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