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<DIV dir=ltr align=left><FONT color=#0000ff size=2 face=Arial><SPAN
class=045583116-13082013>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 <EM>c(s)</EM>
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 <EM>c(s)</EM> 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). </SPAN></FONT></DIV>
<DIV dir=ltr align=left><FONT color=#0000ff size=2 face=Arial><SPAN
class=045583116-13082013></SPAN></FONT> </DIV>
<DIV dir=ltr align=left><FONT color=#0000ff size=2 face=Arial><SPAN
class=045583116-13082013>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 <EM>g(s*) </EM>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).</SPAN></FONT></DIV>
<DIV dir=ltr align=left><FONT color=#0000ff size=2 face=Arial><SPAN
class=045583116-13082013></SPAN></FONT> </DIV>
<DIV dir=ltr align=left><FONT color=#0000ff size=2 face=Arial><SPAN
class=045583116-13082013>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).</SPAN></FONT></DIV>
<DIV><FONT color=#0000ff size=2 face=Arial><SPAN
class=045583116-13082013></SPAN></FONT> </DIV>
<DIV><FONT color=#0000ff size=2 face=Arial><SPAN class=045583116-13082013>John
P.</SPAN></FONT></DIV>
<DIV dir=ltr align=left><BR></DIV>
<DIV lang=en-us class=OutlookMessageHeader dir=ltr align=left>
<HR tabIndex=-1>
<FONT size=2 face=Tahoma><B>From:</B> rasmb-bounces@list.rasmb.org
[mailto:rasmb-bounces@list.rasmb.org] <B>On Behalf Of </B>Walter
Stafford<BR><B>Sent:</B> Monday, August 12, 2013 6:30 PM<BR><B>To:</B> John
Sumida<BR><B>Cc:</B> <rasmb@rasmb.org><BR><B>Subject:</B> Re: [RASMB] MW
calculaiton question<BR></FONT><BR></DIV>
<DIV></DIV>
<DIV>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. <BR><BR>Walter Stafford
<DIV><A
href="mailto:wstafford3@walterstafford.com">wstafford3@walterstafford.com</A></DIV>
<DIV><BR></DIV></DIV>
<DIV><BR>On Aug 12, 2013, at 20:57, "John Sumida" <<A
href="mailto:jpsumida@u.washington.edu">jpsumida@u.washington.edu</A>>
wrote:<BR><BR></DIV>
<BLOCKQUOTE type="cite">
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<DIV class=WordSection1>
<P class=MsoPlainText>Dear RASMB,<O:P></O:P></P>
<P class=MsoPlainText><O:P></O:P></P>
<P class=MsoPlainText>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.<O:P></O:P></P>
<P class=MsoPlainText><O:P></O:P></P>
<P class=MsoPlainText>Objective<O:P></O:P></P>
<P class=MsoPlainText>I am trying to reconcile results obtained using two
different approaches, SEDANAL and SEDFIT.<O:P></O:P></P>
<P class=MsoPlainText><O:P></O:P></P>
<P class=MsoPlainText>Observations<O:P></O:P></P>
<P class=MsoPlainText>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. <O:P></O:P></P>
<P class=MsoPlainText><O:P></O:P></P>
<P class=MsoPlainText>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.<O:P></O:P></P>
<P class=MsoPlainText><O:P></O:P></P>
<P class=MsoPlainText>My question:<O:P></O:P></P>
<P class=MsoPlainText>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.<O:P></O:P></P>
<P class=MsoPlainText><O:P></O:P></P>
<P class=MsoPlainText>Background.<O:P></O:P></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 0.5in; TEXT-INDENT: -0.25in; mso-list: l1 level1 lfo3"><!--[if
!supportLists]--><SPAN style="mso-list: Ignore">1.<SPAN
style="FONT: 7pt 'Times New Roman'">
</SPAN></SPAN><!--[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.<O:P></O:P></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 0.5in; TEXT-INDENT: -0.25in; mso-list: l1 level1 lfo3"><!--[if
!supportLists]--><SPAN style="mso-list: Ignore">2.<SPAN
style="FONT: 7pt 'Times New Roman'">
</SPAN></SPAN><!--[endif]-->The material being studies is a polymer
micelle with a CMC of 14 micrograms/ml. <O:P></O:P></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 0.5in; TEXT-INDENT: -0.25in; mso-list: l1 level1 lfo3"><!--[if
!supportLists]--><SPAN style="mso-list: Ignore">3.<SPAN
style="FONT: 7pt 'Times New Roman'">
</SPAN></SPAN><!--[endif]-->The polymer was run over a series of
concentrations ranging from 0.2 mgs/ml to 1.00 mgs/ml. <O:P></O:P></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 1in; TEXT-INDENT: -0.25in; mso-list: l1 level2 lfo3"><!--[if
!supportLists]--><SPAN style="mso-list: Ignore">a.<SPAN
style="FONT: 7pt 'Times New Roman'">
</SPAN></SPAN><!--[endif]-->Thus under the conditions of the experiment
I am at least 14 times the CMC at the lowest concentration.<O:P></O:P></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 0.5in; TEXT-INDENT: -0.25in; mso-list: l1 level1 lfo3"><!--[if
!supportLists]--><SPAN style="mso-list: Ignore">4.<SPAN
style="FONT: 7pt 'Times New Roman'">
</SPAN></SPAN><!--[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.
<O:P></O:P></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 0.5in; TEXT-INDENT: -0.25in; mso-list: l1 level1 lfo3"><!--[if
!supportLists]--><SPAN style="mso-list: Ignore">5.<SPAN
style="FONT: 7pt 'Times New Roman'">
</SPAN></SPAN><!--[endif]-->From the analysis of raw SV data, SEDANAL
returns an experimental sedimentation coefficient of 7.9s and a calculated
s20w of 8.8s. <O:P></O:P></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 0.5in; TEXT-INDENT: -0.25in; mso-list: l1 level1 lfo3"><!--[if
!supportLists]--><SPAN style="mso-list: Ignore">6.<SPAN
style="FONT: 7pt 'Times New Roman'">
</SPAN></SPAN><!--[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. <O:P></O:P></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 1in; TEXT-INDENT: -0.25in; mso-list: l1 level2 lfo3"><!--[if
!supportLists]--><SPAN style="mso-list: Ignore">a.<SPAN
style="FONT: 7pt 'Times New Roman'">
</SPAN></SPAN><!--[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. <O:P></O:P></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 1in; TEXT-INDENT: -0.25in; mso-list: l1 level2 lfo3"><!--[if
!supportLists]--><SPAN style="mso-list: Ignore">b.<SPAN
style="FONT: 7pt 'Times New Roman'">
</SPAN></SPAN><!--[endif]-->Of these four discrete species, three were
grouped beneath the major peak observed in the initial c(s)
analysis.<O:P></O:P></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 1in; TEXT-INDENT: -0.25in; mso-list: l1 level2 lfo3"><!--[if
!supportLists]--><SPAN style="mso-list: Ignore">c.<SPAN
style="FONT: 7pt 'Times New Roman'">
</SPAN></SPAN><!--[endif]-->The calculated weight averaged s20w for
these three species was 8.1s. <O:P></O:P></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 1in; TEXT-INDENT: -0.25in; mso-list: l1 level2 lfo3"><!--[if
!supportLists]--><SPAN style="mso-list: Ignore">d.<SPAN
style="FONT: 7pt 'Times New Roman'">
</SPAN></SPAN><!--[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.<O:P></O:P></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 0.5in; TEXT-INDENT: -0.25in; mso-list: l1 level1 lfo3"><!--[if
!supportLists]--><SPAN style="mso-list: Ignore">7.<SPAN
style="FONT: 7pt 'Times New Roman'">
</SPAN></SPAN><!--[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.<O:P></O:P></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 1in; TEXT-INDENT: -0.25in; mso-list: l1 level2 lfo3"><!--[if
!supportLists]--><SPAN style="mso-list: Ignore">a.<SPAN
style="FONT: 7pt 'Times New Roman'">
</SPAN></SPAN><!--[endif]-->Estimates of the diffusion coefficient in
SEDFIT and SEDNTERP (and thus presumable also from SEDANAL) are not very
different. <O:P></O:P></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 1in; TEXT-INDENT: -0.25in; mso-list: l1 level2 lfo3"><!--[if
!supportLists]--><SPAN style="mso-list: Ignore">b.<SPAN
style="FONT: 7pt 'Times New Roman'">
</SPAN></SPAN><!--[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).<O:P></O:P></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 0.5in; TEXT-INDENT: -0.25in; mso-list: l1 level1 lfo3"><!--[if
!supportLists]--><SPAN style="mso-list: Ignore">8.<SPAN
style="FONT: 7pt 'Times New Roman'">
</SPAN></SPAN><!--[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. <O:P></O:P></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 1in; TEXT-INDENT: -0.25in; mso-list: l1 level2 lfo3"><!--[if
!supportLists]--><SPAN style="mso-list: Ignore">a.<SPAN
style="FONT: 7pt 'Times New Roman'">
</SPAN></SPAN><!--[endif]-->I understand that this is nonsensical as ff0
cannot be less than 1. <O:P></O:P></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 1in; TEXT-INDENT: -0.25in; mso-list: l1 level2 lfo3"><!--[if
!supportLists]--><SPAN style="mso-list: Ignore">b.<SPAN
style="FONT: 7pt 'Times New Roman'">
</SPAN></SPAN><!--[endif]-->Notably SEDNTERP version 1.09 also
calculates a frictional ratio<1; namely SEDNTERP calculate ffp=0.9449.
<O:P></O:P></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 1in; TEXT-INDENT: -0.25in; mso-list: l1 level2 lfo3"><!--[if
!supportLists]--><SPAN style="mso-list: Ignore">c.<SPAN
style="FONT: 7pt 'Times New Roman'">
</SPAN></SPAN><!--[endif]-->The c(s) analysis in SEDFIT returns an
ff0=1.47.<O:P></O:P></P>
<P class=MsoPlainText><O:P></O:P></P>
<P class=MsoPlainText>Thus to summarize:<O:P></O:P></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 0.5in; TEXT-INDENT: -0.25in; mso-list: l0 level1 lfo1"><!--[if
!supportLists]--><SPAN style="mso-list: Ignore">1.<SPAN
style="FONT: 7pt 'Times New Roman'">
</SPAN></SPAN><!--[endif]-->Relatively similar s20w values are
determined in both SEDFIT and SEDANAL for the same data-set.<O:P></O:P></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 1in; TEXT-INDENT: -0.25in; mso-list: l0 level2 lfo1"><!--[if
!supportLists]--><SPAN style="mso-list: Ignore">a.<SPAN
style="FONT: 7pt 'Times New Roman'">
</SPAN></SPAN><!--[endif]-->8.5s from SEDFIT<O:P></O:P></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 1in; TEXT-INDENT: -0.25in; mso-list: l0 level2 lfo1"><!--[if
!supportLists]--><SPAN style="mso-list: Ignore">b.<SPAN
style="FONT: 7pt 'Times New Roman'">
</SPAN></SPAN><!--[endif]-->8.8s from SEDANAL <O:P></O:P></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 0.5in; TEXT-INDENT: -0.25in; mso-list: l0 level1 lfo1"><!--[if
!supportLists]--><SPAN style="mso-list: Ignore">2.<SPAN
style="FONT: 7pt 'Times New Roman'">
</SPAN></SPAN><!--[endif]-->SEDFIT calculates a MW almost exactly 2
times the value of the MW returned by SEDANAL for the same
s20w.<O:P></O:P></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 0.5in; TEXT-INDENT: -0.25in; mso-list: l0 level1 lfo1"><!--[if
!supportLists]--><SPAN style="mso-list: Ignore">3.<SPAN
style="FONT: 7pt 'Times New Roman'">
</SPAN></SPAN><!--[endif]-->The estimates of diffusion in both programs
are quite similar<O:P></O:P></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 0.5in; TEXT-INDENT: -0.25in; mso-list: l0 level1 lfo1"><!--[if
!supportLists]--><SPAN style="mso-list: Ignore">4.<SPAN
style="FONT: 7pt 'Times New Roman'">
</SPAN></SPAN><!--[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. <O:P></O:P></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 0.5in; TEXT-INDENT: -0.25in; mso-list: l0 level1 lfo1"><!--[if
!supportLists]--><SPAN style="mso-list: Ignore">5.<SPAN
style="FONT: 7pt 'Times New Roman'">
</SPAN></SPAN><!--[endif]-->The frictional ratio calculated in SEDNTERP,
assuming the MW=1.3 MDa, is 1.54<O:P></O:P></P>
<P class=MsoPlainText
style="MARGIN-LEFT: 0.5in; TEXT-INDENT: -0.25in; mso-list: l0 level1 lfo1"><!--[if
!supportLists]--><SPAN style="mso-list: Ignore">6.<SPAN
style="FONT: 7pt 'Times New Roman'">
</SPAN></SPAN><!--[endif]-->There does appear to be heterogeneity in the
major peak observed in the c(s) analysis.<O:P></O:P></P>
<P class=MsoPlainText><O:P></O:P></P>
<P class=MsoPlainText>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.<O:P></O:P></P>
<P class=MsoPlainText><O:P></O:P></P>
<P class=MsoPlainText>Best regards<O:P></O:P></P>
<P class=MsoPlainText>John Sumida<O:P></O:P></P>
<P class=MsoPlainText>University of Washington<O:P></O:P></P>
<P class=MsoPlainText>Analytical Biopharmacy Core<O:P></O:P></P>
<P class=MsoNormal><SPAN
style="FONT-SIZE: 14pt"><O:P></O:P></SPAN></P></DIV></DIV></BLOCKQUOTE>
<BLOCKQUOTE type="cite">
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