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Proteins by LC/MS/MS/ESI
Posted: Tue Oct 02, 2012 1:49 am
by shemesh199
Does anybody have any experience with larger molecules such as proteins using LC Tandem MS ESI. In particular we are looking at a small protein EGF which is around 6kD. Are there any tips with development of an assay for a large compound like this? The instrument we have is an Agilent 6410B QQQ with an ESI source. Does anybody have experience using proteomics grade trypsin for in solution digests? It seems that the peptide sequence for the protein would be N SDSECPLSH DGYCLHDGVC MYIEALDKYA CNCVVGYIGE RCQYRDLKWW ELR.
I would appreciate any comments, suggestions, resources, or references.
Thanks Kindly for the support!
-Best
Re: Proteins by LC/MS/MS/ESI
Posted: Thu Oct 04, 2012 2:45 pm
by ThomasH
Hi,
what's the intention of your analysis? Maybe you can skip the digest...it's really not that large
Re: Proteins by LC/MS/MS/ESI
Posted: Thu Oct 04, 2012 2:50 pm
by shemesh199
Hi Thomas, we are just trying to quantify the amount in our formulation. I was not aware that intact analysis is possible because the protein is 6Kd.
Re: Proteins by LC/MS/MS/ESI
Posted: Fri Oct 05, 2012 2:33 am
by Karen01
Hi Thomas, we are just trying to quantify the amount in our formulation. I was not aware that intact analysis is possible because the protein is 6Kd.
You can get reasonable separations with a 300 angstrom pore reversed phase column in that size range.. many (too many!) years ago (in the 80's I think) I was able to separate iodinated from non-iodinated insulin, mono from di iodinated, and the A and B chain mono iodinated forms from each other... and that was obviously before UPLC and without MS.
So with a little luck you can do it without digestion...
- Karen