parallel Reaction Monitoring (PRM), etc. We transform raw MS data into interpretable biological insights, ensuring accuracy and statistical rigor.
Creative Biolabs is your trusted partner for comprehensive and accurate phosphorylation modification proteomic quantitative analysis. We deliver unparalleled insights into cellular signaling, accelerating your drug discovery, biomarker identification, and fundamental research endeavors. Our commitment to accuracy, depth, and expert interpretation ensures you receive actionable data to drive your scientific breakthroughs. Ready to unlock the full potential of protein phosphorylation in your research? Please contact our team of experts today to receive a customized quote.
Published Data
This research highlights a crucial challenge in interpreting phosphoproteomics data: observed changes in protein phosphorylation often reflect not only actual alterations in phosphorylation status but also shifts in the overall abundance of the protein itself. To address this, the study pioneers a comprehensive approach using mass spectrometry to concurrently measure both protein expression and phosphorylation levels in Saccharomyces cerevisiae (yeast). Through detailed comparisons of yeast mutants lacking key MAPK pathway kinases (FUS3 and STE7) against wild-type strains, the researchers achieved an unprecedented depth of data, quantifying over 4,100 proteins and identifying nearly 12,500 unique phosphorylation sites. This integrated analysis provided a clearer picture of the yeast MAPK pathway, demonstrating how genetic deletions lead to distinct, context-dependent changes in both protein amounts and their phosphorylation states. The study strongly advocates for integrating protein expression data as a standard practice in future phosphoproteomics investigations across various biological systems.
Fig.1 LC-MS/MS analysis of protein phosphorylation.1
FAQs
Q1: How does Creative Biolabs ensure the accuracy of phosphorylation quantification, especially given changes in total protein levels?
A1: Creative Biolabs employs a rigorous normalization strategy. We perform parallel quantitative analyses of both phosphopeptides and their corresponding total protein levels from the same samples. This allows us to accurately distinguish true changes in phosphorylation status from those simply due to altered protein abundance, providing you with highly reliable and biologically meaningful data.
Q2: Can your service identify novel phosphorylation sites, or is it limited to known ones?
A2: Our service excels in both! We utilize advanced DIA mass spectrometry, which is an unbiased approach designed for the comprehensive discovery of novel phosphorylation sites across your entire proteome. For validating or monitoring known sites with high sensitivity, we also offer targeted Multiple Reaction Monitoring (MRM).
Q3: How does Creative Biolabs handle the challenge of phosphate group lability during mass spectrometry?
A3: We overcome this challenge by employing advanced fragmentation techniques like Electron Transfer Dissociation (ETD) and Higher-Energy Collisional Dissociation (HCD) on our state-of-the-art Orbitrap mass spectrometers. ETD, in particular, preserves the labile phosphate group during fragmentation, allowing for more confident and unambiguous localization of phosphorylation sites on serine and threonine residues.
Customer Review
Accurate Quantification
"We previously struggled with distinguishing true phosphorylation changes from protein abundance shifts in our stem cell differentiation studies. Creative Biolabs' rigorous protein normalization strategy for their phosphorylation modification proteomic quantitative analysis service has completely transformed our understanding, providing us with truly accurate quantitative data. This clarity has accelerated our pathway analysis and target validation efforts, making our results much more robust." - Prof. U. Hal***l.
Exceptional Support and Insights
"Creative Biolabs' phosphorylation modification proteomic quantitative analysis service provided not only high-quality data but also deep biological insights into our complex signaling pathway project. Their team helped us map intricate networks and identify key regulatory kinases, far exceeding our expectations compared to previous service providers. The detailed reports and interactive data portal are incredibly user-friendly and have become an essential resource for our lab." - Dr. X. All***n.
References
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Wu, Ronghu, et al. "Correct interpretation of comprehensive phosphorylation dynamics requires normalization by protein expression changes." Molecular & cellular proteomics 10.8 (2011). DOI: 10.1074/mcp.M111.009654. Distributed under an Open Access license CC BY 4.0, without modification.
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