Unfortunately, many biomolecular NMR spectroscopy labs use non-standard methods for determining the 1H, 13C or 15N “zero-point” chemical shift position.
SHIFTCOR uses several simple statistical approaches and pre-determined cut-off values to identify and correct potential referencing, assignment and typographical errors.
The results contain the chemical shift analyses (including lists of potential mis-assignments, the estimated referencing errors, the estimated error in the calculated reference offset (95% confidence interval), the applied or suggested reference offset, correlation coefficients, RMSD values) and the corrected BMRB formatted chemical shift file (see Figure 1 for details).
A key limitation to the SHIFTCOR approach is that requires that the 3D structure for the target protein be available to assess the chemical shift reference offsets.
Given that chemical shift assignments are typically made before the structure is determined, it was soon realized that structure-independent approaches were required to develop.
[5] Several methods have been developed that make use of the estimated (via 1H or 13C shifts) or predicted (via sequence) secondary structure content of the protein being analyzed.
These programs have all been shown to accurately identify mis-referenced and properly re-reference protein chemical shifts deposited in the BMRB,.
As a general rule, PANAV and PSSI typically exhibit a smaller spread (or standard deviation) in calculated reference offsets, indicating that these programs are slightly more precise than either LACS or CheckShift.
Evidently, chemical shift referencing continues to be a significant, and as yet unresolved problem for the biomolecular NMR community.