For cancer gene therapy, a recombinant adenovirus serotype 5 named RPR/INGN
201 has been constructed by susbtitution of the Fl region with human tumor
suppressor gene p53. The protein components of RPR/INGN201 virions were sep
arated by reversed-phase HPLC and were individually identified by electrosp
ray time-of-flight mass spectrometry and N-terminal sequencing, both on int
act proteins and on their proteolytic fragments after trypsin digestion. Tw
enty-five peptide components of the proteome (including fiber) with greater
than 0.25-0.5% contribution to the protein content of the virus were ident
ified and characterized. Fiber was confirmed to be partially glycosylated (
both the non-glycosylated and the monoglycosylated states were identified),
and two proteins were isolated and identified as phosphorylation derivativ
es, namely protein V (non-phosphorylated and monophosphorylated) and protei
n IIIa (mono- and diphasphorylated). This new analytical tool proved to be
very useful not only for refining our current knowledge of the polypeptide
repertoire of purified infectious virions but also for monitoring and very
rapidly identifying structural modifications resulting from changes in the
manufacturing process. It was also used successfully for the characterizati
on of various adenoviral constructs. (C) 2001 Elsevier Science B.V. All rig
hts reserved.