I couldn't agree more, a serious oversight on my part ;-)
Lucky I've got all of my friends in ABRF looking out for me. Indeed, I am
indebted to Mark Hail who had already suggested to me in a private email to
include the +183 modification in Delta Mass, which I have now done.
No real need for references, although if you have got one John, I would
appreciate the details, I've just reproduced the details from the email
discussions.
Regards to all.....Ken
>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
********************************
Ken I. Mitchelhill
The John Holt Protein Structure Laboratory
St. Vincent's Institute of Medical Research
41 Victoria Parade
Fitzroy 3065 Victoria
AUSTRALIA
Telephone: 61-3-9288 2480
Facsimile: 61-3-9416 2676
Email: k.mitchelhill@medicine.unimelb.edu.au (w)
kenandsue@ozemail.com.au (h)
***********************************