in FASP (Filter assisted sample preperation) prosedure, the enzyme to protein ratio needed for digestion of a protein sample is 1:100, if it is used over -use trypsin, what kind of problem can be faced?
Hi there,
If trypsin is used at higher concentration it becomes less specific and tends to cut polypeptides anywhere (it's the same for just any other protease). So tryptic digest analysis becomes more difficult.
Thanks liger for your comment, Do you think we should add N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) to stop the digestion after digestion?
Hi again,
TPCK is a chymotrypsin inhibitor, why adding TPCK to your trypsin digest?
Liger,Sorry it is not TPCK, it is TLCK, on the paper called proteomic analysis of Exosomes from Human Neural Stem Cells by flow fractionation and NanoFlow Liquid chromotography-Tandem Mass Spectrometry by Dukjin, on the sample preperation,TLCK (tosyl-L- lysyl-chloromethane ketone has been added to stop digestion at a 10:1 molar ratio of TLCK : Trypsin attached the paper
Indeed you may stop trypsin digest by TLCK but if you have used too much trypsin it won't change anything to the lack of specificity of the digest.
Does TLCK have an effect on protein expression? Because in the FASP prosedure , it is not added to stop digestion
The possible effect of TLCK would be on tryptic peptide detection/quantification not on protein expression which occurs upstream FASP technique. To answer your question : I don't know but if the addition of TLCK is meant to reverse the effect of too much trypsin it is useless because TLCK won't prevent the excess of trypsin to cut polypeptide anywhere else than at trypsine sites.
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