We are trying to digest a protein (From 12KD to 95 KD ) using Trypsin digestion by MLDI-QTOF ). Trypsin does not digest the protein to peptides. Digestion buffer used 2ng trypisin in 50mM of ammonum bicarbonate ABC ) , Reduction and Alkylation used (10mM of DTT and 50mM Iodoacetamide IAA)....
Does anyone know the possible reasons for incomplete digestion? Please suggest possible solution......Thank you
Do you see anything when you run the sample? You should at least see trypsin autodigestion products. If you are not seeing anything, you have some serious issues. Is the MS working okay? Do you have peptide standards to verify it is all okay? What matrix are you using?
How much of the MassPREP are you using in the digest? How long are you digesting? At what temperature? How are you cleaning up prior to MS?
What you are trying to do is pretty standard, so you need to work out where it is going wrong.
Hi Hatem,
A couple of comments:
1. What is the weight of the protein mix you are digesting? You need to pay attention to Enzyme:Substrate ratio. 2ng of Trypsin is a very small amount of protease. Do you only have ~40 ng of substrate?
2. Efficiency of solution digests depends on the protein(s) you are trying to digest. Some proteins only digest in presence of eg; urea or Guanidium hydrochloride.
3. Post digestion, did you clean up the peptides generated? MALDI-TOF mass spectrometry , to a certain extent, is tolerant to additives and salts but signal is very much reduced and supressed if the peptides are not cleaned up prior to spotting them on the MALDI target.
Best,
Hediye.
Dear Peter G Hains and Hediye Erdjument-Bromage
1- My weight of protein was 100mg =100 micro gram in 1ml + trypsin 20micro gram in 1ml which is the ratio 1:50 w/w.
2 - How i clean up the peptide? after digested I added TFA to qunch and stop the reaction.
3- Incubated overnight 37C
Thank you for help
Dear Hatem,
You can clean up the peptides with a manual "ZipTip" or StageTip which uses Empora C18 disks. I recently found out that the company 3M is no longer supplying the Empora C18 disks which are used to make the Stage Tips. However, you can purchase both "ZipTips" and Stage Tips, ready to use from Millipore and ThermoFisher Scientific, respectively.
Without the clean up step, it is not surprising that you are not seeing a signal during MALDI-TOF MS.
Good luck,
Hediye.
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