Use prOTOF mass spectrometer to measure the tryptic map spectra.
version 20.12.06
1. The prOTOF mass spectrometer should indicate that it is ready for the analysis by a green light, “TOF Vacuum”, on the front panel.
2. Wake up the prOTOF computer by pressing the computer switch button quickly. Login and type a password.
3. Open TOFWORKS. Login as a tofworksuser and type a password.
4. Remove a target cassete from the instrument target chamber. Remove the blank metal MALDI target from the cassette and insert your magnetic target deposited on the thin metal plate (you should find it and leave it in the table drawer) . Be sure to close the camber completely by pressing the top hook all the way down.
5. Define your target either as new or as already existing one. This operation starts the sequence of the target insertion into the instrument. Wait until ready (~ 3-5 min), all for light are green (Instrument status-green, TOF vacuum - green, Plate Loaded - green, Plate Chamber Vacuum - green).
6.Switch on the small tv. In few moments you should see your target on the screen of the tv. Wait until the target is inserted completely into the instrument.
7. File/check plate alignment -> calibrate the plate alignment by marking two dots, X 2/ OK
8. Use the default instrument method to measure the spectra.
9. Give a name for the sample of the current sample spot.
10. Measure the spectrum of a 100 femtomole of a calibration peptide mixture. Acquire (scan the laser spot)/add to sum/ sum + current. Save the spectrum by going to the next layer and pressing “pick peaks”. Peaking peaks deposits the spectrum in the spectra data base.
11. Calibrate the instrument by measuring the spectrum of a peptide mixture. Use m/z values of ions at 1046.542 and 2456.199.
Acquire (scan the laser spot)/add to sum/ sum + current/ pick peaks/ calibrate:
Calibrant – calibrate. Set peaks found at m/z 1046.xxx to correct m/z 1046.5423, and peak found at m/z 2465.xxx to the correct m/z 2465.199. Usually the differences between the old values and the new ones are just few millimasses. Apply to all samples.
12. Go to Acquire data/Acquire (scan laser spot over your sample)/add to sum/ sum + current/ pick peaks/
13. Spectra are saved in the computer database after “pick peaking” procedure.
14. Extract the the collected spectra with the program “prOTOF extractor”. Give the spectra the appropriate names (suggestion: keep the sample position in the file name, example: B01_your file name.txt)
NOTE: Do not connect prOTOF computer to the internet! Use a flash memory to transfer your data.
Spectra processing
1. Use your computer or a nearby computer for analysis of your data. Login as a user and type a password.
Note: this computer is connected to the internet.
2. Use “m/z” program to find and label all ion peaks of interest. Suggestion: use spectrum smoothing procedure (use coefficient 3) and then the spectrum enhancement procedure. Use “A” - automatic mode of peak picking. Check manually whether all peaks are picked properly, and monoisotopic M+H peaks are selected. Add or remove peaks as necessary.
3. Copy the list of peaks in clipboard and then in the “Notepad” program. Save as a text file. Keep the name of the file short, representative of an analyzed spot. Do not use spaces in the file name.
4. These list of peaks may be used for ID of proteins based on the “tryptic mapping”.
Use Xproteo search engine or some other engine of your choice. This pre-screen helps to evaluate the quality of your sample, initial ID of proteins and, in general, the status of your project at this point.
Use MALDI-LTQ mass spectrometer to measure MS/MS spectra on all peaks detected in the tryptic maps.
1 . Copy the files containing the list of masses onto a flash drive. Transfer the data files to the computer of the MALDI-LTQ mass spectrometer.
2. Use AutoMSMS program to generate the script for the LTQ mass spectrometer to perform MS/MS analysis on all peaks from the given data file.
7. Chose the data file(s).
8. Define: num of laser shots per spot 5, if you decide to run the LTQ in completely automatic mode. Or use num of laser shots per spot 100, if you would like to have a manual control over the position of the laser spot on your sample. Leave other paramers as default. Run the program on all selected files. This program will create method files for analysis of each samples. Each method files “knows” what peaks to analyse in each sample and “how” to do it (what is a peak selection widht, activation energy and time etc).
9. Insert your magnetic MALDI target onto the plate holder and insert into of the LTQ mass spectrometer.
10. After all methods are generated run them either one-by one, in a semi-automatic mode usine Tune program. Or use Xcalibur program to define the sequence of analysis of all selected sample spots.
11. After the analysis is complete, generate the “DTA” files from data.raw files. Use the DTA converter program. (Select M0.3 and S0 tags for conversion). Transfer you data to the analysis computer. You can search and identify you proteins based on the accurate m/z of the parent peptides and their MS/MS spectra using several data bases. Use Xproteo, Mascot or some other search engines to ID the proteins.
NOTE: Do not connect vMALDI-LTQ computer to the internet! Use a flash memory to transfer your data.
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