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.