Biology Reference
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Fig. 1 Schematic illustration of different approaches for analysis of the total pro-
teome and the posttranslational modifi cations in Adenovirus using mass spec-
trometry. After growing virus in host cells it is necessary to remove host cell
proteins. Highly purifi ed virus can be obtained by CsCl gradient centrifugation.
Different alternatives are possible for fractionation of proteins, e.g., 1D and 2D
SDS-PAGE before enzymatic cleavage of proteins into peptides. An alternative is
to directly cleave proteins into peptides in solution. At the peptide stage, different
enrichments for PTMs can be applied. The fi nal detection of peptides is per-
formed with mass spectrometry. After database search, protein identities and
possible posttranslational modifi cations are reported. Manual inspection of the
recorded MS/MS spectrum is required for verifying the position of a posttransla-
tional modifi cation in a peptide sequence
Remove as much supernatant as possible. Resuspend each
pellet in 10 mL of 10 mM Tris-HCl, pH 7.9. Spin down at
~250 × g , 4 °C for 5 min. Resuspend again each pellet in 10 mL
of 10 mM Tris-HCl pH 7.9 and freeze four aliquots at −80 °C
until further use.
3.2 Virus Purifi cation
1. Thaw the infected cell solutions at room temperature (we recom-
mend using all four tubes from Subheading 3.1.3 and pooling
them into one highly concentrated virus preparation at the end).
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