[RASMB] graphing AUC data

[John Philo]

Tara,
 
You are making the graphs correctly, but your own statements about your
results show why these lines have different slopes. When you use the sigma
calculated by SEDNTERP using the sequence mass for a trimer, then your graph
will give a line corresponding to that sequence mass. However, you said that
the fit from NONLIN corresponds to a molecular mass that is 4.8% different
from the sequence mass for trimer, and one can easily see a 4.8% difference
in slope on such a graph. So yes, of course, the line using the NONLIN
result goes through the data very well (by definition if it is a good fit),
and the line using the sequence mass won't go through the data as well
because your data don't actually correspond to the molecular mass for pure
trimer.
 
If you are trying to show the expected slopes that correspond to trimer,
dimer, and tetramer then you should use the values based on the known
monomer mass. But fundamentally the graph isn't coming out like the
published results because your data is telling you the sample is not pure
trimer (unless the vbar or density are way off, but presumably you are using
the same vbar as the other labs used).
 
You didn't say whether the NONLIN result is above or below the expected
trimer mass. If it is below, it might simply be because at the concentration
you used there is significant reversible dissociation to monomer (perhaps
the other labs were plotting data taken at a higher concentration, where the
fraction monomer is very low). If the concentrations are similar, and your
mass is low, I'm going to guess your sample is a mixture of trimer and some
denatured 'incompetent monomer' that cannot associate to form trimers. On
the other hand, if NONLIN says the average mass is above that for trimer,
your sample probably contains some aggregate.
 
'Hope this helps,
 
John Philo
Alliance Protein Laboratories

-----Original Message-----
From: rasmb-admin at server1.bbri.org [mailto:rasmb-admin at server1.bbri.org] On
Behalf Of Tara Suntoke
Sent: Wednesday, October 13, 2004 10:48 AM
To: rasmb at server1.bbri.org
Subject: [RASMB] graphing AUC data


Hi all,
I am fairly new to AUC and have a question about Sednterp and graphing AUC
data.  
 
I spun a peptide that is a clean trimer- other people have published the
same result for the same peptide.  I think the data is good for the
following reasons:  the residuals are random, the data fits best to a single
species, and has a square root of variance of 5.9 e-3.  In addition, the
molecular weight I get from the experiment, (24.8kD for the trimer) is
within 4.8% of the calculated MW in Sednterp (based on composition).  
 
I am trying to make a graph of ln(A) vs. r^2, and show that the peptide
corresponds to a trimer and not a dimer or tetramer.  In order to graph the
lines of the dimer, trimer, and tetramer, I used the equation of a line as
follows:
 
ln(A) = (sigma)(r^2) + ln(A1).  I obtained the constant ln(A1) from the
WinNonlin program. 
 
My question is about which value of sigma I should use.  I can use the value
of sigma obtained from Nonlin, which is based on the data.  Alternatively, I
can use the sigma that Sednterp gives me, based on the peptide composition
and buffers.  When making these graphs however, the data fits exactly to the
line for a trimer which uses the Nonlin sigma, and doesn't exactly follow
the slope of the line that uses the Sednterp sigma.  I understand that the
sigma obtained from Nonlin comes from the data, and perhaps that is why it
fits to the data well.  On the other hand, the sigma from Sednterp is
calculated using density and partial specific volumes that are close
estimates, but aren't exact. (at least this is my understanding so far...)
So I am wondering which value of sigma is appropriate to use.
 
I'm not sure whether you will be able to see this at all, but I have
enclosed a PDF to show you the different results I get using the different
sigma values.  In all the papers that use the same peptide, people report
graphs very similar to the lower one, which uses sigma derived from Nonlin.
The data always overlaps the line for a trimer.  However, it isn't clear how
they generated those graphs. 
 
I would greatly appreciate any advice you have.
 
Thanks for your help,
 
Tara Suntoke

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://list.rasmb.org/pipermail/rasmb-rasmb.org/attachments/20041013/e35301f8/attachment.htm>