Is the following reply what you are trying to remember? They show no
modification by PMSF!
Tony
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In response to Ken Mitchelhill's recommendation on the use of AEBSF as a
substitute for PMSF:
We have found that AEBSF modifies many proteins by covalent attachment,
preferentially on Tyr, and to a lesser extent on Lys, His, and the amino-
terminus. These modifications were identified by electrospray MS of the
proteins (adds 183 Da per AEBS-group) and by peptide mapping and MS/MS.
All the proteins we examined were modified after 24 hrs. at 4 C with
1 mM AEBSF in TRIS, pH 8.0. The reaction is 10-20x slower at pH 7; however
AEBSF is quite stable in aqueous solution and the extent of to which the
protein is modified continues to increase for several days. We have seen
the addition of 10 or more AEBS-groups to proteins after prolonged storage.
We found no equivalent modification from PMSF, probably because it degrades
so quickly. We no longer use AEBSF, and urge caution to those who do.
John Stults
Genentech, Inc.
***************************************
At 07:59 AM 7/31/98 -0700, John Philo wrote:
>Kathryn,
>
>Since no one else seems to have replied, I do know that others have seen
>this phenomenon. I remember seeing a poster from a group at Genentech at a
>Protein Society meeting that showed covalent modifications by modest
>concentrations of PEFABLOC, and to some extent by high concentrations of
>PMSF. I believe that was the meeting 3 years ago in Boston, but I
>unfortunately can't remember any of the names or even what the protein was.
>I don't believe the reactions were at serines, though again my memory may be
>wrong about that.
>
>John Philo
>Alliance Protein Laboratories
>
>-----Original Message-----
>From: Association of Biomolecular Resource Facilities
>[mailto:abrf-request@aecom.yu.edu]On Behalf Of Dr K.S. Lilley
>Sent: Tuesday, July 28, 1998 6:45 AM
>To: Recipients of ABRF List
>Subject: Mass spec:PMSF
>
>
>Dear all,
>
>one of my users has recently supplied me with two proteins for mass
>analysis,
>one being the wild type and the other a mutant of a 23kDa protein which
>binds to
>a GTPase causing loss in the latter's activity. In both cases several
>species
>are present differing by multiples of 155Da from the mass deduced from the
>gene
>sequence. The user adds relatively high amounts of PMSF to her final
>protein
>fractions in order to stop proteolysis by a co-purifying protease. We were
>therefore wondering whether the discrepancies seen in the mass analysis
>were
>due to the formation of adducts between PMSF and serine residues within
>these
>proteins.
>
>Has anyone observed anything like this before?
>
>Kathryn Lilley
>
>
>Dr. K.S.Lilley
>Laboratory Manager
>PNACL
>University of Leicester
>Lancaster Road
>Leicester LE1 9HN
>
>tel: 44 116 252 5613
>fax: 44 116 252 5616
>email: ksl@le.ac.uk
>
>
>
>
************************************
Dr. Anthony T. Yeung, Ph.D.
Director, Fannie E. Rippel Biotechnology Facility
Member, Institute for Cancer Research
Fox Chase Cancer Center
7701 Burholme Ave. Philadelphia, PA 19111
Voice: 215-728-2488
FAX: 215-728-3647
email: AT_Yeung@FCCC.edu
http://www.fccc.edu/research/labs/yeung/
************************************