Stable Isotope Labeled Peptides

Creative Peptides is equipped with a strong team of technical personnel and advanced equipment. We have rich experience in research and development and production of new isotope compounds, and constantly breakthrough and innovate to synthesize different types of stable isotopes according to the requirements of our customers, and we always guarantee high quality and efficient service. Creative Peptides can customize peptide products containing different stable isotope atoms according to customers' requirements, and deliver them quickly after completion on time.

What are stable isotope labeled peptides?

Stable isotope labeled (SIL) peptides are chemically synthesized peptides with natural sequences. In stable isotope labeled peptides, one or more atoms in the peptide chain are replaced with the corresponding stable isotope, such as deuterium (2H), carbon-13 (13C), or nitrogen-15 (15N), instead of its common natural isotope. Stable isotope labeled peptides are chemically similar to unlabeled peptides. Labeled and unlabeled peptides exhibit similar behavior in terms of reactivity, solubility, and adsorption.

Fig. 1 Overview of quantification approaches in LC-MS based proteomic experiments. (Mueller, L.N., 2008)

Advantages of stable isotope labeled peptides

Improved sensitivity and specificity: Incorporating stable isotopes into peptides can enhance the sensitivity and specificity of detection methods such as mass spectrometry. Isotope labeling reduces background noise and increases signal intensity, thereby improving the detection limit and accuracy of peptide identification.

Normalization and standardization: Isotope-labeled peptides serve as robust internal standards for normalizing variations in sample preparation, instrument performance, and experimental conditions. They enable standardization across different analytical platforms and laboratories, facilitating reliable and reproducible results.

Absolute quantification: Isotope-labeled peptides allow for absolute quantification of target analytes by comparison with known amounts of labeled standards. This approach bypasses the need for relative quantification methods, providing accurate measurements of peptide concentrations in complex biological samples.

Multiplexing and parallel analysis: Isotope labeling enables multiplexing and parallel analysis of multiple peptides or proteins within the same sample. Different isotopic forms can be synthesized and mixed together for simultaneous detection and quantification, increasing throughput and efficiency in large-scale studies.

Applications of stable isotope labeled peptides

Stable isotope-labeled peptides find applications across various fields in biomedical research, including:

Quantitative proteomics: Isotope-labeled peptides serve as internal standards for accurate quantification of proteins in complex biological samples. They enable precise measurement of protein abundance, aiding in the identification of biomarkers and elucidation of signaling pathways associated with diseases such as cancer.

Metabolic labeling: Isotope-labeled peptides are used in metabolic labeling experiments to track the incorporation of labeled amino acids into newly synthesized proteins. This approach allows researchers to study protein turnover, protein-protein interactions, and post-translational modifications, providing insights into cellular dynamics and disease mechanisms.

Targeted proteomics: Isotope-labeled peptides are used as internal standards in targeted proteomic assays such as selected reaction monitoring (SRM) and parallel reaction monitoring (PRM). These assays enable highly sensitive and specific quantification of predefined sets of proteins or peptides, making them valuable tools for biomarker validation, pharmacokinetic studies, and clinical diagnostics.

Structural biology: Isotope labeling combined with techniques such as nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography enables detailed structural and dynamic characterization of peptides and proteins. By selectively labeling specific amino acids, researchers can probe protein folding, ligand binding, and conformational changes, facilitating structure-based drug design and rational protein engineering.

Pharmacokinetics and drug metabolism: Isotope-labeled peptides are employed in pharmacokinetic studies to assess the absorption, distribution, metabolism, and excretion (ADME) of drugs and therapeutic peptides. By administering labeled compounds to animal models or human subjects, researchers can track their fate in vivo, determine key pharmacokinetic parameters, and optimize drug dosing regimens for efficacy and safety.

Protein-protein interactions: Isotope labeling combined with techniques such as co-immunoprecipitation (co-IP) or cross-linking mass spectrometry (XL-MS) enables the identification and characterization of protein-protein interactions within cellular networks. By labeling interacting partners with different isotopes, researchers can distinguish specific binding events from nonspecific interactions, unraveling the molecular mechanisms underlying complex biological processes.

Protein engineering and biotechnology: Isotope labeling is utilized in protein engineering and biotechnology applications such as protein expression, purification, and characterization. By incorporating isotopically labeled amino acids during recombinant protein production, researchers can facilitate downstream analysis, improve protein stability, and enhance structural studies.

Synthesis steps of stable isotope labeled peptides

At Creative Peptides, we can customize the synthesis of many types of peptides. Based on our specialized capabilities and technologies, we can respond proactively to the changing needs of high-quality stable isotope labeled compounds. Upon completion of the custom synthesis, all stable isotope labeled peptides are subjected to rigorous mass spectrometry and HPLC analysis to determine final purity and ensure that our clients receive only the highest quality peptides. Creative Peptides is a reliable partner to work with to accelerate the pace of achieving your research goals.

• Peptide sequence design: This process involves obtaining the customer's requirements and evaluating and optimizing the designed sequence.
• Isotope labeling selection: Selection is made based on the common stable isotope options below and the feasibility of the solution is evaluated.
• Stable isotope peptides synthesis: After completing the above steps, peptides can be prepared using our specialized isotope labeling techniques. Isotope labeling techniques include, deuterium labeling, carbon labeling, nitrogen labeling and oxygen labeling.
• Labeling efficiency and quality control: Labeling efficiency is the ratio of stable isotopes of the labeled peptide, ensuring high efficiency and high purity of labeling can improve the accuracy and reliability of the experimental results. And after the completion of stable isotope labeling of peptides, quality control is needed to ensure the purity and correct labeling.

Stable isotope options

• ²H: The symbol ²H represents the isotope Deuterium, a stable isotope of hydrogen. It contains one proton, one neutron, and one electron.
• ¹³C: ¹³C stable isotope labeled peptides are peptides that contain a ¹³C isotope, a non-radioactive and stable form of carbon, which is used as a tracer in scientific research.
• ¹⁵N: The naturally occurring nitrogen atom (¹⁴N) in the peptide has been replaced by the stable isotope ¹⁵N.
• ¹⁵N and ¹³C combinations: Refer to molecules that contain both these isotopes.
• ¹⁸O: Refers to a stable isotope of oxygen. It is known as Oxygen-18 and it has 10 neutrons and 8 protons in its nucleus.

Stable isotope labeled peptide modifications

In addition to their fundamental role in structural analysis and quantitative proteomics, SIL peptides can be further enhanced through various modifications, expanding their utility and versatility in biological research. SIL peptide modifications offer researchers the flexibility to tailor peptides to specific experimental requirements, mimic biological conditions, and probe the effects of post-translational modifications (PTMs) on protein function and regulation.

Phosphorylation mimics

One common SIL peptide modification involves mimicking phosphorylation, a ubiquitous PTM that regulates diverse cellular processes. Phosphorylation mimics can be introduced by incorporating phospho-amino acid analogs, such as phospho-serine or phospho-threonine, into SIL peptides. These modified peptides enable researchers to study the effects of phosphorylation on protein-protein interactions, enzyme activity, and cellular signaling pathways.

Acetylation and methylation analogues

Acetylation and methylation are essential histone modifications involved in chromatin remodeling and gene expression regulation. SIL peptides containing acetylation or methylation analogues allow researchers to investigate the interplay between histone modifications, chromatin structure, and transcriptional regulation. These modified peptides serve as valuable tools for studying epigenetic mechanisms underlying development, disease, and cellular reprogramming.

Glycosylation mimetics

Glycosylation is a crucial PTM that modulates protein stability, localization, and function. SIL peptides modified with glycosylation mimetics enable researchers to explore the impact of glycosylation on protein structure and interactions. By synthesizing SIL peptides with glycan analogues attached to specific amino acid residues, researchers can elucidate the role of glycosylation in protein-protein recognition, immune response modulation, and disease pathology.

Ubiquitination and SUMOylation probes

Ubiquitination and SUMOylation are protein modifications involved in protein degradation, signaling, and cellular homeostasis. SIL peptides containing ubiquitination or SUMOylation probes allow researchers to study the dynamics of these PTMs and their functional consequences. By incorporating ubiquitin or SUMO analogues into SIL peptides, researchers can investigate the specificity of E3 ligases, the regulation of substrate recognition, and the crosstalk between different PTM pathways.

Customized modifications

Beyond these examples, SIL peptide modifications can be tailored to mimic a wide range of PTMs and structural features found in proteins. Whether introducing bulky side chain modifications to mimic protein-protein interfaces or incorporating stable isotopes into specific amino acid residues to probe conformational changes, customized SIL peptide modifications offer endless possibilities for designing experiments and addressing biological questions.

Data interpretation and analysis

Creative Peptides provides isotope ratio calculations, metabolic kinetic studies, and pharmacokinetic analyses when using stable isotope-labeled peptides for experiments and analyses to ensure proper interpretation of data and appropriate statistical analysis. Finally, Creative Peptides will deliver the synthesized stable isotope-labeled peptides with associated reports, including purity, quality analysis results, and confirmation of isotope labeling.

• Stable isotope labeled peptide of specified quantity and purity
• HPLC chromatogram
• MS spectra
• Certificate of analysis

Our Services

Creative Peptides provides stable isotope labeled custom peptides for determining protein structure and dynamics or protein-protein interactions because it allows for the doping of NMR active nuclei, which helps to reduce the complexity of spectroscopy and helps researchers to obtain correlations between atoms for more complete structural information.

• Design and synthesize peptides with expected mass differences
• Tracking peptide metabolic pathways and kinetics
• Quantitative analysis, especially in metabolic studies and protein quantification
• Various modifications possible, e.g. disulfide bond formation
• Tracking of intracellular location and migration of proteins
• Cost-effective analytical services

Why choose Creative Peptides?

Precision and accuracy: Our state-of-the-art technology ensures the highest quality stable isotope labeled peptides, delivering precise and reliable results for your experiments.

Customization: We understand that every research project is unique. That's why we offer customizable solutions to match your specific requirements, providing you with the flexibility you need.

Expertise: Backed by a team of experienced scientists and researchers, we offer unparalleled expertise in peptide synthesis and stable isotope labeling, guaranteeing exceptional service and support at every step.

Fast turnaround: We prioritize efficiency without compromising quality. Benefit from quick turnaround times, allowing you to accelerate your research timeline and achieve your goals sooner.

Comprehensive solutions: Whether you're conducting proteomics research, drug development, or metabolic studies, our stable isotope labeled peptides services cater to a wide range of applications, empowering you to explore new avenues of discovery.

FAQ

1. What are the advantages of using stable isotope-labeled peptides?

• Accurate quantification: Stable isotope-labeled peptides serve as internal standards, allowing for precise quantification of target peptides or proteins.
• Improved sensitivity: Isotope-labeled peptides enhance the detection sensitivity and dynamic range of mass spectrometry.
• Multiplexing capability: Different isotopic labels can be used to distinguish multiple samples within a single experiment, enabling high-throughput analysis.

2. How are stable isotope-labeled peptides synthesized?

Stable isotope-labeled peptides are typically synthesized using solid-phase peptide synthesis (SPPS) techniques. During synthesis, amino acids containing stable isotopes are incorporated at specific positions within the peptide sequence. The resulting labeled peptides are then purified and characterized for use in mass spectrometry experiments.

3. What applications are stable isotope-labeled peptides used for?

• Relative protein quantification (e.g., comparing protein expression levels between different samples or conditions).
• Absolute protein quantification (e.g., determining the absolute concentration of specific proteins).
• Targeted proteomics (e.g., quantifying specific peptides or proteins of interest in complex biological samples).

4. Can stable isotope-labeled peptides be customized for specific applications?

Yes, many providers offer customization services where stable isotope-labeled peptides can be synthesized and optimized according to your specific experimental requirements, including peptide sequence, labeling scheme, and purity.

5. What are the typical turnaround times and costs for stable isotope-labeled peptide synthesis services?

Turnaround times and costs can vary depending on the complexity of the synthesis, the quantity and purity of labeled peptides required, and the customization options. It's best to inquire with the service provider for specific details regarding turnaround times and pricing.

6. Are stable isotope-labeled peptides compatible with different mass spectrometry platforms?

Yes, stable isotope-labeled peptides can be used with various mass spectrometry platforms, including triple quadrupole, quadrupole-time-of-flight (Q-TOF), and Orbitrap instruments, among others. Compatibility may vary depending on the labeling scheme and specific experimental protocols.

References

1. Becker GW. Stable isotopic labeling of proteins for quantitative proteomic applications. Briefings in Functional Genomics and Proteomics. 2008, 7(5): 371-382.
2. Kettenbach, A.N., et al. Absolute quantification of protein and post-translational modification abundance with stable isotope–labeled synthetic peptides. Nature Protocols. 2011, 6(2): 175.
3. Ong, S.E., et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Molecular & Cellular Proteomics. 2002, 1(5): 376-386.
4. Mueller, L.N., et al. An assessment of software solutions for the analysis of mass spectrometry based quantitative proteomics data. Journal of Proteome Research. 2008, 7(01): 51-61.