Steve,
I did not calibrate on BSA to get the spectrum shown on the website. The
calibration was done with everyone's favorite calibrant - ultramark. So the
number that I got was what I got. However, I would expect it to be within
0.01% (+/-6 Da) of whatever the actual value really is (whatever that may
be). The sequence that I have for BSA (shown below) calculates to 66463
with no disulfides, or 66433.8 with 14. I have not done any peptide mapping
of BSA, but the number I have gotten by ESI on a number of different
instruments with different (good) BSA samples has always been close to this
value (66430 ish).
Cheers,
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-----Original Message-----
From: Association of Biomolecular Resource Facilities
[mailto:abrf-request@aecom.yu.edu]On Behalf Of Steven Seeholzer
Sent: Friday, February 23, 2001 4:42 PM
To: Recipients of ABRF List
Subject: Re: Big Proteins on MALDI
Hi Mark,
You and others are using a figure of about 16431 or so for the average
molecular
weight of BSA. If you take into account the 16 disulfide bonds then you
should
come to the correct native unmodified molecular weight of 16400.7. This is
the
value that I use. My understanding is that three of the 35 Cys residues are
not
in disulfide bonds and I hope to be corrected if mistaken I have not
validated
this against other known standards as I have for cytochrome c and other
small
proteins nor have I systematically verified the sequence by tandem mass spec
or
peptide mass mapping. I use the ALBU_BOVIN sequence from Swiss-Prot
database. I
wonder what sequence others use.
Best regards to all. See you in San Diego....
Steve...
Mark Hail wrote:
> All
>
> I agree with the others that BSA is not a good calibrant. Here's some
> additional proof. I've put up an ESI spectrum on our website so you can
see
> just how heterogeneous it is. If you want to check it out, go here:
>
> http://www.enovatia.com/discuss/msgReader$153
>
> Mark Hail
> Novatia Corp.
> www.enovatia.com
>
> -----Original Message-----
> From: Association of Biomolecular Resource Facilities
> [mailto:abrf-request@aecom.yu.edu]On Behalf Of Peter Juhasz
> Sent: Thursday, February 22, 2001 8:38 PM
> To: Recipients of ABRF List
> Subject: Re: Big Proteins on MALDI
>
> BSA is also known to be glucated at one or two positions (If I'm correct
> there was a paper from P. Traldi's group in Rapid Communications in Mass
> Spectrometry about five years ago). The highest resolution MALDI mass
> spectra I have seen on BSA clearly showed a relatively sharp peak around
> 66,430 Da and an overlapping "hump" centered about 300 Da higher. You also
> have to take into account various matrix adducts which cannot be resolved
at
> this mass anymore (+207 and +225 for sinapinic acid). Arnie was right
about
> yeast enolase: that one has about the highest mass where high mass
> resolution, limited by the width of the isotope envelope, can be generated
> routinely (unless you are one of the lucky ones having a well
characterized
> F(ab)2 molecule). In my experience mass accuracy is very closely related
to
> mass resolution in TOF. One can't expect to have more than about times
> higher accuracy than mass resolution (i.e. 1:1000=0.1% mass accuracy for
> 1:100 resolution). Anything better should be considered fluke.
>
> ----- Original Message -----
> From: "David A. Schooley" <schooley@med.unr.edu>
> To: "Recipients of ABRF List" <abrf@aecom.yu.edu>
> Sent: Wednesday, February 21, 2001 5:52 PM
> Subject: Re: Big Proteins on MALDI
>
> > At 12:17 PM -0500 2/21/01, Wei Wu wrote:
> > Wei-
> >
> > Note that Swiss Prot lists conflicts in the sequence of BSA
> > that will affect MW. Kathy Schegg here once sequenced a fragment of
> > reduced, carboxymethylated BSA from a digest to test the Procise and
> > protocol. As luck would have it, the fragment she sequenced was one
> > of the ones containing a "conflict", and the Edman sequence agreed
> > with the reported conflict, NOT with the SWISS-PROT sequence.
> > For additional details contact her at schegg@med.unr.edu. It
> > is surprising that they don't correct the original sequence!
> >
> > David
> >
> >
> > >Hi Jim,
> > >
> > >We've had trouble getting the theoretical mass of BSA. From a
> > >thermobioanalysis application note, it's 66431 Da. However, if we go
> > >directly from the sequence in Swiss Prot, the mass is 66433 Da for the
> > >reduced form, and 66398 for the form with disulfides (17 of those). I
> guess
> > >we are dealing w/ the form with disulfides. Yet, if the accuaracy is
0.1%
> > >like Arnie said, it'd be hard to differentiate the two. We also found
> that
> > >if we used the BSA and RNase A to calibrate the double charge peak of
> BSA,
> > >it was off from calculated by either of the numbers above, which
probably
> > >means the BSA we used has a different mass, as we are pretty confident
> about
> > >RNase A.
> > >
> > >Regards,
> > >Wei
> > >
> >
> > --
> > David A. Schooley
> > Dept. of Biochemistry/330
> > Univ. of Nevada
> > Reno, NV 89557
> > schooley@unr.edu
> > tel: (775) 784-4136; fax (775) 784-1419
> > NOTE NEW AREA CODE: Mandatory after 5/15/99
> >
-- Steven H. Seeholzer, Ph.D. ICR Staff Scientist Fox Chase Cancer Center 7701 Burholme Avenue Philadelphia PA 19111voice: (215) 728-1111 fax: (215) 728-3574 email: sh_seeholzer@fccc.edu
trapper
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