RE: +183 Da modification to a recombinant protein

LANGE, GARY W [PHR/1005] (GARY.W.LANGE@stl.monsanto.com)
Tue, 21 Sep 1999 10:40:44 -0500

John's original June 1996 response was so good that I saved it. I found a
plus 183 with something we were working with shortly thereafter. Funny
thing that this has come up again. I am unable to find the N-terminus, of
all things, in an 88Kd protein tryptic digest of a recombinant protein
produced in insect cells. It's blocked, but I've been only thinking about
+42, and +28 when examining the fragments via MALDI. And... the
purification group is using Roche "Complete" which contains Pefabloc
(AEBSF). I had better start looking for a +183 fragment :-)

Does anyone know if you see a characteristic "decay" of AEBSF from a
fragment like you do for phosphorylated or carbamylated fragments in the
reflector mode?

----------------------------------
"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.
--------------------------------
Does anyone have a reference for this that can be forwarded to Ken to be
added to the Delta Mass table? Ken must have missed this one as he was one
of the authors on that string... What would we do without Delta Mass..
Thanks again Ken.

Gary Lange
Monsanto Co.
g.w.lange@monsanto.com
636-737-6602

-----Original Message-----
From: John Stults [mailto:jts@gene.COM]
Sent: Monday, September 20, 1999 4:12 PM
To: Recipients of ABRF List
Subject: Re: +183 Da modification to a recombinant protein

Gejing,

We noticed +183 modifications a number of years ago and investigated them
further. My guess is that AEBSF (aminoethylbenzenesulfonic acid,
PefablocTM)
was used as a serine protease inhibitor in the purification of your protein.
AEBSF reacts primarily with tyrosine phenolic groups, and to a lesser extent
with amino groups. To address the problem, Boehringer Mannheim (now Roche
Molecular Biochemicals) introduced Pefabloc PLUS which includes an
additional
component to compete for these side reactions. In our limited experience
with
Pefabloc PLUS, it reduces the +183 modifications, but does not always
eliminate them. As a result, we prefer PMSF, despite its own set of
drawbacks. We have never found PMSF-induced modification of proteins
(except
trypsin), probably due to its short half-life in aqeous solution.

John

John T. Stults, PhD
Senior Scientist
Protein Chemistry Department
Genentech, Inc.
1 DNA Way, MS #63
South San Francisco, CA 94080
ph: (650) 225-1203
fax (650) 225-5945
email: jts@gene.com