[RASMB] MW calculation question

[Leech, AP]

Hello all,

Further to John P. and Walter's comments, it would be interesting to
know how the DLS and SLS measurements were done and what, if any,
degree of inhomogeneity was apparent in them.

Andrew

On 13/08/2013 18:04, John Philo wrote:
> John S., I agree with Walter Stafford that heterogeneity is causing the
> single-species fit to underestimate the mass, but I don't think there is
> any reason to think the primary cause is heterogeneity of monomer mass
> or conformation. You said yourself that the /c(s)/ analysis gives one
> major peak that is only 80% of the total area. If the sample is only 80%
> pure, why would you expect a single-species fit to give the correct MW?
> If /c(s)/ is giving one narrow main peak then it is not its ability to
> emulate multiple overlapping species that is primarily improving the fit
> quality relative to a single-species fit, it is the addition of the 20%
> of minor species (presumably with significantly different sedimentation
> coefficients).
> The strength of whole-boundary analysis is that it fits all the scans,
> but that means that to get good results it must account for all species
> that are present, whether or not they are things you are interested in!
> That is, a weakness of whole boundary analysis is that it isn't
> particularly easy to exclude impurities, aggregates, fragments or other
> 'junk' from the data analysis, and your results simply demonstrate that
> problem. This is a case where a quick and simple /g(s*) /analysis via
> the dc/dt approach, and then fitting only the main peak of that
> distribution (excluding the junk at high and low sedimentation
> coefficients) would probably correctly identify the MW of your 80% major
> species (but always the best precision should come from whole boundary
> analysis).
> Further, fundamentally your question is an apples vs. oranges
> comparison. You say you want to compare the two different analysis
> programs, but you are trying to compare two fundamentally different
> fitting models. You can easily do a single-species fit in SEDFIT and
> then compare that to the one from SEDANAL (and I'm sure the results will
> be similar).
> John P.
>
> ------------------------------------------------------------------------
> *From:* rasmb-bounces at list.rasmb.org
> [mailto:rasmb-bounces at list.rasmb.org] *On Behalf Of *Walter Stafford
> *Sent:* Monday, August 12, 2013 6:30 PM
> *To:* John Sumida
> *Cc:* <rasmb at rasmb.org>
> *Subject:* Re: [RASMB] MW calculaiton question
>
> I think this was already answered. I think your sample is heterogeneous.
> Small variations on composition (I.e molar mass heterogeneity) will
> cause peak broadening that will appear as diffusion. If you include
> enough data in the fit. It should be apparent in the residuals. C(s)
> will add enough components to account for the heterogeneity (albeit with
> the same f/fo) and should give better residuals. Sedanal will not
> because fitting to a single species (I. E, the wrong model) should not
> give as good of a fit. The heterogeneity will cause Sedanal to return a
> value that is too large and therefore, a molar mass that is too small.
>
> Walter Stafford
> wstafford3 at walterstafford.com <mailto:wstafford3 at walterstafford.com>
>
>
> On Aug 12, 2013, at 20:57, "John Sumida" <jpsumida at u.washington.edu
> <mailto:jpsumida at u.washington.edu>> wrote:
>
>> Dear RASMB,
>>
>> The following was posted on the SEDFIT user list and it has been
>> suggested that I also post this message to the RASMB list.  Thus I am
>> reposting my question to RASMB and am including some edits and
>> additional information for clarity and correctness.
>>
>> Objective
>>
>> I am trying to reconcile results obtained using two different
>> approaches, SEDANAL and SEDFIT.
>>
>> Observations
>>
>> In SEDFIT, analysis of sed velocity FDS data returns an s20w of 8.5s
>> and MW=1.3 MDa for the major peak, comprising >80% of the total
>> loading concentration, determined in a c(s) fit.  Please note after
>> checking with our collaborators this value is consistent with the MW
>> returned from DLS and static light scattering experiments.  Thus the
>> SEDFIT result is consistent with the DLS and static light scattering
>> data which estimate a MW of 1.1 MDa.
>>
>> In SEDANAL, the simplest model necessary to fit the data well was a
>> single species model.  Using this model, analysis of the same data-set
>> returns an s20w of 8.8s, similar to the value calculated in SEDFIT,
>> but a MW of 655 kDa is calculated.
>>
>> My question:
>>
>> Why does the value of the MW returned for SEDANAL and SEDFIT differ by
>> a factor of ~2 when the s20w in each case are similar (8.5s versus
>> 8.8s).  My purpose here is that I believe the apparent difference
>> being returned from these parallel analyses is saying something
>> fundamentally important about the behavior of these materials in
>> buffer and our assumptions thereof.
>>
>> Background.
>>
>> <!--[if !supportLists]-->1.<!--[endif]-->Rotor speed was 30 krpm,
>> vbar=0.917 (measured), solution density = 1.00506 g/cm3 (measured),
>> and viscosity =0.0100281 Poise (measured).  Temperature = 20oC.
>>
>> <!--[if !supportLists]-->2.<!--[endif]-->The material being studies is
>> a polymer micelle with a CMC of 14 micrograms/ml.
>>
>> <!--[if !supportLists]-->3.<!--[endif]-->The polymer was run over a
>> series of concentrations ranging from 0.2 mgs/ml to 1.00 mgs/ml.
>>
>> <!--[if !supportLists]-->a.<!--[endif]-->Thus under the conditions of
>> the experiment I am at least 14 times the CMC at the lowest concentration.
>>
>> <!--[if !supportLists]-->4.<!--[endif]-->A major peak is observed in
>> the initial c(s) distribution comprising >80% of the total loading and
>> the position of this peak (7.7 s-exp; s20w=8.5) does not shift with
>> concentration.
>>
>> <!--[if !supportLists]-->5.<!--[endif]-->From the analysis of raw SV
>> data, SEDANAL returns an experimental sedimentation coefficient of
>> 7.9s and a calculated s20w of 8.8s.
>>
>> <!--[if !supportLists]-->6.<!--[endif]-->A global analysis in SEDPHAT
>> was performed over the entire concentration range, transforming the
>> initial c(s) distribution into a set of 8 discrete species.
>>
>> <!--[if !supportLists]-->a.<!--[endif]-->Four of these 8 species
>> survived a critical chi square analysis suggesting that these four
>> species were important in retaining the quality of the fit.
>>
>> <!--[if !supportLists]-->b.<!--[endif]-->Of these four discrete
>> species, three were grouped beneath the major peak observed in the
>> initial c(s) analysis.
>>
>> <!--[if !supportLists]-->c.<!--[endif]-->The calculated weight
>> averaged s20w for these three species was 8.1s.
>>
>> <!--[if !supportLists]-->d.<!--[endif]-->The  s20w values were checked
>> and confirmed with a manual calculation, SEDNTERP verstion 1.09, as
>> well as the calculate “s(20,w) from s(xp) in SEDFIT.
>>
>> <!--[if !supportLists]-->7.<!--[endif]-->Using the ratio of s/D in the
>> Svedberg equation and values for diffusion estimated by assuming 655
>> kDA, (the MW returned in SEDANAL), I calculate a MW of 655 kDa.  Thus
>> I believe that the value for diffusion being estimated in SEDANAL is
>> consistent with the 655 kDA molecular weight.
>>
>> <!--[if !supportLists]-->a.<!--[endif]-->Estimates of the diffusion
>> coefficient in SEDFIT and SEDNTERP (and thus presumable also from
>> SEDANAL) are not very different.
>>
>> <!--[if !supportLists]-->b.<!--[endif]-->The diffusion coefficient
>> estimate in SEDFIT is 3.19E-7 cm2/sec and the value calculated in
>> SEDNTERP 3.67E-7 cm2/sec (Teller approximation).
>>
>> <!--[if !supportLists]-->8.<!--[endif]-->Using the calculate M(s)
>> function in SEDFIT and providing the experimental sedimentation
>> coefficient noted above I need to input an ff0<1 (0.8967) in order to
>> arrive at the 655kDA MW returned by SEDANAL.
>>
>> <!--[if !supportLists]-->a.<!--[endif]-->I understand that this is
>> nonsensical as ff0 cannot be less than 1.
>>
>> <!--[if !supportLists]-->b.<!--[endif]-->Notably SEDNTERP version 1.09
>> also calculates a frictional ratio<1; namely SEDNTERP calculate
>> ffp=0.9449.
>>
>> <!--[if !supportLists]-->c.<!--[endif]-->The c(s) analysis in SEDFIT
>> returns an ff0=1.47.
>>
>> Thus to summarize:
>>
>> <!--[if !supportLists]-->1.<!--[endif]-->Relatively similar s20w
>> values are determined in both SEDFIT and SEDANAL for the same data-set.
>>
>> <!--[if !supportLists]-->a.<!--[endif]-->8.5s from SEDFIT
>>
>> <!--[if !supportLists]-->b.<!--[endif]-->8.8s from SEDANAL
>>
>> <!--[if !supportLists]-->2.<!--[endif]-->SEDFIT calculates a MW almost
>> exactly 2 times the value of the MW returned by SEDANAL for the same s20w.
>>
>> <!--[if !supportLists]-->3.<!--[endif]-->The estimates of diffusion in
>> both programs are quite similar
>>
>> <!--[if !supportLists]-->4.<!--[endif]-->The frictional ratio ff0 in
>> the SEDFIT analysis is 1.47 whereas the calculated ffp in SEDNTERP
>> based on the observe MW in SEDANAL is <1.
>>
>> <!--[if !supportLists]-->5.<!--[endif]-->The frictional ratio
>> calculated in SEDNTERP, assuming the MW=1.3 MDa, is 1.54
>>
>> <!--[if !supportLists]-->6.<!--[endif]-->There does appear to be
>> heterogeneity in the major peak observed in the c(s) analysis.
>>
>> Thank you in advance for your comments and suggestions.  I apologize
>> for the length of this post, but in fairness, I am attempting to
>> provide information that would enable an informed response.
>>
>> Best regards
>>
>> John Sumida
>>
>> University of Washington
>>
>> Analytical Biopharmacy Core
>>
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>
>
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-- 
Dr Andrew Leech                   *  Laboratory Head
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