2D NMR Processing in Linux and Windows

Processing in Linux:

After all data is transferred from Gambrinus into desired folder on Z Drive - open your desired sample folder. For all commands, do not type "". Everything within "" is command.

1. Building Fid

• a. Right click and select "Open terminal here" and type “bruker” in terminal
• b. Make sure that you are….
  • i. 2D, mode = states, acquisition mode: x=DQD y=Echo-Antiecho
• c. Click "Read Parameters"
• d. Update script (do this after you have read the parameters and nothing is highlighted in yellow)
• e. Save script

2. In terminal

• a. Type “./fid.com” this runs the script
• b. Type “./hsqcproc.com”
• c. Type “nmrDraw” -- this opens NMR draw
• d. Go File --> select file --> hsqc_1.ft2 --> read & draw (the blue = positive, the red = negative or noise)
• e. Then go draw --> estimate noise --> continue --> draw
• f. Use the factor +/- feature to play with noise to get a good level and find the TMSP peak in the upper right corner.
• g. When you locate the TMSP peak, then go mouse --> 2D Location --> z --> right click (this zooms in) --> L --> shift C or file --> calibrate axis --> new ppm value = 0.00 --> apply --> save --> continue x2 --> follow same steps for the y-axis --> f to zoom out

3. Phase it: H gives hydrogen axis, V gives carbon axis. You can access each by pressing H or V key on keyboard.

• a. Play with PO and P1 for both until looks phased. (This is when the yellow quadratic shapes are even on both sides). Make sure this is true for two or so peaks for carbon and hydrogen. Make sure to write down the PO and P1 for each.

4. In Terminal

• a. Type “vi hspqcproc.com”
• b. Type “i” to edit and edit values. Replace PO and P1 values to the manual ones calculated.
• c. Press the escape key and type “:wq!” and enter
• d. Type “./hsqcproc.com”
• e. Type nmrDraw and select file (instructions above) and check to make sure phasing is still accurate. If not, repeat step 3 and 4.

5. Copy "fid.com" and "hsqcproc.com" files into all sample folders and phases all samples. 6. Once all fid files are phased (should be one per sample – best to rename HSQC_1.ft to number related to sample) you need to convert to .nv file

• a. Create a processed folder and put all phased HSQC_.ft files in folder. MAKE SURE ALL FILE NAMES HAVE THE SAME # OF CHARACTERS OR NEXT STEP WILL NOT WORK PROPERLY.
• b. Copy and paste a “./addNMR3.com” file to folder. This can be found in other processed folders. Right click and select "open terminal here"
• c. In the terminal type “./addNMR3.com”

6. Once all files are converted to .nv files, copy and paste results.nv info nvoutput file and reboot in windows.

Processing Spectra in Windows:

1. First open NMRViewJ

• a. Datasets --> open and draw --> open the processed folder you want --> select reults.nv file --> open
• b. Play around with the contour buttons (blue and red arrow at top) to make sure contour is just before noise level
• c. Right click --> attributes --> peak pick --> pick --> delete all peaks in water line and those not on the diagonal. Keep the TMSP peak labeled for reference Use the red and black cross hairs to do this.
• d. Right click --> attributes --> list --> select results --> close
• e. In toolbar, Peak --> show peak table --> peak --> edit --> compress and degap --> okay
• f. In toobar, Analysis --> rate analysis --> in prefix matrix type “Final_” --> check autofit --> click load time file --> open processed folder you want --> select nvoutput folder you working in --> select rate.txt --> open --> measure all (should have as many T columns as number of spectra) --> save table --> title “peak intensities” --> close (If you recieve error message after clicking "measure all" try opening all "Final_.nv" spectra and try again.)
• g. Peak --> file --> write list --> open nvoutput file your working with --> title “peak list” --> close

2. Open an Excel workbook page

• a. Open processed folder your working in, open nvoutput and drag both peak intensities and peak list into excel workbook. Each will generate its own document in excel. Save as "peak list" and "peak intensities" excel workbook document.

The remaining steps only work in the Excel workbook documents.

• b. In peak list, select all --> data --> text to columns --> select delimited and hit next --> select tab and space and hit next --> press and next and finish
• c. Insert a column B and C in peak intensities, copy and paste 1H.W and 13C.W from peak list respectively.
• d. Save all work!

Metabolite Assigning

1. In a web browser, type http://prime.psc.riken.jp/. Click on "SpinAssign"

2. Copy and paste 1H.W and 13C.W into query peaks box. Make sure 1H.W is the left hand column.

• a. 1H tolerance = 0.08, 13C tolerance = 0.25, Solvent = H20/KPi. Make sure spectrum type is ([1H,13C]-2D)
• b. Click “dospinassign” --> at the bottom of the page, click get the "result at tab delimited" --> step in box --> control A (selects all) --> control C (copies all) --> paste in peak intensities workbook in additional sheet
• c. About ¾ down the page of the PRIMe: SpinAssign webpage, follow the same steps from b from above. This will give same info in another format for checks and balances when assigning metabolites.

Tips for assigning metabolites:

1. Find peaks that have unique metabolites (peak with one metabolite assigned to them)

2. Verify that those peaks exist

• a. For E. Coli --> check the E. Coli Metabolome datebase (http://ecmdb.ca/)

3. Go through and categorize Nucleic Acids, Fatty Acids, Amino Acids, etc and continue labeling from the categories.