Hello, we have a peptide where amide peaks are broad and there is no hint of observable coupling with HA protons (no discernible multiplet structure - even with line-sharpening apodization). Is there a method to measure them anyway? Will E.COSY type of experiments still be helpful to obtain the couplings?
Are there homonuclear (1H) two-dimensional experiments that allow estimation of scalar coupling by the intensity of cross-peak without resorting to heteroatom isotope labeled samples? Pseudo-3D experiments?
Are there ready pulse sequences in the standard libraries or elsewhere that can help here?
Four solutions come to mind, two of which necessitate 15N labeling, but the other two may be used with an unlabeled peptide.
First, there's the 3D HNHA experiment, in which the 3JHN-HA is measured by comparing the intensity of the amide "diagonal" peak to the intensity of the HN-HA crosspeak: Vuister, G. W.; Bax, A. Journal of the American Chemical Society 1993, 115, 7772-7777.
Second, there's a 2D HMQC derivative in which peaks in two spectra, taken with and without 1HN decoupling, are integrated and compared: Ponstingl, H.; Otting, G. Journal of Biomolecular NMR 1998, 12, 319-324.
Third, Frank Delaglio has a module for NMRPipe called ACME that extracts scalar coupling constants from 2D COSY spectra. I understand it uses a combination of linefitting and intensity measurement. See: http://spin.niddk.nih.gov/NMRPipe/
Fourth, I've had good results by fitting crosspeaks in constant time COSY (CT-COSY) spectra, which are absorptive in F1, but dispersive in F2. This experiment is actually really nice as a substitute for the DQ-COSY, but it's also easy to extract individual F2 traces and fit them to a dispersive shape to extract coupling constants. A bit labor intensive if you wish to measure several dozen or more coupling constants, but not too bad for a little peptide. (1) Girvin, M. E. Journal of Magnetic Resonance Series A 1994, 108, 99-102.