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.
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.
Protein De Novo Sequencing Services
Creative Proteomics is equipped with a high-quality Orbitrap Fusion Lumos mass spectrometer and has established a comprehensive database, which enables us to provide high-quality sequencing services of protein full sequence to a wide range of customers. For different experimental needs, we also provide Edman degradation method to determine up to 67 amino acid sequence lengths at the N-terminal end of proteins.
Whole protein de novo sequencing based on Orbitrap Fusion Lumos mass spectrometer complemented by Edman degradation.
After the customer sends the protein product to Creative Proteomics, Creative Proteomics will first identify and fragment the protein sample. We will use six commonly used proteases including Trypsin, Chymotrypsin, Asp-N, Glu-C, Lys-C and Lys- to obtain as A wide variety of fragmented sequences were obtained to achieve full protein sequence coverage. Sequence information from peptide mass spectrometry sequencing is spliced by PEAKs Studio (protein product) and PEAKs Ab (antibody product) software with manual assistance to achieve correctly spliced protein or antibody primary sequences. For more information on de novo amino acid sequencing, please refer to the antibody de novo sequencing service.
Advantages of de novo sequencing technology
(1) Determination of the amino acid sequence of unknown peptides from scratch, independent of known peptide sequence databases.
(2) Ability to localize the post-translational processing or chemical modification of side chain motifs without the restriction of N-terminal closure.
(3) Enables the identification of non-standard amino acids.
(4) Improved detection sensitivity.
- Nano HPLC- 7T solariX XR FTICR-MS (equipped with ECD)
- Nano HPLC- Orbitrap Fusion™ Lumos™ Tribrid™ MS (equipped with ETD)
- Normal amount: 500ug/protein
- Minimum amount: 200ug/protein
- Determine the sequence of protein by HPLC-MS.
- 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.