Hi,
Any suggestion about inefficient digestion by BEH particle Enzymate online digestion column?
Low flow (100ul/min, increased temperature up to 40 C, varying protein concentrations were tested so far. We intend to digest the target protein in its native form therefore partial denaturation was omitted using guanidin/urea/TCEP etc...We did not evaluate the POROS particle-based low-pressure online digestion, we have only the BEH Enzymate option and we wish to conduct the study under the mild back pressure.
Do I need to pressurize the column up to the 5.000-10.000 psi range or should we denature and reduce the target protein for proteolytic cleavage? How can we obtain the proteolytic peptides, any suggestion would be highly appreciated.
Thanks in advance...emir
Many proteins are inherently resistant to proteases, especially those that normally function in the presence of protases. So your native cleavage strategy may not work.
Some proteins are resistant to pepsin, especially if they are low in aromatic AA, but may be susceptible to other proteases. You may know trypsin and chymotrypsin are popular for protein fragmentation.
You might try varying your pH, but your target protein may denature and/or precipitate, especially near pI. Likewise, the column may perform better under higher pressure, but high pressure will at least partially denature your target protein.
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