In the vast field of life science research, proteomics, as a key tool to reveal the mysteries of life, is increasingly attracting widespread attention from scientific researchers. Proteomics aims to comprehensively analyze the structure and function of all proteins in organisms, and differential protein screening is one of the crucial research contents. It can help us identify proteins whose expression changes significantly under different physiological or pathological conditions, thereby revealing the key roles of these proteins in biological processes, disease occurrence and drug response.
Fully automatic pipetting workstation
1. Sample preparation 1. Sample collection: According to the research purpose and sample source (such as tissue samples, serum or cell culture supernatant, etc.), collect protein samples under different conditions or tissues. 2. Protein extraction: Choose an appropriate protein extraction method, taking into account the complexity of the sample and the range of protein abundance, to extract high-quality proteins. 3. Quantification and preprocessing: Quantify the extracted protein samples to ensure that the same amount of protein is used in the experiment. Then preprocessing steps are performed, such as removing low-abundance proteins, enriching specific types of proteins, etc., to improve the detection sensitivity of differential proteins.
2. Protein separation 1. Select separation technology: Use separation technologies such as gel electrophoresis (such as SDS-PAGE, two-dimensional gel electrophoresis 2-DE) or liquid chromatography (such as HPLC, ion exchange chromatography, reverse phase chromatography) to separate Proteins separate into distinct bands or peaks. 2. Operation of the fully automatic pipetting workstation: (1) Set the pipetting program: According to the experimental needs, set the pipetting program on the fully automatic pipetting workstation, including pipetting volume, pipetting speed, pipetting times and other parameters. (2) Automatic pipetting and separation: The fully automatic pipetting workstation automatically completes pipetting and separation operations according to the set program, ensuring the accuracy and consistency of each experiment.
3. Mass spectrometry analysis 1. Selection of mass spectrometry technology: Use mass spectrometry technology (such as tandem mass spectrometry MS/MS, liquid chromatography mass spectrometry coupled with LC-MS/MS) to identify and quantify the separated proteins. 2. Sample processing and injection: The separated protein samples are properly processed, such as enzymatic hydrolysis, derivatization, etc., and then loaded into the mass spectrometer for injection. Fully automated pipetting workstations can automate this step to ensure accuracy and consistency in sample processing. 3. Mass spectrometry data collection: The mass spectrometer scans and analyzes the injected protein sample and collects mass spectrometry data.
4. Data analysis 1. Mass spectrometry data analysis: With the help of database search, spectral library matching and quantitative software and other tools, mass spectrometry data are analyzed and identified to determine the identified proteins. 2. Differential protein screening: (1) Data preprocessing: Preprocess mass spectrometry data, such as denoising, normalization, etc., to improve the accuracy of data analysis. (2) Statistical analysis: Use statistical methods (such as t test, analysis of variance, partial least squares regression, etc.) to compare protein expression levels between different conditions and identify differential proteins. The highly accurate and consistent data provided by the fully automated pipetting workstation helps improve the accuracy of statistical analysis. (3) Multiple hypothesis testing correction: When performing differential protein screening, multiple hypothesis testing correction, such as the BH FDR correction method, is usually required to reduce false positive results. 3. Functional annotation and enrichment analysis: Functionally annotate differential proteins to understand their roles in biological processes and related metabolic pathways. At the same time, bioinformatics analysis such as Pathway enrichment analysis of differential proteins and construction of differential protein interaction networks were conducted to gain a deeper understanding of the role of these differential proteins in biological processes.
5. Results verification and follow-up research 1. Verification of differential proteins: Verify the expression level and function of the selected differential proteins through other experimental methods (such as Western blot, immunofluorescence, etc.). 2. Follow-up research: Carry out follow-up research based on the screened differential proteins, such as exploring their role in the occurrence and development of diseases, searching for potential drug targets, etc.
With the help of a fully automated pipetting workstation, differential protein screening experiments have become faster, more accurate and more reliable. Researchers can quickly identify proteins whose expression changes significantly under different physiological or pathological conditions, and then delve into the key roles of these proteins in biological processes, disease occurrence, and drug responses. This not only provides new ideas and methods for the diagnosis and treatment of diseases, but also opens up new avenues for new drug development and biomarker discovery.
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