Each step of the de novo amino acid sequencing method requires expertise and experience in order to get the best results quickly (Figure 2).
- Sample preparation. Ensure the sample purity by first running SDS-PAGE. More specific and purpose built purification methods will get the best downstream results.
- Digestion. Use pepsin, trypsin or other enzymes to ‘cut’ the protein into peptides for analysis. Pay attention to the quality of the enzyme used for digestion. Also consider using multiple enzymes.
- LC-MS/MS. HPLC separates peptides, which are then fed into a mass spectrometer for analysis. It is essential to use a mass spectrometer built for proteomics. Care must be taken in the fragmentation method for the MS2, and in the selection window to ensure optimal coverage.
- Peptide de novo sequencing. Interpret each mass spectrum to determine the sequence of each peptide. Software is available for this purpose, but the latest, most advanced algorithms get answers faster with less ambiguity. Expert human interpretation may still be required post software analysis.
- Sequence assembly. Construct the full length protein sequence from the peptide sequences. Look for as much overlap as possible. Expert human interpretation may still be required post software analysis.
Figure 2. Infographic providing an overview of the methodology behind de novo amino acid sequencing.
While ESI-based MS instruments initially contained one mass analyzer and one ion detector, nowadays, ion cells are typically flanked by the ion source and the ion sensor and sandwiched between electrodes that can modulate the frequency and voltage to ‘select’ a window for the desired m/z ratio and then redirect the flow of ions for additional fragmentation, for instance.
The ion source, and mass analyzers and ion detectors are all kept under vacuum. An example of an ion cell that can act as either or both a mass analyzer and an ion detector is the ion trap.
As peptides travel between mass analyzers and ion detectors, collisions can fragment these peptides further via high-energy collision dissociation (HCD), or electron-transfer high-energy collision dissociation (EThcD). All of the internal systems feed into the instrument control. Essentially, at any of these points, data can be detected such that the user may select a specific range of ions and record the spectra of these fragments for data analysis.
A mass spectrometer that comprises different sequential cells with mass analyzer and ion detector capabilities is referred to as a tandem mass spectrometer. Mass spectrometers used to only be able to house a mass analyzer and an ion detector. However, mass spectrometry technology has evolved to such a sophisticated level that current tandem mass spectrometers house many more components than original instruments, conveniently fitting in one corner of a laboratory.