De Novo Protein Sequencing

A: The fundamental distinction between protein sequencing and gene sequencing lies in their respective levels of examination. Protein sequencing is primarily concerned with the amino acid level, while gene sequencing focuses on the nucleotide level. In situations where the nucleotide sequence is unknown or no reference database is available, protein sequencing can independently provide the complete amino acid sequence of the protein, thereby offering potent support for related research.

A: De novo sequencing employs algorithms for protein sequencing, directly inferring peptide sequences from ion information in mass spectra. Conversely, the Edman degradation method is typically suitable for peptides that consist of 15-20 amino acids and requires a high purity level for the sample, usually around 97% or above. Moreover, this method necessitates protein digestion, peptide purification, and individual, sequential machine tests. Therefore, the time and cost investments for monoclonal antibody sequencing through Edman degradation are comparatively substantial.

Contrasted with the traditional Edman method, mass spectrometry-based de novo sequencing displays superior efficiency, throughput, and cost-effectiveness. Given the known amino acid sequence of a protein, de novo sequencing can reveal new protein variants, which could emanate from unknown mutations, splice junctions, and various post-translational modifications, thus providing a more comprehensive picture of monoclonal antibody sequences.

A: Protein de novo sequencing is irrespective of the size or length of a protein, accommodating a wide range of sample types. This includes, but is not limited to, monoclonal antibodies (mAbs), Fab/Fc fragments, bispecific antibodies (bsAbs), multispecific antibodies, recombinant proteins, peptides, fluorescence-labeled antibodies, and cross-linked antibodies. Essentially, the technique is extraordinarily versatile, applicable to virtually all types of protein samples irrespective of their size and complexity.

A: Indeed, both antibodies cross-linked to beads and those labeled with fluorescent markers such as FITC, Cy5, and PE are compatible with de novo sequencing processes, demonstrating the versatility of this approach.

A: The standard requirement for protein de novo sequencing is a sample quantity of 100µg with a purity level exceeding 90% coupled with a proven track record of successful sequencing with at least 20µg. It is important to emphasize that protein purity considerably influences the outcome of the sequencing process. If the sample contains impurities such as antibody proteins, homologous proteins, or Bovine Serum Albumin (BSA), these could interfere with the sequencing process, thereby potentially compromising the experimental outcome.

A: In antibody sequencing, the required sample volume varies depending on the sequencing type. For monoclonal antibody sequencing, as long as the purity of the antibody meets the requirement, 100 μg of antibody protein would suffice for sequencing. However, the scenario for sequencing mixed antibodies in serum is more complicated and requires a specific pre-initial assessment, to ascertain the appropriate sample volume.

A: Our sequencing service employs comprehensive de novo sequencing to deliver a complete amino acid sequence report. This includes not only heavy and light chains, but also encompasses both the variable and constant regions.

A: For protein solubilization, our recommended buffer solutions typically involve PBS or Tris buffer systems. If the sample contains elements such as glycerol, BSA, detergent, or salt, we'll undertake appropriate purification techniques to mitigate their influence, thereby ensuring they do not adversely affect the sequencing output.

A: During the data analysis phase, potential post-translational and glycosylation modifications are taken into account. Therefore, such modifications do not affect the sequencing results of the sample itself.

A: Utilizing advanced data analysis software, we are capable of identifying a range of common types of post-translational modifications, further pinpointing the corresponding modification sites. This provides substantial support for studying the functionality of proteins and their regulatory mechanisms within biological organisms.

A: The main equipment utilized in de novo protein sequencing comprises Thermo Fisher's tribrid ultra-high resolution mass spectrometers, specifically the Orbitrap Fusion and Orbitrap Eclipse models. With the continuous advancement in mass spectrometry technology, Creative Proteomics platform is actively committed to introducing more high-end devices to meet the diverse requirements of our clientele, thereby ensuring the delivery of premium quality service.

A: The de novo protein sequencing report comprehensively features the following elements: (1) the full depiction of the protein amino acid sequence, inclusive of antibody heavy and light chain sequences, as well as constant and variable regions; (2) comprehensive molecular weight validation data, achieved through the comparison of observed molecular weight with theoretical sequence molecular weight, enabling the confirmation of sequence accuracy; (3) the portrayal of peptide coverage maps of an entire sequence, ensuring amino acid residue support from more than ten distinct peptide variants at each position; (4) the demonstration of secondary mass spectrograms supporting antibody variable region peptides; (5) the provision of a credibility analysis for I/L identification.

A: To date, we have successfully completed thousands of projects. The accuracy of an individual amino acid sequence can reach 100%, with each amino acid in the sequence report being corroborated by as many as 30 or more different overlapping peptide spectra.

A: Absolutely, protein sequencing can pinpoint if the peptides being identified derive from humans or microbes. This discrimination is achieved through the analysis of species-specific unique peptides.