Hi! I study the effect of formulation excipients on protein degradation pathways and was looking disulfide-mediated aggregation by comparing reduced/non-reduced SDS page samples. I am using TCEP as my reducing agent. I noticed that in every reduced sample (lane closest to ladder (left), lane furthest from ladder (right) is exactly the same but WITHOUT reducing agent) I detect a new band between 20-25kDa. My protein of interest is between 45-50 kDa. I also see the supposed dimer around 90kDa disappear in the TCEP reduced samples which is what I would expect. I initially suspected that the emergent band could be the result of TCEP hydrolysing the peptide bond of my protein somehow but don't see this being reported in the literature. I analysed the reduced and non-reduced samples via LCMS and oddly don't see much difference between them. Any input would be greatly appreciated!!
I should also note that my protein is NOT a dimer.
So your results are completely the wrong way round yes? Have you tried DTT which is a stronger reducing agent? In your LC-MS analysis you are reducing and trytic digesting, classic bottom up proteomics mapping of peptide fragments? You could use MALDI-ToF MS which looks directly at your protein without tryptic digestion. I am at Cambridge in the Vet School doing precisely this on COVID-19 proteins so we can run your protein on our MALDI ToF in 5mins with and without TCEP/DTT.
So your results are completely the wrong way round yes? Have you tried DTT which is a stronger reducing agent? In your LC-MS analysis you are reducing and trytic digesting, classic bottom up proteomics mapping of peptide fragments? You could use MALDI-ToF MS which looks directly at your protein without tryptic digestion. I am at Cambridge in the Vet School doing precisely this on COVID-19 proteins so we can run your protein on our MALDI ToF in 5mins with and without TCEP/DTT.
Hi,
i think it could really be artificial cleavage of your protein by TCEP. I also observed that already and maybe have a look to that paper:
Article A Tris (2-Carboxyethyl) Phosphine (TCEP) Related Cleavage on...
Don´t know about your protocol but you should not heat your sample when using TCEP. If you heat it, better use DTT.
Looking at your gels careful I think the faint bands at 90kD is part of your protein. Could your study protein consist of 2-3 subunits (90Kd and 40 and/or 42 Kd) held together by non covalent bonds hence the SDS separation? However these subunits themselves are cleaved in the primary aminio acid structures but are held together by disulphide bridges. Thus effectively two chains are being reduced: 90Kd protein to to 48 - 50KD and the 42-45KD band is reduced to 20-24Kd.
What is your detection system in the SDS-PAGE, as the band intensity may not reflect actual relative quantity of different proteins/peptide/subunits but affinity for your detection system?
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