Add 30 µL of 100 mM acetic acid.
Spot 0.5 µL of beads with 0.5 µl of MALDI matrix onto the MALDI target for MALDI-MS analysis.
Resume the carboxypeptidase Y reaction by repeating Steps 16-19.
Mass Spectrometry
Perform MALDI analyses on the samples (from Steps 11, 14, and 20) using a delayed-extraction TOF mass spectrometer.
TROUBLESHOOTING
Problem: The CRC leaks during rotation.
[Step 9]
Solution: Seal the bottom of the CRC with Parafilm.
Problem: Peptide detection is below the level of MALDI-MS sensitivity.
[Step 21]
Solution: MALDI-MS sensitivity is significantly reduced in the presence of glycerol. Ensure that enzyme solutions containing no glycerol.
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Anyone using the procedures in this protocol does so at their own risk. Cold Spring Harbor Laboratory makes no representations or warranties with respect to the material set forth in this protocol and has no liability in connection with the use of these materials. Materials used in this protocol may be considered hazardous and should be used with caution. For a full listing of cautions regarding these material, please consult:
Proteins and Proteomics, A Laboratory Manual, by Richard J. Simpson, © 2003 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, p. 478-481. |
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