Stable Isotopes in Proteomics Analysis
What is Proteomics?
Proteomics refers to the study of cell, tissue or organism protein composition and its changing law, protein post-translational modification and protein-protein interactions on the basis of protein biodiversity at a large scale level, so as to reveal the laws and mechanisms related to disease occurrence, development and drug therapy. With the rapid development of mass spectrometry and chromatography-mass spectrometry coupling technology, proteomics analysis technology has become more and more widely used in the identification of unknown proteins, analysis of protein structure, quantification of target proteins, as well as biotechnology drug research and development, quality control, and in vivo pharmacokinetic studies.
What is the Proteome?
The proteome refers to the complete set of proteins that can be expressed by an organism's genome. Unlike the genome, which is relatively stable, the proteome is dynamic and varies with time, developmental stage, and environmental conditions. The proteome includes not only the types of proteins present but also their quantities, modifications, and interactions, providing a comprehensive snapshot of cellular functions and states.
Proteomics Analysis
Proteomics analysis technology is now usually based on mass spectrometry and bioinformatics principles of high-throughput analysis methods, rapid and efficient detection, identification and quantification of protein samples in the composition, quantity and interactions and other basic information, and thus reveal the function and regulation of proteins in the life of the body and other biological characteristics. Mass spectrometry-based proteomics analysis technology is of great significance to identify and analyze intact proteins or enzymatically cleaved peptides, and to carry out the research and application of cellular, tissue or organismal protein composition and its changing law, post-translational modification of proteins as well as protein-protein interactions.
Proteomics Analysis Strategies
There are three main proteomics analysis strategies, including Bottom-up - peptide level proteomics, Top-down - complete proteomics, and Middle-down - subunit level proteomics.Bottom-up is currently the most widely used, and Top-down and Middle-down are the better analysis strategies complementing Bottom-up. down is a better complementary analytical strategy to Bottom-up.
The analytical tools used in proteomics can be used for molecular weight determination, amino acid sequence, peptide profile, disulfide bonds, post-translational modifications (N-glycans, O-glycans, glycoforms, modification sites), and analysis of impurities (truncated bodies, mutants, isoforms, aggregates, and residual proteins in host cells) of recombinant protein drugs. Depending on the characteristics of the analytes and the performance of the instrumentation, one of the three strategies, Bottom-up, Top-down or Middle-down, or a combination of two or three of the three strategies, can be selected to carry out the research and analytical testing. The combination approach can integrate and analyze the analysis results, and the advantages of multiple strategies complement each other to obtain more reliable and accurate information on molecular weight determination, amino acid sequences, peptide profiles, post-translational modifications or impurities.
At present, the analysis of protein advanced structure, also known as traditional structural biology, is also included in the scope of proteomics research, which is mainly carried out by using X-ray analysis technology, nuclear magnetic resonance spectroscopy and transmission electron microscopy analysis technology to carry out the structural analysis of proteins and protein complexes, and the study of protein-protein and protein-activated molecule interactions.
Uses of Stable Isotopes in Proteomics Analysis
Stable isotopes play a pivotal role in the field of proteomics by providing robust methods for quantifying and comparing protein levels across different biological conditions. These isotopic labels offer enhanced accuracy and sensitivity in protein analysis, enabling researchers to uncover detailed information about protein dynamics, interactions, and modifications.
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Quantitative Protein Analysis
Stable isotopes are widely used for quantitative proteomics, enabling precise measurement of protein abundances in different samples.
- Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC): Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC) is a metabolic labeling strategy for in vivo labeling of peptides that has the advantage of being straightforward to implement, quantitatively accurate, reproducible, and useful in measuring relative protein abundance, visualizing protein aggregation, and calculating protein turnover rates.
- Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) and Tandem Mass Tags (TMT): iTRAQ and TMT are chemical tags used to label proteins or peptides with isobaric reagents that differ in their mass only due to the presence of multiple atoms of a stable, nonradioactive isotope(s). To put it simply, these tags enable multiplexed analysis so that multiple samples can be compared in a single mass spectrometry run. This is because the reporter ions produced by these transformative reagents have unique mass signatures that allows you to quantify proteins relative to one another across different conditions or time points.
Protein-Protein Interaction Studies
Stable isotopes facilitate the study of protein-protein interactions (PPIs) by allowing for quantitative assessment of interaction changes under different conditions.
- Cross-Linking Mass Spectrometry (CL-MS): CL-MS employs stable isotope-labeled cross-linkers, which are used to covalently link the proteins that interact. In addition, this method is also useful for identifying sites of interaction and protein complex formations. Moreover, the incorporation of stable isotopes into cross-linkers allows accurate quantification of cross-linking efficiencies and interaction dynamics thereby offering functional inference related to protein structural organization
Post-Translational Modifications (PTMs) Analysis
Stable isotopes are instrumental in studying post-translational modifications (PTMs) by providing quantitative information on modification sites and their functional implications. Key methods include:
- Isotope-Coded Affinity Tag (ICAT): ICAT utilizes stable isotope-labeled tags that bind specifically to cysteine residues in proteins. By comparing the abundance of labeled and unlabeled peptides, researchers can identify and quantify disulfide bond formations, a common PTM. This method aids in understanding the role of cysteine modifications in protein function and stability.
Metabolic Labeling and Tracking
Stable isotopes are used in metabolic labeling experiments to trace protein synthesis and degradation. Key techniques include:
- Metabolic Labeling with Stable Isotopes: In this approach, cells or organisms are fed with stable isotope-labeled nutrients, leading to the incorporation of isotopes into newly synthesized proteins. This method allows researchers to track protein dynamics, study metabolic fluxes, and investigate the effects of different treatments on protein synthesis and turnover.
Types of Stable Isotopes in Proteomics Analysis
Carbon-13 (13C) is a stable isotope of carbon with an additional neutron, resulting in a mass difference compared to the more common carbon-12 (12C). In proteomics, 13C abeling is used to trace the incorporation of carbon into proteins, facilitating the measurement of protein turnover rates and metabolic fluxes. For instance, 13C -labelled amino acids are incorporated into proteins during cell growth, allowing for detailed studies of protein dynamics and synthesis rates.
Deuterium (2H) is an isotope of hydrogen with one neutron, making it twice as heavy as the most common hydrogen isotope (1H). Deuterium labeling is often used in hydrogen-deuterium exchange mass spectrometry (HDX-MS), a technique that provides information about protein structure, dynamics, and interactions. By substituting hydrogen atoms with deuterium, researchers can study protein folding, conformational changes, and binding interactions with high sensitivity.
Nitrogen-15 (15N) is a stable isotope of nitrogen with an additional neutron. 15N labeling is employed in proteomics to enhance the resolution of peptide identification and quantification. By incorporating 15N-labelled amino acids into proteins, researchers can distinguish between peptides derived from different samples based on their mass differences. This method is particularly useful in studies of protein synthesis, turnover, and post-translational modifications.
Oxygen-18 (18O) is a stable isotope of oxygen with two additional neutrons compared to the more common oxygen-16 (16O). 18O labeling is used in proteomics to study protein phosphorylation and other post-translational modifications. By incorporating 18O into proteins during enzymatic reactions, researchers can differentiate between phosphorylated and non-phosphorylated forms,
Advantages of Stable Isotopes in Proteomics Analysis
The use of stable isotopes offers several advantages in proteomics analysis:
- Enhanced Sensitivity and Accuracy: Stable isotopes improve the detection limits of mass spectrometry, allowing for more accurate quantification of proteins and their modifications.
- Quantitative Precision: Isotopic labeling provides precise quantitative measurements of protein abundance and dynamics, enabling accurate comparisons between experimental conditions.
- Minimized Matrix Effects: Stable isotopes reduce interference from complex sample matrices, leading to more reliable and reproducible results.
- Multiplexing Capability: Stable isotopes allow for the simultaneous analysis of multiple samples or conditions, increasing the efficiency of proteomics experiments.
