Protein ID Solutions Sample Prep Guide
63
In-Gel Tryptic Digestion with Agilent Kit
6
Troubleshooting
Tips
Missed
cleavages
Trypsin is a serine protease that specifically cleaves peptide bonds at the
carboxyl side of lysine and arginine residues; however, cleavage can be blocked
or slowed by proximal acidic, aromatic, or proline residues with proline having
the most significant effect. Peptide fragments with one missed cleavage are
common and should be taken into consideration during mass analysis.
Autolysis
products
The modified trypsin provided in this kit displays only limited autolytic
activity that should not interfere with mass spectral analysis. Trypsin
fragments of mass 842.51 (m/z, [M+H]
+
) and 2211.10 (m/z, [M+H]
+
) will be the
most common using standard conditions and can be used as internal
standards.
Staining method
The Protein In-Gel Tryptic Digestion Kit is designed for colloidal Coomassie or
fluorescent dye-stained acrylamide gel slices. For protein bands stained with
MS-compatible silver stains or reversible zinc staining, alternative destaining
procedures will be required [3, 4].
Problem
Cause
Solution
Incomplete digestion
Insufficient enzymatic
activity
Increase incubation time.
Ensure gel slice was dry before addition of enzyme. A dry gel slice will
pull trypsin into gel slice and increase hydration volume.
Enzyme is losing activity
Use a new trypsin stock aliquot.
Incorrect pH
Ensure gel slice has been completely destained and trypsin working
solution has been diluted with digestion buffer.
Residual SDS
Ensure gel slice has been completely destained.
Poor mass spectrum
Concentration or
detection limits of
application
Ensure sample is within the detection limit of the specific downstream
application; concentrate digest with Cleanup C18 Pipette Tips
(5188-5239).
Note: Limits vary considerably based on application and
instrumentation.
Interfering agents
Clean up digest with Cleanup C18 Pipette Tips (5188-5239).