Experiments and Spectra

About

An NMR experiment is the process of data collection on a particular sample on a particular NMR spectrometer under a particular set of conditions, such as temperature, pH etcetera.

The result of an NMR experiment is an NMR spectrum. The raw NMR spectrum is typically time domain data and is thus called a time domain spectrum. Typically, NMR time domain data are transformed into frequency domain data using a Fourier Transform, leading to a frequency domain NMR spectrum.

The Fourier Transformation can be performed with different settings in order to obtain different spectra with different features but originating from the same raw NMR data. For example, one may want to perform 2 transformations leading to 2 spectra: One with resolution enhancement to separate partly overlapping peaks, and one with signal to noise enhancement to observe peaks that are just above the noise level.

Experiments in CcpNmr Analysis

In CcpNmr Analysis, an Experiment is a container in the program that allows the user to describe the NMR experiment and its conditions, e.g.:

- Name of an experiment
- The type of experiment
- Experimental details
- Chemical shift references
- NMR instruments

The user can edit each of these by modifying the Experiment parameters.

Experiments in CcpNmr Analysis are also containers of Spectra (as well as spectrum attributes) and it is important to clearly distinguish Experiments and Spectra (Figure 1). In CcpNmr Analysis, Experiments describe the NMR experiment, whereas Spectra are the actual pointers to the resulting NMR data stored on disk.

Attributes of Experiments

Two important attributes of Experiments are worth mentioning here:

- The Experiment Type
- The Shift List

Spectra in CcpNmr Analysis

In CcpNmr Analysis, a Spectrum is a header that points to a block of data on the disk. Since multiple spectra can be created from a single set of raw data, Analysis can group the spectra that originate from the same experiment into a single Experiment. For example, a 15N-HSQC Experiment in an Analysis project can contain 2 spectra originating from the same NMR experiment but processed with different settings.

Spectra in CcpNmr Analysis have their own specific parameters that are independent from the Experiment parameters, e.g.:

- Display options
- Referencing
- Tolerances
- File details
- Data locations

Attributes of Spectra

An important attribute of a spectrum is that it has peak lists associated with it. The peak list will contain all the peaks that have been identified in the spectrum. A spectrum can have multiple peak lists associated with it, so the user can group peaks into different lists according to their method of detection or creation, assignment status etcetera. For example, one can create one synthetic peak list from chemical shifts, and another peak list for peaks that are picked in a spectrum using automated peak picking.

Figure 1. The relation between Experiments and Spectra in Analysis.

Experiment types

In order to analyse and interpret NMR data, the user needs to know the experiment type: for example whether this was a NOESY or a TOCSY experiment. CcpNmr Analysis uses the Experiment Type for analysing NMR data (semi-)automatically, or to help the user in manual analysis. The experiment type describes the magnetization transfer pathway that was used to record the NMR data, and thus provides information on:

- what kind of NMR resonances or nuclei can be observed
- what the relationships between the different resonances are, i.e. how the resonances in different experimental dimensions are connected.

Full Type

In order to interpret the information present in an Experiment, the user needs to set a Full Type for the experiment, which is a formalism that is used to define the magnetization transfer pathway unambiguously. The experiment formalism is very abstract, may be difficult to use, and requires in depth understanding of the formalism. A detailed explanation of the Experiment Type formalism is described in the paper A nomenclature and data model to describe NMR experiments.

A simple example of the Full Type description is that of a 15N-HSQC or HMQC experiment, which is notated as H[N], where the capitalization indicates the observed nuclei H and N, and the square brackets [] that it is an ‘out and back’ transfer pathway, with magnetization starting on H and being transferred to N and back to H.

Type Synonyms

Since the Full Type selection is difficult to use in practice, Analysis uses Type Synonyms to simplify the correct selection of a Full Type. Type Synonyms are commonly used names for certain experiment types and help the user to filter a number of Full Types. Since there is no clear definition of naming experiments with common names, the Type Synonyms should not be seen as the common name, but rather as a guide to selecting the correct Full Type.

A simple example of a Type Synonym is 15N-HSQC/HMQC, with a Full Type H[N], which can be used for both 15N-HSQC as well as 15N-HMQC experiments.

Experiment dimension

The dimensions of a multi-dimensional NMR spectrum can be indicated in various ways, for example by:

- their detected nuclei, e.g.:
  - 1H, 13C, 15N
  - 1H, 1H, 15N
  - ...
- notations like:
  - F1, F2, F3
  - X, Y, Z
  - 1, 2, 3
- or combinations thereof, e.g.
  - F1: 1H, F2: 13C, F3: 15N

All of these dimensions refer to one of the detected dimensions in the pulse sequence of the experiment that was used. Since different users have different setups for recording and processing NMR data, these experiment dimensions can be in a different order than the reference experiment dimensions in the Experiment type that describes the actual experiment.

In CcpNmr Analysis, a parameter called Experiment Dimension is used to indicate the dimensions in terms of numbers. Depending on the order of the data, these numbers can be different for the same observed nucleus. For example, a 3D-CBCA(co)NH can be processed such that its experiment dimensions are:

- 1: for the amide H dimension
- 2: for the 13Cα and 13Cβ dimension
- 3: for the amide N dimension

but also can be processed with dimensions 1 and 3 swapped:

- 3: for the amide H dimension
- 2: for the 13Cα and 13Cβ dimension
- 1: for the amide N dimension

Reference experiment dimension

In CcpNmr Analysis, an experiment type has reference dimensions associated with it, one for each detected dimension in the pulse sequence of the experiment that is described by the experiment type. Each reference dimension thus refers to the detected nuclei in each evolution period of the NMR experiment, and has a pre-defined number assigned to it.

For example, in CcpNmr Analysis, the 3D-CBCA(co)NH is described by:

- Experiment Prototype H{CA|Cca}CONH
- Reference experiment h{CA|Cca}coNH
- The reference experiment dimensions being
  - 1: for the 13Cα and 13Cβ dimension
  - 2: for the amide N dimension
  - 3: for the amide H dimension

Dimension mapping

In order to interpret the data derived from a spectrum correctly, CcpNmr Analysis requires that the Experiment Dimensions in the experiment are mapped correctly to the Reference Experiment Dimensions. For example, the mapping of a 3D-CBCA(co)NH experiment with Experiment Dimensions:

- 1: for the amide H dimension
- 2: for the 13Cα and 13Cβ dimension
- 3: for the amide N dimension

to the above Reference Experiment Dimensions would look as follows:

In general, when the different dimensions of an experiment are unique, CcpNmr Analysis will detect the correct dimension mapping automatically. In cases where different dimensions in a single experiment have the same type of isotopes, the correct dimension mapping can be deduced from the Atom types associated with the reference dimensions in the Experiment types.

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