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
>
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