Self Service MALDI Operation

  • Reserve MALDI on Collab:
  • Create a project in LabLink:

    the number of samples is the number of spots you use on the plate, excluding the calibration standard.
  • Bring samples, sample plate, pipet tips, supplies and reagents to Mass Spectrometry laboratory, room 1105 Jordan Hall (access card required). Standard matrix solutions and protein calibrants are provided.
  • Mix 1-2 µL of sample with an equal volume of matrix solution (normally sinapinic acid (SA) for proteins >4000, cyanohydroxycinnamic acid (CHC) for peptides <4000) in a tube or on plate, and load 1-2 µL total volumeon the sample plate. Include a standard (peptide up to 4000, prot 1 for 4000-20,000, prot 2 for >20,000). Leave to dry. Return standards to freezer.
  • On computer for MALDI, look on the spreadsheet to verify that there is a vacuum reading for the day, and that the current vacuum is less than 1 x 10-6 (flexControl-Statustab-click on word Vacuum).
    On Sample Log tab of spreadsheet, enter the number of samples you are running and your name.
  • After your samples are dry, open the sample compartment; do not open earlier because the instrument should be under vacuum unless loading or removing a plate. Use either the green button on instrument or icon in flexControl program to open the compartment; IN PROGRESS light illuminates. After you hear a click, you can open the sample compartment lid.
  • Remove plate from compartment and place in storage box and gently lower your plate into the holder.
  • Gently close lid and load plate by pressing green button or clicking icon in flexControl software-IN PROGRESS light illuminates.
  • Wait for vacuum to reach less than 1 x 10-6 typically within 4 minutes (flexControl-Statustab-click on word Vacuum). If the vacuum is not reaching this level, contact staff to clean the sample compartment seal.
  • When the vacuum has reached the target level, on the diagram of your plate, click the spot with the standards.
  • Select the method for your samples (Method button)-each group should have their own folder of methods. Decline the option to save method.
  • Click the Start button-there may be a delay while voltages increase. If there are no peaks, and only a flat line, you probably need more power; a 10% increase in power level(slider to right of image of the sample plate) is usually appropriate if you see only a flat line.
  • A signal intensity of 1000 to 2000 is believed optimal; higher power can reduce resolution, cause small shifts in observed mass, induce dimer and adduct formation and give inaccurate intensity readings.
  • When you get a suitable spectrum, save it using the Save As button.
  • The comment boxes are text fields that can be used for information about the sample and conditions.
  • Selecting a processing method from the drop down list in the Process with box may simplify operations later in the flexAnalysis program
  • Sample name will be used as a file name, so there are restrictions on the characters used in this field.
  • Use the Browse and New Folder buttons to make a data path of the form:
  • D:\Data\year\monthyear\nyymmdd\lab name.
  • To calibrate, click the Smooth tool in the middle top row of the screen, then click the Calibration tab, which should show the appropriate standards. Click on a calibrant protein in the list, then click to the left of the peak; if the vertical line changes from red to green, the peak was recognized as a standard. If the line does not change color, possibly the signal intensity is too low.
  • Repeat for other calibrants, then click the Apply button and the green lines should all be on the center of a peak.
  • Then move to a sample and take a spectrum. Save all spectra with recognizable peaks, in case your sample does not give strong signals. You can open the data in the analysis program either by setting the option in the Save as screen, or by clicking the Load last saved spectrum button beneath the word Compass in flexControl program..

Data analysis with flexAnalysis program

  • Click the Find mass list icon (single peak icon) below the word Annotation.
  • If there are too many or too few peaks, click the Edit Method Processing Parameters icon (below the word Window). Go to Signal to Noise Threshold box, and increase the number to reduce the number of peaks, or decrease it to detect more peaks and click Find mass list icon again.
  • Display choices are on the View menu. In Overlaymode all spectra use the same intensity range. List can show multiple spectra in individual windows with different intensity scales. Right click the display area and then List windows to change the number of windows.
  • To put data in a form for presentation, use the Print icon or press Ctrl-P to print a spectrum to a pdf file- you will have to navigate to your data folder when saving it. Before printing, often you need to increase the range of the intensity scale so that there is room to print the mass of the largest peak.
  • If you want the data as a text file which can be plotted in Excel or other program, go to File-Export-Mass Spectrum. In Excel, open text files, and choose delimited by space character. If you need this data immediately, we prefer that you email it to yourself rather than use a flash drive.

End of session

  • When you have finished, remove your plate; reloading the plate you removed is optional, close the sample compartment lid and press the Load button to evacuate the sample compartment. Please wait a few minutes to verify that the sample compartment has evacuated by switching to the vacuum display (flexControl-Status tab, click Vacuum).
  • Check that you recorded the samples you ran on the SampleLog spreadsheet.