Why N-terminal (Edman) sequencing ?
N-terminal protein (Edman) sequencing remains a reliable method for obtaining N-terminal sequence of intact proteins.
Top-down MS methods which also allow analysis of intact proteins requires highly purified protein, does not allow determination of first 7-10 residues and is not compatible with SDS gel electrophoresis. In some cases, TOP-down MS can provide N-terminal sequence of terminally blocked proteins and sometimes C-terminal sequence. Moreover, the method is not robust enough to be used routinely.
Bottom-up MS methods require protein processing (cleavage) before analysis making it difficult to find and analyze actual N-terminal peptide.
- Sensitivity: 5-10 pmol
- Purified protein or a mixture 2-3 proteins can be identified
- N-terminal sequence up to 50-70 residues, in most cases 7-10 residues are sufficient for protein identification
- Compatible with SDS-PAGE (after blotting to PVDF membrane)
- Isobaric (Ile/Leu) and near isobaric (Gln/Lys) residues are reliably determined
- Requires free N-terminal amino group (unblocking is possible in some cases)
- Analysis is essentially de novo (does not depend on sequences data base).
ISD MALDI-TOF MS:
- 10-50 pmol
- Purified protein at high concentration (~1 mg/ml)
- Determines N- and C-terminal sequence up to 50-70 residues. First 7-10 residues are not easily determined
- Does not depend on the status of N-terminal amino group
- Not compatible with SDS-PAGE
- Not quantitative
- Isobaric (Ile/Leu) and near isobaric (Gln/Lys) residues are not determined
- X-Pro bond is not fragmented
- Analysis is database dependent
Automated Edman protein sequencing is especially valuable for characterization of recombinant proteins and determination of proteolytic sites in proteins.
It is complimentary to MS methods.
Combination of Edman automatic sequencing and MS based methods for protein analysis is the advantageous because the methods are complimentary: some analytical questions are easily answered by Edman sequencing and for other questions, MS is more appropriate.
The summary above is from the Association of Biomolecular Resource Facilities (ABRF) Protein Sequencing Research Group study reported in 2009 at the annual meeting. The purpose of the study was to investigate traditional and alternative methods for obtaining the N-terminal sequence of a protein.