N- and C-terminal sequences are important structures and functional parts of protein and polypeptide, and they may play decisive roles in the biological function of protein. The main methods for protein C-terminal sequencing include carboxypeptidase, chemical and tandem mass spectrometry, and the common methods for N-terminal sequencing include Edman degradation sequencing and mass spectrometry. Each of these methods has the respective advantage and shortcoming. Therefore, the combination of a variety of different sequencing methods can adapt to the requirements of multiple protein sequencing. For example, Edman degradation may not well solve the problem of blockage and protein modification on the N-terminal end. This drawback can be overcomed by mass spectrometry analysis. MtoZ Biolabs uses both Edman degradation system and MALDI-TOF for N/C sequencing of biopharmaceutical products. This combination of two methods ensures accurate analysis of N-terminal blockage and modified protein N/C terminal.
Disulfide bond (S-S bond) is formed by oxidation of the sulfhydryl group (-SH) on two cysteines in the protein. It is an important post-translational modification of protein. Disulfide bond is essential for protein molecules to maintain the correct advanced structure and maintain protein bioactivity. The distribution of disulfide bonds in antibody drugs is a direct structural characteristic of the drugs. Therefore, confirmation of disulfide bonds plays a very important role in the confirmation process of antibody drug structure. MtoZ Biolabs has developed a high-resolution mass spectrometry, coupled with pLink-SS software, to provide our customers with accurate analysis of disulfide bonds and free cysteines. Our sample preparation steps have also been optimized to prevent in vitro exchange of disulfide bonds, and maintain native structure.
Application of Disulfide Bond Analysis
(1) Identification of the number of disulfide bonds in the biopharmaceuticals and free cysteines
(2) Identification of the position of disulfide bonds in the biopharmaceuticals
The mutation of the end of the antibody drug is divided into the N terminal and the C terminal variations. The common variation at the N end is usually due to incomplete processing of the N- terminal signal sequence, resulting in the N- terminal methionine residue being not removed or the N- terminal Glu and Gln residues partially or completely forming pyroglutamic acid. The common mutations at the C end are sequence truncation, such as the deletion of the C terminal Lys residue of the heavy chain in the monoclonal antibody, which is also known as K deletion. These changes in the protein terminals may occur in the fermentation conditions, or caused by condition change in the protein purification buffer. For example, it has been reported that the monoclonal antibody derived from B cell hybridoma and CHO cell transfected tumor have a very significant variation in the proportion of C- lysine deletion. Another case is that when a monoclonal antibody is being modified, the ratio of lysine to the C-terminal end also changed significantly. These two common cases reveal the importance of analyzing the c-terminal variation. In the ICH Q6B guideline, C terminal K deletion detection is mandatory. The methods of IEF, cIEF, ion exchange chromatography and LC-MS are common in analyzing the variation of lysine at the C-terminal end. MtoZ Biolabs uses LC-MS for antibody C-terminal variation analysis, including detection of the proportion of K deletion in the C-terminal end of antibody and other types of truncation on antibody C-terminal.
Reports
• Experiment procedures
• Parameters of liquid chromatography and mass spectrometer
• MS raw data files
• Antibody c-terminal variation results
• Bioinformatics analysis
In recent years, the use and number of biotherapeutics has increased significantly. For these protein-based therapies, the quantitation of aggregates is of particular concern, given their potential effect on efficacy and immunogenicity. This need has renewed interest in size-exclusion chromatography (SEC). SEC is a chromatographic method, which acts as a sieve and separates molecules in solution by their size, and in some cases molecular weight. Molecular sieves are materials containing precise and single tiny holes, which can be used to adsorb gases or liquids. SEC has long residence time for small molecular weight compounds into gel pores, whereas large molecular compounds are washed away earlier. The advantage of molecular sieve chromatography is that it can efficiently process at least 1nmol of target protein. The separation time is also very short, generally within 3 hours, and the chromatographic separation peak obtained at the same time is also smaller.
Advantages of SEC in Protein Purification
The liquid chromatography is divided into positive and reverse phase chromatography according to the relative polarity of the mobile phase and the stationary phase. Reversed-phase liquid chromatography (RPLC) refers to chromatographic method that uses a hydrophobic stationary phase. RPLC has the characteristics of high resolution and high repeatability, therefore it has been widely used in protein and polypeptide analysis, esp. for the separation of small molecular weight proteins and protein fragments with molecular weight <20000 Da. MtoZ Biolabs has established a platform with both SEC and RPLC technologies, to meet the requirements of analyzing various biopharmaceutical samples.
Reports
• Experiment procedures
• Parameters of liquid chromatography
• Protein purity results
• Bioinformatics analysis
In the life of an organism, the function of proteins is achieved by the interaction between proteins and proteins. Therefore, the study of new proteins can provide new directions for the study of the potential function of proteins.
MtoZ Biolabs has advanced native MS analysis platform, Exactive Plus EMR mass spectrometer and Protein Deconvolution 3.0 software for analyzing native structure of biopharmaceuticals.
"Molecular mass is the most fundamental characteristics for proteins and peptides. It directly reflects the structural integrity of proteins, and hence it is commonly applied in identification of proteins and peptides. To better assist the scientific research of our clients, MtoZ Biolabs provides macromolecular mass measurement service based on Bruker ultrafleXtreme™ MALDI TOF/TOF, and high-resolution protein mass measurement service via nano LC-MS. This service can be applied to accurately analyze the molecular mass, subunits, and dimerization/polymerization of multiple biotechnological molecules, including proteins, peptides, antibodies, and vaccines, etc.
Application
• Measurement of the molecular mass of protein/peptide/antibody/vaccine, etc.
• Analysis of number of subunits in protein sample
• Analysis of dimerization/polymerization of protein sample
• Evaluation of sample purity and protein/peptide contaminants
Reports
• Experiment procedures
• Parameters of liquid chromatography and mass spectrometer
• MS raw data files
• Peptide identifications and intensity
• Protein identifications and intensity"
"MtoZ Biolabs utilizes 2D-nano LC-MS/MS technology for protein identification. In our general workflow, protein complexes are separated in 1D/2D gel and the target protein is then digested into peptide fragments, followed by HPLC separation and tandem MS analysis. We use Scaffold and Mascot software for analyzing peptide MS data, and ensuring confident protein identification.
Reports
• Experiment procedures
• Parameters of liquid chromatography and mass spectrometer
• MS raw data files
• Peptide identifications and intensity
• Protein identifications and intensity
"In the life of an organism, the function of proteins is achieved by the interaction between proteins and proteins. Therefore, the study of new proteins can provide new directions for the study of the potential function of proteins. In many protein tests, pull-down assays are often used to test protein-protein-interaction. Pull-down's experiment uses highly purified and enriched bait protein to capture the targeted proteins that specifically interact with the bait protein in the cell, and greatly improve the efficiency of identification of new target protein. The specific steps of pull-down's experiment are to express the bait protein modified with poly-His, or Biotin tags. Using immobilized affinity ligand to link the bait protein to the solid-phase matrix, and then mix the bait protein with the protein sample. In this process, targeted protein specifically interacts with the bait protein, and thus, the targeted proteins are isolated from the protein sample mixture. This process can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins. Identification of the target protein is then processed by the mass spectrometer. Using CST pull-down assay kit, combined with advanced UPLC-MS platform, MtoZ Biolabs provides quick and accurate pull-down protein analysis service.
Reports
• Experiment procedures
• Parameters of liquid chromatography and mass spectrometer
• MS raw data files
• Peptide identifications and intensity
• Protein identifications and intensity"
"As continuous development of Antibody-drug conjugates (ADCs) and the improving needs for precise determination of drug structure, drug characterization technology has also been constantly updated. When the traditional mass spectrometer technology cannot fully meet the demand for precise determination of complex drugs, the technology of non-variable character spectrum (Native MS) has emerged. Native MS has wide application, such as measurement of drug to antibody ratio (DAR) of ADCs. ADCs drugs are divided into two kinds of drugs, which are lysine and cysteine conjugated drugs. Determination of lysine-conjugated drugs is often based on the traditional RPLC/MS technology, and the protein needs to be measured under degeneration condition. However, degeneration condition can result in the separation of heavy and light chains of cysteine-conjugated drugs, thus, cysteine-conjugated drugs must be measured by native MS technology.
MtoZ Biolabs has advanced native MS analysis platform, Exactive Plus EMR mass spectrometer and Protein Deconvolution 3.0 software for analyzing native structure of biopharmaceuticals. With fast scanning speed, high sensitivity and wide detecting range, Exactive Plus EMR mass spectrometer enables accurate analysis of the structure and topology of protein and protein complexes in native conditions. Other than these advantages, Exactive Plus EMR mass spectrometer also provides the best solution for analyzing peptide and small molecules. "
Protein primary sequence determines the advanced structure of a protein, influencing a protein’s function. Therefore, it is essential to analyze the sequence of a protein, to verify the integrity and correct translation of a protein sample. MtoZ Biolabs has developed a comprehensive protein sequencing platform to meet various needs of our clients, from N-terminal and C-terminal sequencing to full-length, de novo sequencing and protein sequence analysis. Our featured services are presented below, but if you have any special requirement, you are welcome to contact us for custom service.
"Accurate determination of protein sequences is fundamental in the development of commercial monoclonal antibodies, vaccines, and diagnostic kits. Traditional sequencing methods uses database searching for protein sequencing, and has limitations for sequencing the unknown proteins, which may not be included in the database. De novo sequencing is a novel sequencing methods, with no need for database searching, and therefore, is the optimal method for analyzing unknown proteins and antibodies. Based on the world's most advanced mass spectrometry instrument, Obitrap Fusion Lumos, and combined with rich experience in bioinformatics analysis, MtoZ Biolabs has established a whole new generation of de novo sequencing platform that can achieve accurate sequence analysis of monoclonal antibodies and proteins, independent of any database searching. The primary sequence of proteins and antibodies and any mutations on the sequence can be analyzed quickly and accurately, without any prior information of the protein.
Advantage of de novo sequencing:
• Advanced high-resolution mass spectrometry technology
• 100% protein/antibody sequence coverage
• 95% accuracy for sequencing the first amino acid
• Minimum amount of sample required"
"MtoZ Biolabs uses the most advanced mass spectrometer-Orbitrap Fusion Lumos, for de novo sequencing service. Coupled with our professional bioinformatics analysis and reverse sequence confirmation, high-quality sequence analysis is guaranteed. Our de novo sequencing service is also well-suited for analyzing multiple kinds of antibodies, including IgM and IgG antibody subtypes, and antibodies modified with FITC, biotin, and Alexa, etc.
http://www.mtoz-biolabs.com/protein-de-novo-sequencing.html"
"Monoclonal antibody plays a key role in the development of biopharmaceuticals and diagnostic kits. The primary structure of the antibody, especially the amino acid sequence of the CDR region, is the core of antibody’s biological function. Accurate and rapid analysis of its complete sequence is of great importance for characterization of antibody products. Analysis of the primary antibody sequence is the basis for antibody development and humanization for developing antibody-based drugs. However, due to the variation and modification of antibody products, the sequence information of most antibodies is often not included in the available databases. De novo sequencing technology uses advanced data processing algorithm for sequence analysis, has a superior advantage versus database dependent traditional methods. Antibody sequence can be accurately analyzed using the de novo sequencing method, with no need for database searching.
Based on the world's most advanced mass spectrometry instrument, Obitrap Fusion Lumos, and combined with rich experience in bioinformatics analysis, MtoZ Biolabs has established a new generation of antibody de novo sequencing platform to achieve accurate analysis of the primary antibody structure. This platform is suitable for sequence analysis of different subtype antibodies, such as IgG and IgM, and different types of antibodies, including fluorescently conjugated antibodies, immobilized antibodies, and antibodies from different species. "
"Protein N-terminal is the start point of the expression of a protein. It also influences the subcellular distribution, degradation, and the turnover rate of a protein. Thus, sequence analysis of the N-terminal of a protein is very important for studying the function of a protein. Aimed to provide comprehensive sequencing service, MtoZ Biolabs has developed a sophisticated N-T sequencing platform consisting of Edman sequencing and protein N-T de novo sequencing, with their advantages complementary to each other.
In this service, protein sequence is not restricted by sample types, N-terminal blockage, and PTMs. Protein N-T sequences can be accurately analyzed by Edman sequencing, whereas, the N-terminal blockage and PTMs can be analyzed by mass spectrometry."
Protein integrity is a crucial charateristics for protein, as protein truncation or microheterogeneity can lead to malfunction of protein. It is even more critical for the quality tests of biopharmaceutical products, such as antibodies and vaccines. Thus, analysis of the C-terminal sequence of protein products is necessary for evaluating the quality and function of protein products. MtoZ Biolabs have developed a LC-MS/MS system, which is suitable for analyzing both N-terminal sequence and C-terminal amino acid sequence and PTMs. We strictly follow the ICH Q6B Guidance to fulfill our customers’ needs.
Edman sequencing method analyzes the protein N-terminal sequence in sequential Edman reactions. While mass spectrometry technology has been commonly used for protein analysis, Edman sequencing is still a powerful and irreplaceable method for protein N-terminal sequencing. As a well-established sequencing method, Edman sequencing provides more accurate protein sequence data, compared to MS. MtoZ Biolabs is aimed at providing reliable protein analysis service. Our protein/peptide N-terminal sequence analysis is performed by SHIMADZU PPSQ-33A Analyzer, equipped with the newest HPLC analysis system.
"""Full-length sequence information is essential for studying the integrity and correct translation of biopharmaceuticals. In general, protein sequence analysis service provided by MtoZ Biolabs includes three steps: digestion of proteins into smaller peptide fragments; tandem LC-MS/MS analysis of peptides; MS/MS data analysis. Traditional protein sequencing only uses trypsin for digestion, which renders the final protein sequence coverage of around 60%.
To ensure 100% protein sequence coverage, MtoZ Biolabs uses up to 6 kinds of protein enzymes for protein digestions, eliminating any missing peptides.
Applications
• Determination of full-length sequence of Proteins/Peptides/Antibodies/Vaccines
• Analysis of PTMs & chemical modifications on protein
• Verification of the correct translation of Recombinant Proteins
Visit http://www.mtoz-biolabs.com/protein-full-length-sequencing.html for service details.
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"Although hybridoma cells are immortalized, extensive passage of hybridoma cells may cause mutations on antibody genes, and hybridomas may loss their specificity to produce the correct antibody. Therefore, sequence information of antibody is of extreme importance for stable quality of antibody in large-scale industrial manufacturing. To solve this problem, MtoZ Biolabs has developed a PCR based antibody sequencing workflow, coupled with degenerate primer design. Compared with the mass spectrometry-based antibody sequencing method, this PCR-based antibody sequencing method is much more cost effective and less time consuming. To better meet your research needs, we provide a full-package service, covering the whole analytical steps involving antibody sequencing. You can simply send us your hybridoma cells, and we will perform all the following analytical experiments.
Visit http://www.mtoz-biolabs.com/pcr-based-antibody-sequencing.html for service details.
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"Proteomics is a large-scale study of proteins in a certain organism. Depending on specific research objective, there are many analytical techniques for proteomics study. To better meet our clients’ needs, MtoZ Biolabs is proud to offer 5 quantitative proteomics service, including Label-free, iTRAQ/TMT, SILAC, MRM, and SWATH. We also provide custom proteomics service for specific project. You are welcome to contact us for more detailed information on project design and sample requirements.
Service Category:
label-free proteomics
iTRAQ/TMT
SILAC/Dimethyl
SWATH
MRM
Visit http://www.mtoz-biolabs.com/quantitative-proteomics.html for service details. "
"Label-free analysis is a powerful technique for identifying and quantifying relative changes in complex protein samples. In this method, samples are run individually and identified by MS/MS information. Relative quantitation of different samples is then analyzed by aligning chromatographic peak areas of precursor ions between various runs. The most distinctive advantage is that it quantifies proteins without any use of labels, and can analyze unlimited number of samples. Since Label-free is relatively simple and easy compared to other proteomics techniques, it has been widely used for proteomics study and biomarker discovery. MtoZ Biolabs is proud to offer Label-free quantitative proteomics service with faster simpler results. We can also perform MS3 analysis to ensure higher analytical accuracy of qualification and quantitation.
Visit http://www.mtoz-biolabs.com/label-free.html for service details. "
"Isobaric Tag for Relative Absolute Quantitation (iTRAQ) and Tandem Mass Tags (TMT) are two similar quantitative proteomic techniques developed by AB SCIEX and Thermo Fisher, respectively. The most distinctive advantage of these two techniques is that they can label up to 10 different samples and compare the relative proteomic change through LC-MS/MS analysis. Therefore, these techniques are widely applied to study proteomic changes under different treatments or in different development stages. MtoZ Biolabs is proud to offer iTRAQ/TMT analysis service to meet your research needs. On the basis of these two well-established techniques, we can also provide MultiNotch MS analysis service through MS3 or proton-transfer reactions (PTR) to further increase the accuracy of the proteomic quantitation.
Visit http://www.mtoz-biolabs.com/itraq-tmt.html for service details. "
"Similar to SILAC technique, Dimethyl labeling is also an isotopic labeling method. However, Dimethyl labeling is a chemical labeling process, and labels lysine or the N-terminal amino acid residues on peptide, instead of labeling proteins. Except for the difference in labeling method, SILAC and Dimethyl labeling technique have comparable sensitivity and accuracy, and are being more and more widely used in relative proteomics studies.
MtoZ Biolabs has developed a specialized platform, equipped with Q Exactive HF (Thermo Fisher), Orbitrap Fusion, and Orbitrap Fusion Lumos mass spectrometers equipped with Nano-LC for SILAC/Dimethyl analysis service.
Visit http://www.mtoz-biolabs.com/silac-dimethyl.html for service details. "
"Sequential Window Acquisition of all Theoretical fragment ions (SWATH) is a brand new mass spectrometry technology co-developed by Dr. Ruedi Aebersold research group (ETH Zürich) and AB SCIEX. SWATH data is an independent acquisition (DIA) technique. Unlike data dependent acquisition technique, which only selects certain MS spectrum for MS/MS detection, SWATH analyzes all MS and MS/MS spectra, allowing detection of virtually all detectable proteins in a sample. In SWATH technology, MS spectra are divided into several narrow windows and peptides in each window are sequentially analyzed equally, followed by MS/MS analysis. SWATH technique combines the high-throughput quality of shotgun proteomics with high accuracy comparable to MRM. To better meet our clients’ needs, MtoZ Biolabs offers a comprehensive SWATH service, including optimized sample preparation, high-quality SWATH mass spectrometry analysis, etc.
Visit http://www.mtoz-biolabs.com/swath.html for service details. "
"Multiple Reaction Monitoring (MRM) is a quantitative method for analyzing targeted proteomics. Based on the sequence information of targeted proteins, MRM selectively analyzes proteins correlating with the targeted peptide signals, eliminating signals from interfering peptides. MRM has the highest accuracy among all proteomics quantitation methods, enabling absolute quantitation of protein with reference to internal standard, and analysis of up to 200 proteins at the same time. MtoZ Biolabs is proud to offer an all-inclusive MRM service. Just tell us your project objective and send us the protein sample, we will perform all the corresponding experiments, and provide the most professional analytical service to meet your specific needs.
Visit http://www.mtoz-biolabs.com/mrm.html for service details. "
