What is Peptide Mapping?
Peptide mapping is a powerful analytical technique used to unravel the intricacies of proteins. It involves digesting a protein using enzymes like trypsin, Lys-C, Glu-C, or chymotrypsin. This enzymatic digestion breaks the protein into smaller fragments known as peptides. These peptides are then separated by HPLC and analyzed via mass spectrometry, providing critical insights into the protein's primary structure and composition.
Why Peptide Mapping?
Peptide mapping plays a pivotal role in protein characterization and provides detailed information about the primary amino acid structure of proteins. Further, peptide mapping is indispensable for confirming identity, providing primary structural insights, and ensuring quality assurance and quality control (QA/QC) of biopharmaceutical such as monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs). More specifically, peptide mapping can be used for:
- Confirming the Primary Sequence:Peptide mapping can confirm the primary sequence of the protein and identify any errors or misincorporations in the amino acid sequence, which can affect the protein function and stability. These errors can occur due to mutations, transcription errors, or translation errors, and they can have significant implications for the quality of biopharmaceutical products.
- Identifying Post Translational Modifications:Peptide mapping can identify post translational modifications (PTMs) of the protein, such as phosphorylation, deamidation, oxidation, ubiquitination, nitrosylation, methylation, acetylation, lipidation and glycosylation. PTMs are changes in the protein structure that regulate its function and have various implications for research and drug development. For example, PTMs can affect the protein activity, interactions, localization, stability, and signaling.
- Mapping Disulfide Bonds:Peptide mapping can map the location of disulfide bonds in the protein, which are covalent bonds that stabilize the protein structure and influence its folding and activity. Disulfide bonds are formed between cysteine residues in the protein sequence, and they can be intra-chain or inter-chain. Peptide mapping is used to determine the number and position of disulfide bonds by comparing the mass spectra of reduced and non-reduced peptides.
- Detecting Impurities:Peptide mapping can detect impurities or contaminants in the protein sample, even at very low concentrations. Impurities can arise from various sources, such as degradation products, host cell proteins, endotoxins, or residual solvents. Peptide mapping can identify these impurities by comparing the mass spectra of the sample with a reference standard or a database. Impurities can affect the safety and efficacy of biopharmaceutical products, so it is important to monitor them during development and manufacturing.
LC-MS Advantages
LC-MS-based peptide mapping offers unparalleled specificity and detailed structural information.
Specifically:
- High Sensitivity: LC-MS-based peptide mapping excels in detecting even trace amounts of peptides and proteins. This high sensitivity is invaluable for the analysis of samples with limited availability or when identifying impurities at low concentrations.
- Accurate Mass Measurement: Accurate mass determination is crucial for confident identification of peptides and post-translational modifications. Advanced instruments, such as high-resolution mass spectrometers, provide precise mass measurements, enhancing the reliability of results.
- Relative Quantitative Analysis: The technique facilitates relative quantitative analysis, enabling comparisons of peptide or protein abundances across different samples. This is essential for understanding changes in expression levels or the relative abundance of post-translational modifications in various conditions.
- Robust Across Different Matrices: LC-MS-based peptide mapping is a versatile and robust method applicable across diverse sample matrices. This adaptability is particularly beneficial in proteomics, where samples and research questions can vary widely.
- Detection of Diverse PTMs: LC-MS-based peptide mapping can identify and quantify a wide range of post-translational modifications (PTMs) not easily detectable by other methods, including phosphorylation, ubiquitination, methylation, acetylation, and sulfonation. This capability aids in the comprehensive characterization of proteins.
Our Expertise
At Emery Pharma, we leverage our extensive experience in LC-MS-based peptide mapping studies. We use state-of-the-art technology, including the Orbitrap Exploris™ 240 Mass Spectrometer, to deliver precise and reliable results. Whether you need detailed characterization of your protein, identification and quantification of post translational modifications, or detection of impurities, we can provide you with the best solution for your needs. We offer customized services, comprehensive reports, and data analysis/interpretation. Contact us today to find out how we can help you with your peptide mapping project.
Example :
The following spectra were obtained from a peptide mapping project on the NIST Monoclonal Antibody Reference Material 8671. All results were obtained here at Emery Pharma.
LC-Chromatogram after tryptic digest:
Sequence Coverage Map after tryptic digest:
Scientific Solutions in Action: Three Real-Life Case Studies from Emery Pharma
Case # 1
Emery Pharma was approached by a client planning a clinical study involving patients with severe arthritis. Our role was to analyze synovial fluid samples collected from these patients to uncover proteomic insights. Upon receiving the samples, our team designed and implemented a robust analytical workflow capable of processing up to 30 samples simultaneously.
The process involved enzymatic digestion of the samples using trypsin, followed by liquid chromatography- high-resolution mass spectrometry (LC-HRMS) analysis. Utilizing our state-of-the-art Thermo Exploris 240 system and its advanced data processing software, we generated detailed proteomic profiles for each sample. This approach enabled the identification and quantification of a wide range of proteins present in the synovial fluid, providing valuable data to support the client’s clinical research objectives.
Case # 2
In another real-world example, Emery Pharma collaborated with a gene therapy company focused on gene editing. Their objective was to confirm the success of gene modifications, whether through suppression of protein expression or editing a gene to induce production of an alternative protein. Our team supported both approaches by developing a tailored analytical workflow. We received routine samples from the client and provided rapid turnaround to meet their R&D timelines. The core of our approach involved classic peptide mapping using LC-HRMS with the Thermo Exploris 240 Orbitrap platform.
Leveraging both in silico tools and experimental confirmation, we were able to precisely evaluate the protein expression profiles, helping the client verify the outcomes of their gene editing strategies with confidence.
Case #3
Emery Pharma was engaged by a client conducting a comprehensive clinical study involving a therapeutic cocktail of six distinct antibodies administered to patients over a two-year period. The primary objective was to develop a method for quantifying each antibody in patient blood samples collected at various time points.
Initially, we designed individual Lys-C based digestion workflows for each antibody. However, the client ultimately requested a unified, single-run method to quantify all six antibodies simultaneously-an ambitious task given the complexity of the mixture, the need for high specificity, and challenges surrounding critical reagent availability.
Our team began by performing detailed peptide mapping for each antibody using high-resolution LC-HRMS. We identified unique peptides for each antibody, peptides that were not only distinct from one another but also absent from the native human plasma proteome. These peptides were then synthetically produced and used to create reference standards.
Building on this foundation, we transitioned the assay to a triple quadrupole mass spectrometry platform, enabling simultaneous quantification of all six antibodies in a single analytical run. This streamlined approach met the client’s needs for high-throughput, precision, and reproducibility and the work has since been published.
Top 15 Frequently Asked Questions on Peptide Mapping:
Peptide mapping is a powerful analytical technique used to assess the primary structure of proteins. It involves enzymatically digesting a protein (typically with trypsin) into smaller, well-defined peptide fragments. These peptides are then separated by liquid chromatography (LC) and analyzed using detection methods such as mass spectrometry (MS) or ultraviolet (UV) detection.
The resulting chromatographic and spectral profile serves as a molecular fingerprint of the protein. Peptide mapping is critical for:
- Verifying protein identity
- Detecting amino acid sequence variants
- Monitoring post-translational modifications (PTMs) such as oxidation, deamidation, or glycosylation
- Mapping disulfide bonds
- Identifying impurities or degradation products
It is a cornerstone method in the development, manufacturing, and quality control of biopharmaceuticals and is routinely used to ensure consistency across production batches.
Conclusion
Peptide mapping is a critical tool for scientists, pharmaceutical developers, and regulatory agencies to ensure the identity and integrity of therapeutic proteins. At Emery Pharma, we perform these analyses using qualified equipment, established workflow with the highest level of precision and care. Our commitment to scientific rigor and customer service is at the core of everything we do.
Our team brings extensive experience across a broad range of applications, proteins derived from gene editing, supporting clinical studies with proteomics, and the characterization of proteins from alternative food and novel protein sources. We invite you to contact us to schedule a consultation and explore how we can support your project.