Mass Spectrometry (MS)-Based Sequencing Technology

Mass spectrometry (MS)-based analysis is the preferred method for determining protein quality and structure. MS sequencing refers to the use of MS technology to quickly cleave peptides and determine the amino acid sequence of the primary structure in protein samples. Thanks to decades of experience in developing and executing MS sequencing, CD Formulation uses MS sequencing technology to provide reliable analytical support for the development and characterization of your protein and peptide drugs. These supports cover all aspects from the early R&D stage to late product release testing, ensuring that you obtain high-quality analytical data during the pharmaceutical process to support the effectiveness and safety evaluation of biopharmaceuticals.

About Mass Spectrometry (MS) Sequencing Technology

The core of MS sequencing technology is to decompose protein samples into short polypeptide fragments and accurately measure the mass of these fragments to infer the amino acid sequence and structural information of proteins. It involves ionization, accelerating charged particles into the mass spectrometer, and detecting them based on deflections proportional to the mass-to-charge ratio (m/z). Although traditional protein sequencing methods (such as Edman degradation technology) can systematically eliminate amino acid residues from the amino terminus of peptides to maintain the integrity of proteins. However, the technology is not efficient enough and cannot sequence larger proteins. As a more efficient and flexible alternative, MS sequencing can process multiple proteins at the same time, especially for complex protein samples. Its advantages lie in its high throughput, high sensitivity and ability to analyze complex mixtures. It can also provide information about peptide modifications, such as phosphorylation, glycosylation, etc.

Fig. 1 A typical experimental workflow for protein identification and characterisation using MS/MS data. (Cottrell JS, et al., 2011).

Our Services Related to Mass Spectrometry (MS) Sequencing

Thanks to decades of experience in supporting protein/peptide biopharmaceutical development and manufacturing using MS sequencing technology, our team of highly qualified experts offers a range of MS sequencing-related services to accelerate the implementation and success of your projects.

Our experienced scientists are able to use the most advanced and cutting-edge MS technologies to efficiently identify the characteristics of target proteins, analyze their modification status, monitor consistency, and provide strong data support to meet regulatory requirements.

Typical MS techniques include bottom-up and top-down approaches. Bottom-up MS techniques involve proteolytic digestion followed by analysis, while top-down MS allows for the characterization of intact proteins and their modifications in one step.

Utilizing best-in-class MS sequencing technology, we support the following protein and peptide characterization programs, including but not limited to:

Protein Identification

• Peptide mass fingerprinting: Used to determine the mass of peptides obtained from digested proteins, allowing protein identification via database searches.
• De novo sequencing: For proteins with no available sequence information, MS sequencing can determine the amino acid sequence directly from protein fragments.
• Amino acid sequence confirmation: Used to determine the amino acid sequence of protein drugs, especially useful for the identity of biologics produced by recombinant DNA technology.

Protein Quantification (Absolute Quantification)

MS sequencing can provide quantitative data on protein drug concentrations in complex biological samples by using stable isotope labeling or standard addition methods.

Protein Aggregation Assessment

It is important to understand the aggregation state of protein drugs because aggregation can lead to altered pharmacokinetics, reduced efficacy, or increased immunogenicity. Our scientists use MS sequencing to detect and quantify protein aggregates, which is critical for assessing the safety and efficacy of protein and peptide biopharmaceuticals.

Protein Stability and Degradation Assessment

MS can be used to study the stability of protein drugs under different conditions (e.g., temperature, pH, light) by monitoring changes in protein mass over time, allowing identify cleavage sites and degradation pathways, which is critical for optimizing formulation and storage conditions.

Biosimilarity Assessment

For biosimilars, MS sequencing is essential when comparing the structure and composition of biosimilar protein drugs to their reference products, including evaluating the presence of PTMs, aggregate formation, and overall protein integrity.

Our Procedure for Mass Spectrometry (MS) Sequencing

Fig. 2 Workflow for MS sequencing. (CD Formulation)

• Sample preparation (reduction and alkylation of proteins): This step aims to reduce disulfide bonds in proteins while preventing the generation of free thiols through alkylation.
• Enzyme digestion: Specific proteases (trypsin, chymotrypsin, Asp-N, Glu-C, Lys-C, and Lys-N) are used to cleave the target protein to obtain a mixture of peptides. The selection of enzymes is based on theoretical protein sequences and knowledge of enzyme digestion.
• Peptide separation and peptide detection (LC-MS/MS): Ions are analyzed using a mass spectrometer. The mass spectrometer measures the mass-to-charge ratio (m/z) of the ions and generates a mass spectrum showing the relative abundance of different ions.
• Qualitative analysis and quantitative identification: The obtained mass spectrometry data is analyzed, and specific software is usually used for peptide identification and quantitative analysis. Or, it can be compared with a database to determine the amino acid sequence and source protein of the peptide.

Advantages of Our Mass Spectrometry (MS) Sequencing

• High sensitivity, allowing detection of low abundance molecules.
• Full sequence analysis, using multiple enzyme digestions to ensure full sequence analysis of the entire protein and peptide from the N-terminus to the C-terminus.
• High accuracy, each amino acid position in the sequence is supported by more than ten different peptides, ensuring that each amino acid has strong ion fragment peak evidence in MS/MS for inference, and can accurately identify compounds, such as PTMs.
• Wide applicability, allowing the detection of different types of protein drugs, including monoclonal antibodies, bispecific antibodies, recombinant proteins, etc.
• Fast analysis, MS technology can be performed quickly, enabling high-throughput analysis.
• Label-free, unlike some sequencing methods, MS-based techniques generally do not require label molecules, simplifying sample preparation and reducing detection time.

Custom Mass Spectrometry (MS) Sequencing Services

Why Choose Our Mass Spectrometry (MS) Sequencing Technology?

• Our MS sequencing technology has excellent sensitivity, allowing the detection of low-abundance protein and peptide molecules, and is ideal for studying complex biological samples.
• We use the latest advances in MS technology and software for accurate and reliable data analysis, ensuring the highest quality standards.
• Our team consists of experienced professionals who provide expert guidance throughout the sequencing process, from sample preparation to data interpretation.
• We have accumulated decades of expertise and successful project experience using MS sequencing technology to support the development of protein/peptide biopharmaceuticals.
• Our laboratories are equipped with state-of-the-art MS equipment and technology to support the characterization of any type of protein and peptide.
• We provide customized services to meet specific research needs.
• We provide clear and comprehensive reports to help you easily interpret and utilize data for protein and peptide product development.

Publication

CD Formulation aims to provide a powerful analytical tool for determining protein quality and structure. Please feel free to contact us if you are interested in our services. Learn how our MS sequencing technology can support the smooth implementation of your protein/peptide biopharmaceutical program.

How It Works

STEP 1

Submit a service request describing your specific research or development need.

STEP 2

We'll email you to provide your quote and confirm order details if applicable.

STEP 3

Execute the project with real-time communication, and deliver the final report promptly.