In the field of life sciences research, the study of proteins and their post-translational modifications (PTM) is crucial for understanding the complex molecular mechanisms behind various cellular processes. Creative Proteomics based on data-independent acquisition (DIA) for phosphoproteomics analysis, can help researchers delve deeper into the complexity of the proteome. Our service provides effective technical support for phosphoproteomics analysis and plays an important role in helping researchers advance the field of DIA-based proteomics research.
DIA proteome analysis is a robust and versatile method for the comprehensive study of the proteome within biological samples. It involves the systematic identification and quantification of a wide range of proteins present in a given sample. DIA does not rely on precursor ion selection, making it highly suitable for quantitative proteome analysis. This service enables researchers to gain a holistic view of the proteome's composition, facilitating the discovery of biomarkers, elucidation of cellular pathways, and in-depth characterization of protein profiles.
Phosphorylation is undoubtedly the most important form of post-translational modification ubiquitous in prokaryotic and eukaryotic cells, which can lead to changes in protein structure, activation/inhibition of protein activity, and promotion/prevention of protein-protein interactions. Phosphorylation is at the core of many biological processes, including the regulation of signal transduction, gene expression and cell division. In humans, aberrant phosphorylation of proteins is commonly associated with a number of diseases such as cancer[1]. Therefore, a better understanding of protein phosphorylation will help deepen our understanding of important cellular processes, better understand disease mechanisms, and drive the discovery of effective therapies and new biomarkers.
DIA phosphoproteomics analysis is an extension of DIA proteome analysis specifically tailored to the comprehensive study of phosphorylated proteins and their PTMs. By combining the power of DIA with phosphopeptide enrichment techniques, this service allows for the systematic identification and quantification of phosphorylated proteins in complex biological samples. This in-depth analysis is crucial for unraveling the intricacies of signaling pathways, drug development, and biomarker discovery in the context of diseases.
DIA proteome analysis relies on a data-independent approach, wherein all precursor ions within a specified mass-to-charge (m/z) range are fragmented in a systematic manner. This fragmentation produces a series of fragment ions, enabling the accurate identification and quantification of peptides and proteins. The method typically involves liquid chromatography-mass spectrometry (LC-MS/MS) to separate and analyze peptides, followed by database searches for peptide identification. The use of stable isotope labeling or label-free quantification techniques further enhances the accuracy and reliability of protein quantification.
Phosphoproteomics analysis builds upon the principles of DIA proteome analysis and incorporates phosphopeptide enrichment strategies. These strategies, such as immobilized metal affinity chromatography (IMAC) or titanium dioxide (TiO2) enrichment, selectively isolate phosphorylated peptides from the complex mixture of proteins. The enriched phosphopeptides are then subjected to LC-MS/MS analysis, enabling the identification and quantification of phosphorylated proteins. Advanced software tools are used to interpret the resulting mass spectra, facilitating the mapping of phosphorylation sites and the determination of phosphorylation stoichiometry.
Our Phosphoproteomics Service offers high sensitivity detection and accurate quantitation of protein phosphorylation and identification of phosphorylation sites. Using Our advanced Discovery Proteomics Platform, we can detect phosphorylation on a large scale from complex biological samples to provide a comprehensive overview of signaling events and pathway activity regulation.
We can analyze up to 9,000 proteins per sample under different conditions, and identify and localize protein phosphorylation sites with or without enrichment approach prior to mass spec analysis. Our Discovery Proteomics Platform is ideal for complete phosphorylation analysis from minimal sample volume. We also offer highly multiplexed targeted proteomics with absolute quantification for customized panels of proteins.
Summary of MS-based phosphoproteomics experiments.
Based on our special protein extraction technology, we can quickly extract proteins from various samples and design personalized experimental schemes according to different experiment purposes. Specific requirements are as follows:
Sample Type | Protein | # of Cells | Animal Tissue | Plant Tissue | Blood | Urine | Serum | Microbes |
Quantify | 100 ug | 1×107 cells | 1 g | 200 mg | 1 mL | 2 mL | 0.2-0.5 mL | Dry weighed: 200 mg |
Reference:
Non-small cell lung cancer (NSCLC) remains a major global health concern, necessitating a deeper understanding of its molecular mechanisms. Phosphoproteomic and proteomic approaches offer valuable insights into the intricate signaling networks associated with NSCLC, potentially unraveling key biomarkers and therapeutic targets.
The study incorporated diverse samples, including NSCLC cell lines (PC9, CL68, H3255, CL141, and H1975), MDA-MB-231 breast cancer cell line, and clinical tissues from NSCLC patients. Cell lines were subjected to specific treatments to enhance tyrosine phosphosite abundance. Tissue samples were collected post-surgery from 25 NSCLC patients, ensuring a representative cohort for in-depth analysis.
Chemicals and Materials:
Cell Culture and Lysis:
Lung Cancer Tissue Collection and Lysis:
Protein Digestion:
LC-MS/MS Analysis:
Data Processing and Protein Identification:
Pipeline for hybrid spectral library construction and phosphosite quantification.
Phosphoproteome Spectral Library Construction: Generated a hybrid library from DDA of fractionated cell line and tissue samples.
Proteome Spectral Library Construction: Constructed a proteome library from lung cancer tumor tissues and NSCLC cell lines.
Differential Phosphoproteomic Analysis: Identified dysregulated phosphosites between NSCLC EGFR-TKI-sensitive and -resistant cell lines.
Tissue vs. Normal Comparison: Detected significant changes in phosphosite abundance between NSCLC tumor tissues and adjacent normal tissues.
Pathway Enrichment Analysis: Performed KEGG pathway analysis, revealing altered signaling pathways in NSCLC.
Comparison of quantification performance in DDA and DIA using cell lysate.
Phosphoproteome profiling of lung cancer tissues.
Reference:
4D Proteomics with Data-Independent Acquisition (DIA)
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×Specializing in proteomics, Creative Proteomics offers cutting-edge protein analysis services. Our distinctive approach revolves around harnessing the power of DIA technology, enabling us to deliver precise and comprehensive insights that drive advancements in research and industry.