Chemical Shift Referencing and Offsets (tof, dof, dof2)


In order to properly reference NMR spectra in nmrPipe or FELIX you need to know the chemical shifts of the center (carrier) frequencies in the 1H, 15N, and 13C dimensions. These are obtained by properly referencing a 1D 1H spectrum using internal DSS. Once this is done, the chemical shifts of the carriers based on the currently set values of dof and dof2 can be calculated. In addition, the appropriate values of dof and dof2 can be calculated to set the carriers at known chemical shift values.

1H Referencing

First obtain a proper reference in the 1H dimension with a 1D spectrum using an internal reference (DSS) (preferred method). If necessary, one could also use the water line at, e.g.4.7 ppm, but this is not as accurate and is no longer acceptable.
  • use the water presat experiment (the one you use for finding tof) to collect a 1D proton spectrum.
  • place the cursor on the DSS peak, type ‘nl’, and type 'rl(0p)'.
  • type “dscale” and “full f ds” to check the referencing
  • type “centersw” to move the cursor to the exact center of the spectrum
  • finally, type “cr?” and note the center position in hertz, or note the chemical shift (in ppm units) in the center of the spectral display window
  • write this number down - you will need it below for setting the 15N and 13C referencing. Note the tof - it must be same in the spectra obtained below.

Heteronuclear Referencing (N15 / C13)

  • read in the pulse sequence (e.g., hnco) and collect the 1st 1D increment using the tof determined above.
  • set the cursor to the center of the spectrum by typing “f, full, centersw”.
  • set the chemical shift of this point by typing “rl(center value in ppm or hertz determined above)”, e.g. rl(4.78p) or rl(1500). [Note: do not forget to append the 'p' to indicate that the value is in ppm; or omit the p to specify hertz].
  • find the proper dof or dof2 value by executing one of the following macros and then setting dof or dof2 to the returned value.
  • repeat this for the 3rd dimension if present. These ppm values will be correct values to use for referencing the spectrum in nmrPipe and nmrView.
  • “dof2cal(118)” for N15 to find the dof2 value corresponding to 118 ppm (as an example)
  • “dofcal(174)” for C13 to find the dof value corresponding to 174 ppm (as an example

Displaying Referenced Spectra

This step is not necessary and is only useful for displaying the spectrum in Vnmr with proper axis units. After running and processing the multidimensional experiment, do the following to display the correct referencing in Vnmr.
  • make sure the axis parameter is set correctly for the type of experiment. (For example, axis = 'p2' for Nhsqc; axis = 'pd' for Chsqc; axis = 'pp2' for a noesyNhsqc; axis = 'pd2' for hnco.)
  • now set the display reference by using one the following macros.
  • ncalsw1 - for N15 in 2nd dimension
  • ncalsw2 - for N15 in 3rd dimension
  • ccalsw1 - for C13 in 2nd dimension
  • ccalsw2 - for C13 in 3rd dimension

Determining the reference after the fact

You can often determine the correct center frequency of data that has been measured at some earlier time if the data was referenced with DSS prior to data collection as described above. To find the H1 center frequency, which is already properly referenced, do the following
  • centersw
  • cr? (to find center in hertz) or read the ppm value from bottom of graph window
  • To get the other dimensions use one of the following macros. You can give the macros an argument (in Hz) or use the default values of dof/dof2. See below for more detail.
  • dof_ppm - macro for C13 dof
  • dof2_ppm - macro for N15 dof2
  • If the data was not referenced as described in “1H Referencing” above, then you must compare peak positions with a spectrum that has been properly referenced.
  • If there are no identical peaks to compare with a properly referenced spectrum, then you will have to calculate the referencing using the following equation
  • [dof(2) - dof(r)] / dfrq = ppm(2) - ppm(r)
  • where dof(r) and ppm(r) are the dof and carrier ppm of the referenced spectrum, dof(2) is the dof of the spectrum not referenced, and ppm(2) is the carrier ppm that you want to know. Warning: this may not be valid if dfrq has changed by very much.

Setting Chemical Shift referencing in nmrPipe

Use the ‘varian’ program from nmrPipe and set the values for the 1H, 13C, and 15N center (carrier) ppm values to those determined above. These values can only be applied to spectra that have been collected as described above and where proper referencing to internal DSS has been done.
Data collected in the past without referencing to DSS can only be properly referenced by comparison of the chemical shifts to those found in properly referenced spectra. 1H and 15N chemical shifts can be obtained from a 2D 15N HSQC spectrum. 1H, 15N, and 13C shifts are easily obtained from properly referenced HNCO. The systematic deviation found in each dimension can be added to the old data to properly reference the spectra.


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Setting the 15N carrier (center) frequency

dof2cal: used to calculate what value of dof2 will give a desired chemical shift before you collect a spectrum. The dof2 appropriate for a specific chemical shift is calculated using the macro dof2cal(ppm) where ppm is the numeric value of the chemical shift where you want to place the carrier, e.g. dof2cal(119). Note this macro assumes that the referencing in the proton dimension is done properly. Do not use this macro while acquiring data.
dof2_ppm: used to calculate the chemical shift (in ppm) of the center of the N dimension after you have aquired a spectrum. Calculate the chemical shift of the carrier in the 15N dimension using the macro dof2_ppm. If an argument is given, e.g. dof2_ppm(2500), the chemical shift is returned for this offset (The offset is in Hz, the value returned is in ppm). If no argument is given, the chemical shift is returned for the value of dof2 currently specified in the parameter list. Note this macro assumes that the referencing in the proton dimension is done properly and that the spectrometer frequency has not changed. Do not use this macro while acquiring data.
To check the spectrometer frequencies open up the procpar file with an editor and find the value dfrq2. Compare this to the value returned by the VNMR command spcfrq.

15N reference

Once the 1H dimension is properly referenced, the 15N chemical shift reference can be calculated from the tof and dof (or dof2) and the spectrometer frequencies using the ratio of the zero frequencies for 1H and 15N (0.101329118 using DSS and liquid NH3, respectively. See J. Biomolecular NMR 12, 1-23, 1998 for IUPAC-IUBMB-IUPAB recommendations, and Cavanagh's book "Protein NMR Spectroscopy", p. 176). Collect the first 1D increment (ni=0, phase=1) for a 15N HSQC spectrum using the same tof in the proton dimension as for the 1H reference spectrum above. Of course the DSS line cannot be observed in this spectrum and cannot be used to set the 0 ppm point. Set the cursor to the center of the spectrum by typing rfl=0, rfp=0, cr=sw/2. Set the chemical shift of this point to the value determined in the 1H 1D spectrum above by typing rl(center value in ppm or hertz determined above), e.g. rl(4.73p) [Note: do not forget to append the 'p' to indicate that the value is in ppm] or rl(1500) [Note: no unit to specify hertz]. This establishes the proper reference parameters, including reffrq, for 1H and 15N referencing from the 1H spectrum.
ncalsw1 - Type ncalsw1 to set the calibration for the 15N dimension in a 2D experiment (e.g. 15N hsqc) where 15N is the second dimension. Do not use this macro while collecting data as it moves the tof.
ncalsw2 - Type ncalsw2 to set the calibration for the 15N dimension in a 3D experiment (e.g. HNCO) where 15N is the third dimension. Do not use this macro during data acquisition.

Setting the 13C carrier (center) frequency

dof_ppm: Type dof_ppm(offset) where offset is the numeric value of dof or the variable name [e.g., dof_ppm(dofcacb)]. The value returned is the chemical shift of the carrier (center) frequency in the second dimension. If no argument is given, e.g. dof_ppm, the currently specified dof is used.
dofcal: If you want to know what offset to use for a specific 13C chemical shift, use the macro dofcal(ppm) where ppm is the value of the chemical shift that you want the carrier to be at.

13C reference

Once the 1H reference is established, the 13C chemical shift reference can be calculated from the tof and spectrometer frequencies using the ratio of the zero frequencies for 1H and 13C (0.251449530 using DSS for 1H and 13C. See J. Biomolecular NMR 12, 1-23, 1998 for IUPAC-IUBMB-IUPAB recommendations, and Cavanagh's book "Protein NMR Spectroscopy", p. 176). Collect a the first 1D increment for a 3D spectrum (e.g. HNCO) using the same tof in the proton dimension as for the 1H reference spectrum above. Set the cursor to the center of the spectrum by typing rfl=0, rfp=0, cr=sw/2. Set the chemical shift of this point to the value determined in the 1H 1D spectrum above by typing rl(center ppm value determined above), e.g. rl(4.73p) (Do not forget to append the 'p' to indicate that the value is in ppm). This establishes the proper reference parameters, including reffrq, for 1H and 13C referencing from the 1H spectrum.
ccalsw1 - Type ccalsw1 to set the calibration of the 13C dimension in a 2D or 3D experiment where 13C is displayed in the second dimension (e.g. C HSQC). If ccalsw1 is not followed by an argument, the dof value is used for setting the 13C scale. If ccalsw1 is followed by an argument, e.g. ccalsw1(56), the value in parentheses is used to set the center frequency in the 13C dimension (used when the carrier is shifted in the pulse sequence either explicitly or with shifted pulses).
ccalsw2 - Type ccalsw2 to set the calibration of the 13C dimension in a 3D experiment where 13C is displayed in the third dimension. Can be used with or without an argument as described for ccalsw1.


Macros

The vnmr macros used above are macros developed for us and are not part of the normal vnmr distribution
dofcal, dof2cal, dof_ppm, dof2_ppm, ccalsw1, ccalsw2, ncalsw1, ncalsw2