MALDI with short peptides

Mass spectrometry of the histone tail peptides is easy with the Voyager MALDI machines located either in the Mass Spec facility over in the basement of MCB or on the 4th floor in the Loo lab. We can use the Loo lab machine if we ask but there is no formal arrangement over cost etc.

Preparation of sample

Sample should not contain any salt so should be resuspended in water after HPLC purification and drying. Sample can be used straight from the HPLC.

Very small quantities (~4 pmol/ul) can be measured.

Make a solution of 50:50:0.1% H2O:ACN:TFA this can be stored at RT.

For each run make a fresh matrix solution using about 500ul H2O:ACN:TFA with about 0.5mg of alpha-cyano-4-hydroxycinnamic acid powder added. Shake on tumbler for about 5-10 minutes to dissolve - some undissolved material is not a problem.

Spot about 0.4ul of your sample onto a well on the 100 well plate. Pipette 1ul of the matrix solution onto the spot and mix up and down several times. Repeat for each sample. Allow samples to dry at room temperature.

Take plate to MALDI machine.

Open the Voyager Data Discovery software.

Eject the plate arm with the hand icon. Slide the plate into the holder until you hear the click. Do not allow the arm to be up for more than a couple of minutes. Select the default plate (KFF Gold Plate - downstairs; Plate1 - upstairs) and click load plate. The arm will lower and the software will be unresponsive for a while.

Once loaded you should see the TV screen settle down to a spot indicated on the 100 well plate outline. Click to go to your first sample spot.

Open an Instrument Settings File. Open the D:\Voyager Data\Grunstein\Methods\ACTH_Linear method (downstairs). This will load the settings for the machine.

Chose a location to save the data - eg. D:\Voyager Data\Grunstein\PlateX\YOU_X_01 using the top left data panel. Unselect the autosequence.

Change the number of shots to be about 250. Check the Voltage is about 1900.

To fire the laser use the joystick left hand button. Once firing move the aim point around with the stick until a good signal is achieved (top right number in the current spectrum graph). A good signal is around 4000-5000. If you max out to 10e4 you should lower the voltage. If you get poor signal try moving around, upping the voltage, or remaking your sample with fresh matrix.

When done - click the top right button to save the data. Move to the next spot, change the output name - YOU_X_02 - then fire again.

When all samples are done eject the plate, slide it out of the holder, then 'load no plate'.

Close the data discovery and open the data browser.

Open your datafiles 8 at a time. Set the printer to Portrait. Maximize each graph in turn and go to 'print view'. print all views prints all on one page very small.

Close both programs - check the machine is ok, sign the sheet.