What is Proteomics?
Proteomics not only focuses on the individual proteins but also aims to understand their dynamic behavior and interactions within complex biological systems. Proteins are the workhorses of living organisms, performing a myriad of functions, including catalyzing chemical reactions, transmitting signals, providing structural support, and regulating gene expression. By studying the proteome, which represents the complete set of proteins expressed by a cell, tissue, or organism, proteomics provides a holistic view of the intricate protein networks that underlie cellular processes and contribute to the overall functionality of biological systems. Through proteomics, researchers strive to unravel the intricate language of proteins, uncover their roles in health and disease, and pave the way for targeted interventions and personalized medicine.
Select Service
Strategies and Scope of Proteomic Analysis
Proteomic analysis employs a range of strategies to investigate the proteome. Let's take a closer look at two prominent approaches: discovery proteomics and targeted proteomics.
Discovery Proteomics
Discovery proteomics aims to comprehensively identify and characterize proteins within a given sample. This approach often utilizes mass spectrometry, a powerful analytical technique that measures the mass-to-charge ratio of ions, to identify and quantify proteins. By comparing the acquired data with protein databases, researchers can unveil the identity and abundance of proteins present in the sample.
Targeted Proteomics
In contrast to discovery proteomics, targeted proteomics focuses on the quantitative analysis of specific proteins or protein modifications. This approach utilizes techniques like selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) to precisely measure the abundance of target proteins. Targeted proteomics is particularly useful for studying protein biomarkers and their potential roles in diseases.
Research Content in Proteomics
Proteomics encompasses several key areas of study, including protein identification, protein quantification, protein modification profiling, and protein-protein interactions.
Protein Identification
Protein identification involves the determination of protein identities within a given sample. Mass spectrometry plays a crucial role in this process by enabling researchers to analyze the unique peptide sequences generated from protein digestion. By comparing these peptide sequences to protein databases, scientists can confidently assign specific protein identities. This scientific approach facilitates the understanding of protein composition and aids in advancing various fields, such as medicine and agriculture.
Select Service
Protein Quantification
Quantitative proteomics serves as a powerful tool for comparing protein abundances across diverse samples or experimental conditions. It enables researchers to gain a deeper understanding of the intricate interplay between proteins and their roles in biological systems. Isotope labeling techniques, such as stable isotope labeling by amino acids in cell culture (SILAC), tandem mass tagging (TMT) or isobaric tags for relative and absolute quantification (iTRAQ), play a vital role in achieving precise and reliable protein quantification.
In addition to the labeled quantitative methods described above, label-free quantitative proteomics can also be used. Label-free quantification has the advantage of not requiring complex sample processing or the introduction of external markers. They provide a relatively fast and cost-effective way to compare changes in protein abundance across samples or conditions. However, the accuracy and sensitivity of label-free quantitative methods may be somewhat limited compared to labeled quantitative methods.
Protein Modification Profiling
Post-translational modifications (PTMs), such as phosphorylation, acetylation, and glycosylation, are pivotal for regulating protein function and cellular processes. They introduce chemical alterations to proteins, expanding their functional repertoire and impacting various biological phenomena. Proteomics has emerged as a powerful tool for investigating and comprehensively characterizing these modifications on a global scale.
By employing advanced analytical techniques, proteomics enables the identification and quantification of PTMs across diverse protein populations. Mass spectrometry-based approaches, combined with enrichment strategies and bioinformatics analyses, facilitate the discovery and mapping of PTMs in a high-throughput manner. This comprehensive exploration of PTMs offers valuable insights into the intricate landscape of protein regulation.
Protein-Protein Interactions
Understanding protein interactions is essential for deciphering the intricate molecular networks within cells. Proteomics offers valuable tools, including chemical crosslinking mass spectrometry (XL-MS) or pull-down assay, to identify and characterize protein-protein interactions. By mapping these interactions, researchers can unravel the complex web of protein networks that drive cellular functions.
Techniques and Analytical Workflow in Proteomics
The following are the key techniques and analytical workflow commonly employed in proteomics research:
a) Sample Preparation:
Protein Extraction: Proteins are extracted from the biological sample using various methods such as cell lysis, tissue homogenization, or subcellular fractionation.
b) Protein Separation:
- Gel Electrophoresis: Proteins are separated based on their size using techniques like sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional gel electrophoresis (2DE).
- Liquid Chromatography: High-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS) techniques are used for protein separation based on various properties such as charge, hydrophobicity, or affinity.
c) Protein Identification:
- Mass Spectrometry (MS): Proteins are fragmented into peptides, and the resulting peptide fragments are analyzed by mass spectrometry. Techniques like matrix-assisted laser desorption/ionization (MALDI) or electrospray ionization (ESI) are used to ionize and analyze the peptides.
- Database Searching: The acquired mass spectrometry data is compared against protein databases using algorithms like SEQUEST, Mascot, or X!Tandem to identify the proteins.
d) Quantitative Analysis:
- Stable Isotope Labeling: Isotope labeling techniques such as stable isotope labeling by amino acids in cell culture (SILAC), isobaric tags for relative and absolute quantification (iTRAQ), or tandem mass tags (TMT) are used to compare protein expression levels between samples.
- Label-Free Quantification: Proteins are quantified based on the intensity of their peptide signals in mass spectrometry data, without the use of exogenous labels.
- Data Analysis: Statistical and bioinformatic tools are used to analyze and compare protein expression levels across different samples or conditions.
e) Functional Analysis:
- Protein-Protein Interactions: Techniques like co-immunoprecipitation (Co-IP), yeast two-hybrid (Y2H) assays, or affinity purification followed by mass spectrometry (AP-MS) are employed to study protein-protein interactions.
- Post-Translational Modifications (PTMs): Mass spectrometry-based techniques can identify and characterize various PTMs, such as phosphorylation, acetylation, ubiquitination, or glycosylation, which play crucial roles in protein function and regulation.
f) Validation and Verification:
- Western Blotting: Specific proteins of interest are detected and quantified using antibodies in Western blot analysis.
- Targeted Proteomics: Techniques like selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) are used to quantify specific proteins or peptides of interest with high sensitivity and accuracy.
Applications of Proteomics
Proteomics has broad applications across various scientific disciplines and industries. Here are some notable areas where proteomics plays a crucial role:
Disease Research
Contribute to the understanding of disease mechanisms, the discovery and identification of diagnostic biomarkers, and the development of targeted therapeutic approaches Discover key proteins associated with specific diseases by studying protein expression patterns in healthy and diseased tissues and gain insight into disease progression and response to therapy.
Drug Discovery and Development
Proteomics aids in the discovery and validation of potential drug targets. By identifying and characterizing proteins involved in disease pathways, researchers can develop targeted drugs that selectively modulate protein function. Proteomics also helps assess drug efficacy, monitor drug-protein interactions, and investigate drug resistance mechanisms.
Agricultural and Food Science
Proteomics is employed in agricultural and food science to enhance crop yield, improve nutritional content, and ensure food safety. It enables the identification and characterization of proteins associated with crop traits, such as stress tolerance or disease resistance. Proteomic analysis also helps detect food contaminants, allergens, and adulterations.
Environmental Research
Proteomics contributes to environmental research by assessing the impact of pollutants on organisms and ecosystems. It helps identify biomarkers of exposure and evaluate the effects of environmental stressors on protein expression and function. Proteomics also aids in monitoring the health of ecosystems and assessing the success of environmental restoration efforts.
References
- Carnielli, Carolina Moretto, et al. "Combining discovery and targeted proteomics reveals a prognostic signature in oral cancer." Nature Communications 9.1 (2018): 3598.
- Greco, Todd M., Michelle A. Kennedy, and Ileana M. Cristea. "Proteomic technologies for deciphering local and global protein interactions." Trends in Biochemical Sciences 45.5 (2020): 454-455.
- Kwon, Yang Woo, et al. "Application of proteomics in cancer: Recent trends and approaches for biomarkers discovery." Frontiers in Medicine (2021): 1644.
You may be interested in:
