Chenomx NMR Suite is available for:

• Windows XP SP3 / Vista / 7 (32 and 64-bit)
• Mac OS X 10.5 / 10.6 (64-bit)
• Linux x86 (32 and 64-bit)

Chenomx NMR Suite requires that you have the latest Java VM installed. You can download that here.

Chenomx NMR Suite currently supports raw data in these formats:

• Varian (fid)
• Bruker (fid)
• JEOL (.jdf)
• NMRPipe (.fid)

and processed data in these formats:

• Varian (phasefile)
• Bruker (1r)
• JEOL (.jdf)
• NMRPipe (.ft2)
• JCAMP-DX (.jdx, version 5.1 and higher)

The Chenomx Compound Library supports spectra at 400, 500, 600, 700, and 800 MHz.

The latest version of Chenomx NMR Suite can handle saved data from all previous versions of the software.

The Chenomx Compound Library is based on the characteristics of DSS, since its chemical shift does not change with pH; the chemical shift of TSP does change with pH. When you select TSP as a CSI, your spectra are rereferenced as if they had been acquired using DSS to allow accurate comparison with the compound library. Following this adjustment, the chemical shift for TSP is not exactly 0 ppm.

We recommend that you add a supported CSI to all samples intended for use with Chenomx NMR Suite (currently, we support DSS, TSP and formate). You should consider adding a CSI to any new samples that you intend to analyze using Chenomx NMR Suite. That being said, you can still extract some information from existing spectra of samples with no CSI.

When you open a spectrum of a sample that contained no CSI, the software has no reference with which to calibrate the converted spectrum; you need to explicitly specify a reference for the spectrum in order to complete the conversion.

First, you need to set the location of the CSI peak such that 0 ppm is close to where it should be based on visible features in the spectrum. Start by selecting DSS as a CSI during import (this is the default option). When the import is complete, and the spectrum appears, use the CSI Editor to place the red peak as close as possible to where you want 0 ppm to be. As you move the red peak, the whole x-axis (horizontal) scale updates to reflect your changes, so you can check the current setting against known positions of other peaks in the spectrum, too. When you have the spectrum referenced to your satisfaction, you can save it, and proceed with profiling.

When you have manually referenced spectra using this technique, please remember:

1. Absolute compound concentrations measured in Profiler will be inaccurate, but relative concentrations will still be reasonably accurate. For example, if you measure acetate at 1 mM and alanine at 2 mM, those numbers will not necessarily reflect the 'real' concentrations of those compounds in the sample. However, it would be fair to say that alanine is present at twice the concentration of acetate.
2. The transform windows may not be optimal for all compounds; you may not be able to move a cluster to exactly where you would like it to be. To fine tune your manual referencing, you can click Jump to Processor, tweak the CSI position in the CSI Editor, and then click Jump to Profiler to continue profiling.

When you import spectra of samples that do not contain a supported CSI (DSS, TSP or formate), keep the default CSI settings of DSS at 0.5 mM. This ensures a common starting point for all of your spectra.

In the absence of a supported CSI, you will need to set reasonable values for the position (chemical shift), width and height (intensity) of the CSI peak using the CSI Editor in Processor.

1. Referencing the chemical shift to another compound requires knowledge of the expected chemical shift of the alternate compound. Basically, zoom in as far as possible while keeping both the red CSI peak and the alternate peak visible. Then, click and drag the red triangle on the horizontal axis until the scale lines up properly with the alternate peak. For example, if the alternate peak should have a chemical shift of 2.34 ppm, drag the red triangle until 2.34 on the horizontal scale lines up with the alternate peak.
2. To set the width, start by setting the CSI width to about 1.2 Hz. Most of the compounds in the Chenomx library were originally fit using spectra with about this linewidth, so this is a good starting point for many high-quality spectra. If you realize while analyzing the spectrum in Profiler that most compound signatures are considerably narrower or wider than the corresponding features in the spectrum, switch to Processor (use the Jump to Processor feature), adjust the CSI width accordingly, and switch back to Profiler (the Jump to Profiler feature lets you do this with the click of a button).
3. Height is the easiest of the three. When you are manually referencing a spectrum, do not modify the height of the CSI peak away from its starting value.

Remember, this technique will not allow accurate absolute quantification, but you will still be able to identify compound. Relationships among concentrations within the same sample will work (you should be able to tell that creatinine was twice the concentration of alanine in sample X), but comparisons across multiple samples will generally not work (you can not necessarily say that alanine appeared in sample X at twice the concentration that it did in sample Y).

In addition to setting the chemical shift, the CSI in Chenomx NMR Suite allows calculating concentrations of compounds in your compound library. The 'extra' information that you need to enter about the CSI is what allows these calculations to occur.

Calculating the absolute concentration of any compound based on its signal intensities in an NMR spectrum requires the following information:

1. The number of protons contributing to the signal from the compound
2. The number of protons contributing to the signal from the reference compound
3. The intensity of a signal from a reference compound
4. The concentration of the reference compound
5. The intensity of a signal from the compound

When you are using Chenomx NMR Suite, item #1 is stored in the compound signatures distributed in the Chenomx Compound Library, or any signatures that you might create yourself using Spin Simulator and Compound Builder.

#2 is built into the definitions for the supported CSI compounds (DSS, TSP and formate).

You define #3 and #4 when you process a spectrum in Processor, via the CSI editor. You set #3 when you fit the red CSI line to the spectrum using the CSI Editor, and #4 when you enter a CSI concentration in mM.

Finally, you manipulate #5 as you fit the compound in Profiler.

In short, the concentrations that you determine in Profiler depend on the number of protons (as you might expect), but they also necessarily depend on the concentration of the reference compound (as may be less obvious). It is not possible to calculate absolute concentrations using NMR without involving the concentration of a reference compound (in Chenomx NMR Suite, the CSI).

The problem described appears in Chenomx NMR Suite v6.1 or earlier. All subsequent releases should handle MestReNova JCAMP files natively.

If you are using an affected version of Chenomx NMR Suite, you can adjust JCAMP files to allow them to be opened. Simply open the file in a text editor (e.g., Notepad, Wordpad, vim, Emacs, etc.), and replace all instances of 'NMRSPECTRUM' with 'NMR SPECTRUM' and 'NMRFID' with 'NMR FID' (i.e., insert a space after 'NMR'). You can use the relevant search and replace functions in your text editor as needed.

If you have a large number of affected JCAMP files, you can use tools like grep (Linux and Mac) or grepwin (Windows) to change the text strings in all of the files in a single operation. Please be sure to backup your data before attempting any large scale changes of this nature.

Chenomx NMR Suite does not support reading arbitrary text files as spectra. We have no knowledge of any third-party products that produce .cnx files from arbitrary text files. However, the JCAMP-DX file format is stored as plain text, and Chenomx NMR Suite does supports importing JCAMP spectrum files.

Chenomx NMR Suite does not currently support deleting regions of the spectrum other than the water region.

The CSI lineshape is the key to matching library signatures to patterns in your experimental spectrum. The more closely your CSI settings in Processor match a spectrum, the better the library compounds will match while profiling the spectrum.

Specifically, when you make changes to your spectrum in Processor (like adding or changing line broadening), you need to update your CSI settings to reflect the changes. If you are using DSS or TSP as a CSI, you can usually just switch to the CSI Editor in Processor and click 'Find Automatically'. If the automatic method does not work, simply adjust the red CSI peak to better fit the spectrum, exactly as you would adjust peaks and clusters in other modules.

Profiler can measure concentrations up to 5000 mM, or 5000000 μM. If you are using mg/dL as your concentration units, the maximum concentration will vary depending on the molecular weight of the compound, but is equivalent in every case to 5000 mM.

In short, no, you cannot change the line width of the signature line in Profiler.

The line width of the signature lines in Profiler is directly calculated from the line width of the CSI that you set in Processor, and is applied equally to every compound that you fit in Profiler. Thus, it is not possible to set different line widths for different compounds.

You may get better results with a different CSI (we recommend DSS), or if you are working with samples that have some protein content, you may want to try filtering your samples using a 3kDa molecular weight cutoff filter to remove the proteins. Also, some variation in line widths may occur due to varying shimming technique during data acquisition, especially if more than one person is involved in acquiring the spectra.

You should select a normalization method based on the nature of the dataset that you are binning. Use total area to reduce the influence of dilution effects among the samples in your dataset; this is the most common scenario. If you can assume that dilution effects are insignificant, use standardized area.

Profiler works exclusively with clusters (groups of peaks), not individual peaks. It is possible to see cluster centers in ppm using Profiler. Simply select a cluster and hover the mouse cursor over it; the display in the top right corner of the spectrum includes the center of the selected cluster.

You can measure area under a cluster using the Select Region tool; double-click on the spectrum, then click and drag to select a region. The areas appear in the top right corner of the spectrum. 'Spectrum Line Area' indicates the area under the black line (acquired spectrum), while 'Sum Line Area' indicates the area under the red line (your current analysis of the spectrum).

You must decide for yourself what minimum threshold you will consider acceptable for profiling. There is no minimum level of absolute peak heights that applies to every sample. As a rule of thumb, if the peaks are small enough that you question whether the compound is present at all, you will not obtain reliable concentration measurements for that compound.

The simplest possibility to consider is that the compound in question is just not present. Having excluded that, for example by supplementary analysis confirming that the compound is indeed present, there are some other considerations.

If you are running reconstituted samples in pure D₂O, Profiler may erroneously assign some compounds a maximum concentration of zero. In pure D₂O, exchangeable protons are completely replaced by deuterium, effectively removing the signal for those protons from the NMR spectrum by reducing its intensity to near zero. When calculating maximum concentrations, Profiler only considers values that do not allow any cluster of a compound to exceed the measured intensity of the spectrum. If an expected cluster is effectively zero intensity, Profiler will predict a maximum concentration of zero for that compound.

Two other factors can also influence Profiler's calculation of maximum concentration. Incorrect CSI settings can reference the spectrum to the wrong chemical shift or result in Profiler incorrectly interpreting intensity ratios with which it calculates all compound concentrations. Also, incorrectly setting the spectrum pH can result in clusters or transform windows starting in the wrong positions, meaning that Profiler will not use the correct spectrum locations in calculating maximum concentrations.

To add compounds to the library, you need to acquire spectra of the compounds under conditions similar to those you expect to encounter in your experiments. Once you have these spectra:

1. Process the spectra in Processor and save them as .cnx files
2. Overlay a .cnx file in Spin Simulator and fit it (see the Spin Simulator tutorial in the User Guide for details). Save the simulation as a .xss file.
3. Import the .xss file in Compound Builder, and overlay the .cnx file. Refine the fit to match the acquired spectrum (see the Compound Builder tutorial in the User Guide for details). Save the signature as a .xcpd file.
4. Import the .xcpd file using Library Manager. Make sure that the new compound appears in at least one Compound Set.
5. Use the new signature in Profiler. Make sure that at least one compound set containing the new compound is selected to see the compound in Profiler.

There is no method available to import Bruker sbase data into a Chenomx library. The Chenomx library format is not simply a collection of raw spectra, but rather a collection of mathematical models (called compound signatures). The models are based on raw spectra, but contain additional information that allows calibrating the models to better match experimental line widths, solution pH, ionic strength, and so on. The process outlined in the tutorial sections of the user guide (http://chenomx.com/support/support.php?pageID=58) will help you apply this process to your own compounds, but creating new compound signatures cannot readily be automated, as it requires informed human input.

If you have access to the original spectra, you can create your own compound signatures using the Spin Simulator and Compound Builder modules, as described in the user guide tutorials. You can create a 'quick and dirty' set of signatures by generating clusters in Compound Builder, but signatures created this way will be significantly less flexible than properly simulated and calibrated signatures in fitting arbitrary spectra. We recommend using them only in limited testing scenarios, and not for production analysis.

Another option is to let us prepare proper compound signatures via contract services (http://chenomx.com/services/services.php). You can send pure samples of the compounds, or spectra that you have acquired, and have us prepare signature files for you to add to your library.

We recommend (3-trimethylsilyl)​propanesulfonic acid (DSS) as a CSI, at a concentration of about 0.5 mM in the analyzed sample. You may also want to include [difluoro​(trimethylsilyl)​methyl]​phosphonate (DFTMP) as a pH indicator, at a concentration of about 2 mM in the analyzed sample.

You will get the best results analyzing spectra acquired with NMR parameters similar to those used to build the metabolite library. The Chenomx metabolite library was acquired between pH 4 and 9 at a temperature of 298 K (25 ^oC), with an acquisition time of 4 s and recycling delay of 1 s. The recycle delay includes a 990 ms saturation pulse on water. Our 1D pulse sequence is NOE-based, with a short mixing time of 100 ms. The mixing time also includes a water saturation pulse. The length of the proton 90° pulse (pw) was calibrated to maximize the intensity of the DSS peak. Pulse sequences for Varian and Bruker spectrometers are available on request.

Chenomx NMR Suite supports three compounds for use as a CSI: DSS, TSP and formate. The CSI serves not only as a shift reference, but also a line shape reference; the shape of the CSI peak is used to correct shimming issues in spectra and to calculate expected line widths for all compounds. The CSI peak is also used as an internal standard for quantification.

It is possible that DSS or TSP can bind to certain species, typically proteins, causing broadening of the DSS/TSP resonance and complicating the calculation of expected line widths. Methods of compensating for this binding effect are available. For serum samples, ultrafiltration using 3 kDa molecular weight cutoff filters can remove proteins and large lipids, reducing or eliminating their binding effects on DSS and TSP.

The large humps that you see in the NOESY spectra are due to macromolecules in the samples (mostly proteins and lipids). Alternative pulse sequences such as CPMG or J-resolved spectroscopy can help to remove these humps, but the effects of the proteins on the signals of small molecules will still be present. Most notably, you will need to adjust the linewidth of DSS or TSP to obtain reasonable fitting results, as both DSS and TSP bind to protein.

For more details on preparing blood serum and plasma samples for analysis using Chenomx NMR Suite, please refer to our application note.

The most common cause of very broad peaks in a plasma sample is high protein content. Even when you use special pulse sequences like CPMG to reduce the appearance of protein signals, some compounds will still bind to the protein, resulting in broadening of specific signals from the affected compounds. We strongly recommend microfiltration of plasma samples using a 3 kDa molecular weight cutoff filter to remove proteins before acquiring spectra. See here for more details.

Note: Coordinate this with FAQ 4 on the current website

The volume we typically need for NMR analysis is 630 uL. Depending on the sample preparation protocol, we try to maximize the concentration of metabolites in each matrix. For urine samples we need approximately 700 μL. For serum we need approximately 1 mL before ultrafiltration. For cell extracts, we normally recommend at least 8 million cells per mL cell density.

We recommend that you ship samples to Chenomx via overnight service by a courier. You should properly package the samples, including:

• Watertight Primary Receptacles
• Watertight Secondary Receptacles
• Absorbent Material
• Sturdy Outer Packaging (Styrofoam cooler)
• Pack in dry ice

Results from our standard MetaProfile Service include:

1. Targeted Profiling Report

   • Summary statistics on compound concentrations
   • Per sample pages outlining concentration deviations from the mean
   • Per compound pages highlighting differences across samples for a specific compound

2. Excel Spreadsheet

   • Compound IDs and concentrations in a table format

3. CNX files

   • Spectra and compound profile used by Chenomx NMR Suite