[RASMB] Reproducibility in densitometry measurement

Tue Sep 30 13:46:50 PDT 2014

John, to back up a bit, do you actually know what fraction of this
glycoprotein is carbohydrate? If so, why not simply calculate the vbar,
using the values discussed in Lewis, M. S. and Junghans, R. P. (2000).
Ultracentrifugal analysis of molecular mass of glycoproteins of unknown or
ill-defined carbohydrate composition. Methods Enzymol. 321, 136-149? This
has always worked pretty well for me.

Yes if you assume values for the dn/dc of the carbohydrate you could in
principle use the synthetic boundary experiment to determine the dn/dc and
the fraction carbohydrate by getting the protein concentration from the OD.
But I would use the OD as measured by a bench-top spectrophotometer rather
than from the XL-I. For the carbohydrate dn/dc in 2001 we suggested using
the value for beta-cyclodextrin (0.145 as measured by Brent Kendrick at
Amgen), but frankly whether that is really a relevant or accurate estimate
is hard to know. 

John

From: John Sumida [mailto:jpsumida at u.washington.edu] 
Sent: Tuesday, September 30, 2014 11:41 AM
To: jphilo at mailway.com; rasmb at list.rasmb.org
Subject: RE: [RASMB] Reproducibility in densitometry measurement

Dear John,

Thank you for your response and suggestions.  We will try to work at higher
protein concentration, though we are somewhat sample limited.

Since you raise the question of knowing dn/dc and since the protein is
glycosylated, for us knowing the correct value for dn/dc is a problem. A
colleague of mine and I thought about comparing the interference
determination of the synthetic boundary with the absorbance signal obtained
in the same experiment.  I have an extinction coefficient determined for the
sequence composition (as per Gill et al. Analytical Biochem 1989) so we
could determine the protein concentration (O.D. < 1.00).  I could also
assume that the protein portion of dn/dc = 0.187, and that portion
corresponding to sugar was 0.146, (which I understand to be the value for
dextran).  

Since the interference measurement will be sensitive to glycosylation
whereas the absorption is not, I should be able to estimate a value for
dn/dc that better reflects the degree of glycosylation by determining the
relative contribution of the sugar component to dn/dc?

Thanks again for your time in considering my question and your willingness
to share your knowledge on RASMB.

Best regards,

John Sumida

University of Washington

From: RASMB [mailto:rasmb-bounces at list.rasmb.org] On Behalf Of John Philo
Sent: Tuesday, September 30, 2014 10:12 AM
To: 'John Sumida'; rasmb at list.rasmb.org
Subject: Re: [RASMB] Reproducibility in densitometry measurement

John, it has been a long, long time since I last did any of these
measurements, so regrettably I don't remember any details, but I do remember
all too well that it takes quite a while to figure out how to load the cell
reproducibly without any bubbles, and that to get reproducible results you
really have to obsessive about doing everything exactly the same way. 

If someone failed to clean your cell well and you've got some junk stuck on
the surfaces that might explain your problems, so perhaps you should try a
more aggressive cleaning.

Remember that the protein samples will always behave differently with regard
to bubbles because the protein itself lowers the surface tension.

But I definitely agree with Holger that you are using quite low protein
concentrations. Back in my Amgen days we would typically use concentrations
of at least several mg/mL, i.e. an order of magnitude higher than you are
attempting. 

Holger is also quite right that accuracy for the protein concentrations is
often a limiting factor. Determining the extinction coefficient via the
synthetic boundary approach assumes you know the correct refractive
increment, and that will vary significantly depending on the buffer
composition.

John

From: RASMB [mailto:rasmb-bounces at list.rasmb.org] On Behalf Of John Sumida
Sent: Tuesday, September 30, 2014 9:06 AM
To: 'HGSR (Holger Martin Strauss)'; rasmb at list.rasmb.org
<mailto:rasmb at list.rasmb.org> 
Subject: Re: [RASMB] Reproducibility in densitometry measurement

Dear Holger,

Thank you for your detailed comments.  Our experience with degassing the
sample is consistent with yours: does not appear to make much of a
difference.  We have not tried removing the syringe during measurement -
Anton Paar rep recommended that we leave it in the plug during the sample
measurement.  We will try removing the syringe and see if that helps.

What concentration range to you normally use for your measurments?

Thank you for taking the time to consider my question.

Best regards,

John Sumida.

From: RASMB [mailto:rasmb-bounces at list.rasmb.org] On Behalf Of HGSR (Holger
Martin Strauss)
Sent: Tuesday, September 30, 2014 1:29 AM
To: John Sumida; rasmb at list.rasmb.org <mailto:rasmb at list.rasmb.org> 
Subject: Re: [RASMB] Reproducibility in densitometry measurement

Dear John,

Your observations sound very familiar to our experiences with the DMA5000
and I would love to also hear from other users. Some comments from our
experiences:

-      It is an extremely sensitive instrument requiring a corresponding
experimental standard (which is higher than with a 4000 or 4500)

-      Make sure that the calibration is exactly as the measurement. For
example, don't leave the syringe in the plug under the
measurement/calibration. It will affect the resonator, at the 5th and 6th
decimal place. Likewise with the plugs or tubing at the other end. We fill
our samples, and then remove every tubing, plug, etc. That's also how we
calibrate, but others might have found a better way.

-      Gas bubbles can be a problem. Make sure that the temperature of the
samples and solvent is similar to your measurement temperature before you
dilute and fill them. Likewise, degass preferably at the measurement
temperature. We use the small vacuum pump from our ITC-instrument, but in
our hands, degassing did not make a huge difference. You can also degass the
solvent before you dilute the protein, which can be done more thoroughly
than with a protein in it. Any other experiences out there?

-      0.1-0.3 mg/mL is a rather low concentration and small changes in the
concentration due to adsorption of your sample to surfaces (from syringes,
etc) will have a relatively large effect. But there might be a reason for
the rather low concentration.

-      Do you determine the density increment or the vbar? A single
solution/solvent pair is sufficient for vbar and the highest concentration
usually is the most precise measurement. You can of course increase the
precision of your parameter by repeated measurements.

-      I'm not aware that a particular salt or concentration can have an
effect - but this is more testimony to my ignorance (and bliss, for that
matter).

-      Accuracy of the concentration measurements is a huge issue and my
impression is that the DMA5000's precision is better than the accuracy of
most concentration measurements. Are you using interference optics for the
concentration determination? Are you sure that the additional sample
handling steps do not change your concentration? Can you run N-determination
or AA-analysis in parallel (though they also have their issues)?

-      Finally, instruments from a certain period have a CPU-board that will
give out after a certain time (in our case, after the warranty expired). I
seem to be remember that our measurements were less stable in the weeks
before the instrument became unusuable, but I have no documentation to this
end. Check with your local AntonPaar subsidiary.

-      

There is of course the experimental alternative of using 18OH2, which avoids
the issue of concentration accuracy. But you will still need accuracy in
your density and s-values. Moreover, defining the solution conditions wrt
species concentrations becomes more of an issue.

Cheers, Holger

From: RASMB [mailto:rasmb-bounces at list.rasmb.org] On Behalf Of John Sumida
Sent: 30 September 2014 00:50
To: rasmb at rasmb.org <mailto:rasmb at rasmb.org> 
Subject: [RASMB] Reproducibility in densitometry measurement

Dear RASMB,

We are trying to perform a measurement of v-bar using an Anton Paar DMA5000.
The measurement is of a glycosylated protein.  The densitometer was
calibrated against a pure dI water standard and we've resorted to air checks
between measurements to control for our cleaning protocol: 3 x detergent
wash, 3 x dI water rinse, 3 x ethanol rinse, and air drying for 4 minutes or
until the reading on the densitometer is 0.0012 g/cm3.

We determined the extinction coefficient by synthetic boundary and believe
we have accurately measured the concentration of our samples.  Stock sample
was dialyzed for a little over 48 hours against PBS and dilutions of the
stock into dialysate were made in order to generate four solutions ranging
from 0.1 to 0.3 mgs/mL.

We are getting a values ranging between ~0.60 - 0.70, and are observing that
the density measurements are not reproducible beyond the fourth decimal
place.  

The reason for the lack of reproducibility appears to be that over time,
while the sample is in the U-tube, small gas bubbles are observed inside the
tube, either due to auto-hydrolysis of the solution or due to the small
vibration of the tube or something else I am not aware of.  We do not
observe this for water alone.  Samples were degassed, each for 10 minutes.

My questions: 

Has anyone observed similar issues when performing this measurement?

Is there an optimal buffer system  for this measurement - perhaps at a salt
concentration less than 100mM?

Is there, perhaps, a subtlety in this measurement that I need to be aware
of?

Thank you for any and all comments returned.  They are greatly appreciated.

Best regards

John Sumida

University of Washington

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