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produced by CsCl gradient centrifugation. In this way, the virus is
separated from host cell proteins. Further, viral structural proteins
are extracted from the virus and cleaved into peptides. For exam-
ple, in phosphoproteome analysis, enrichment of phosphopeptides
before mass spectrometric analysis improves the results by reduc-
ing the competition from non-phosphorylated species [ 13 ] and is
a requirement for detecting phosphorylation sites in complex sam-
ples as cell lysates [ 14 ]. If studying a fractionated part of the cel-
lular proteome PTMs, e.g., acetylation and methylation, and to
some extent also phosphorylations, it can be analyzed without pre-
ceding enrichment [ 15 ]. The same principle applies to analysis of
the relatively small viral proteome. Analysis of peptides carrying a
PTM with MS requires detection of a mass shift corresponding to
the added modifi cation, e.g., +79.98 Da for phosphorylation, and
localization of the PTM site in the peptide chain. Technically, this
requires high mass accuracy and resolution to detect the mass shift
and tandem MS analysis (MS/MS) of the precursor ion to localize
the site of modifi cation. With the aid of search engines and data-
bases the recorded MS/MS spectra are converted to peptide
sequences, with possible PTMs, that also are matched to protein
identity. We have used mass spectrometry and shown that there are
at least 29 different sites of phosphorylation in 11 different pro-
teins in the adenovirus type 2 (Ad2) particle [ 16 ]. In agreement
with previous studies [ 17 - 21 ] we showed that the pIIIa protein is
the most extensively phosphorylated protein and for this protein
12 sites of phosphorylation were identifi ed [ 16 ]. Below we present
the key steps for proteome analysis of adenovirus by LC-MS sum-
marized in a protocol.
2
Materials
For quality of chemicals, see Note 1 .
1. HeLa spinner cells (ATCC/LCG).
2. Medium for cell growth: Minimal Essential Medium (MEM),
spinner modifi cation supplemented with 10 % fetal bovine
serum, 1 % Penicillin-Streptomycin (PEST), 1 % L -glutamine.
Store prepared medium at 4 °C (up to 2 months) and warm to
37 °C in water bath upon use. To obtain a virus preparation,
around 5 L of medium is needed.
3. Medium for infection: MEM, spinner modifi cation supple-
mented with 1 % PEST and 1 % L -glutamine. Store prepared
medium at 4 °C (up to 2 months) and warm to 37 °C in water
bath upon use.
4. Adenovirus (ATCC/LCG).
2.1 Adenovirus
Production
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