Couldn't you use a standard MS technique like MALDI-TOF or ESI-TOF (no
need for MS/MS) to get the percentage fairly reliably? People in the
NMR field do this all the time to estimate the percent incorporation of
deuterium (or 15N or 13C, etc).
% incorporation =
(Experimental Protein Mass - Calculated All-Sulfur-Met Mass) /
(Calculated All-Seleno-Met Mass - Calculated All-Sulfur-Met Mass) * 100
Of course, this percentage will be the ensemble average of all the
molecules the mass spectrometer actually sees, and it tells you nothing
about the SeMet incorporation on a site-specific basis, but that
shouldn't necessarily a fatal problem for crystallographic purposes. I
assume you simply want to see whether you have a high percentage of
selenium incorporation for experimental phasing purposes. In this case,
whether the exact percentage is 94% or 96% isn't so important in
comparison to discovering whether the incorporation is only 50% when you
thought your methionine auxotroph would give you > 90% incorporation.
The above method should help you determine that reliably enough. Unless
there's some fatal flaw the MS experts can clarify?
Good luck,
Matthew
---
Matthew J. Whitley, Ph.D.
Research Associate
Angela Gronenborn Lab
Department of Structural Biology
University of Pittsburgh School of Medicine
On 11/2/2015 7:00 PM, CCP4BB automatic digest system wrote:
> Date: Mon, 2 Nov 2015 21:13:23 +0000 From: Christian Roth
> <[log in to unmask]> Subject: Re: SeMet incorporation Hi, you
> might be able to say if there is SeMet incorporation or not, but you
> won't get a percentage. Standard MS is not quantitative. Another
> problem might be that you don't "see" the right peptides or some
> peptides simply don't fly with the SeMet or the other way around.
> Cheers Christian Am 02.11.2015 um 13:51 schrieb Reza Khayat:
>> >Hi,
>> >
>> >I would like to determine the percentage of SeMet incorporation into my proteins via MS. Is MALDI MS/MS sufficient for this? The company I have outsourced the experiment to is indicating that it is not, but to my recollection it is. Thanks for any advice.
>> >
>> >Best wishes,
>> >Reza
>> >
>> >Reza Khayat, PhD
>> >Assistant Professor
>> >City College of New York
>> >Department of Chemistry
>> >New York, NY 10031
>> >
>> >
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