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Home > Services > Other Services > De Novo Peptides/Proteins Sequencing Service

De Novo Peptides/Proteins Sequencing Service

The sequence of peptide/protein is important to study the biological  function of the peptide/protein. However, complete characterization of  peptides/proteins, including post-translational modifications (PTMs), sequence  mutations and variants, is very challenging. There are two approaches to  determine the sequence of peptide/protein by mass spectrometry: database search  and de novo sequencing. Database search approach compares acquired mass spectra  to a database of known protein sequences to identify the protein sequences. De  novo sequencing is a process in which amino acid sequences are directly  interpreted from tandem mass spectra without the assistance of a database.

Peptide/Protein De Novo Sequencing

Although database search identification of proteins by mass  spectrometry is well established, the method does not apply if the protein  sequence does not exist in the current database. Therefore, de novo sequencing is  the only method for identifying novel peptides, unsequenced organisms, and antibodies  drugs, which database search methods were not able to detect. However, de novo  sequencing poses more challenging than the traditional database search  approach, such as, ambiguous assignments of fragment ions, insufficient product  ions generated in incomplete fragmentation leading to low sequence coverage and  difficulty in distinguishing ion series, notably N-terminal from C-terminal  MS/MS product ions (b ions from y ions).

At Creative Proteomics, we tackle these challenges by using the  following four strategies. Firstly, Fourier transform ion cyclotron resonance  mass spectrometry (FTICR-MS) has the highest mass resolution and accuracy to  avoid the false positivity led by low mass resolution and accuracy. With 7T  solariX XR FTICR-MS, the mass resolving power can research 10,000,000. As the  mass accuracy increases, the resulting assignments become increasingly  confident. Secondly, both bottom-up and top-down mass spectrometry is used to  analyze the same sample. Bottom-up allows us to use different enzyme digestions  to generate overlapping peptides while top-down mass spectrometry offers intact  mass data and provides protein fragmentation details. By combing these data,  the peptide/protein sequence can be confirmed in a better way. Thirdly, four types of fragmentation  techniques: collision induced dissociation (CID), electron transfer  dissociation (ETD), electron capture dissociation (ECD) and high energy  collisional dissociation (HCD) are employed for peptide/ protein fragmentation,  which could provide more fragment ions from the same peptide/protein and these  complementarity data will sure to improve the ion assignment. For example, it  would be possible to distinguish C-terminal (z•) from N-terminal (c’)  ECD product ions based on the ratio of prime to radical ion abundance in ECD vs  activated-ion ECD (AI-ECD) MS/MS product ion mass spectra. Finally, we also  provide chemically derivation to identify ion series. For example, introduce  bromide on the C-terminus by oxazolone chemistry can enable identification of y  ions because of the distinct bromide isotope peaks. Similarly, introduction of guanidination  can increase the possibilities of identification and the selectivity.

Technology platform

  • Nano  HPLC- 7T solariX XR FTICR-MS (equipped with ECD)

  • Nano  HPLC- Orbitrap Fusion™  Lumos™ Tribrid™ MS (equipped with ETD)

Sample Requirement

  • Normal  amount: 500ug/protein

  • Minimum  amount: 200ug/protein

Summary

  • Determine  the sequence of protein by HPLC-MS.

Report

  • A  detailed technical report will be provided at the end of the whole project,  including the sample preparation, HPLC-MS/MS parameters.

  • The  sequence (FDR<1%) of the protein, molecular weight will be reported in the  report.

Ordering Procedure:

Ordering Procedure


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