Biology Reference
In-Depth Information
Before entering the analytical column for LC-MS analysis,
samples need to be free from salt, large polypeptides and deter-
gents. Large polypeptides can be easily removed by fi ltering the
sample through a membrane cut-off fi lter. Detergents are com-
monly used for effi cient protein extraction, but even small amounts
of detergent in the sample disturb the LC-MS analysis (
see
Note 3
about detergent removal). Instead of detergents, chaotropes can
be used for protein extraction. The classical example is concen-
trated urea (8-10 molar), which can be removed from the sample
together with salts. Desalting is then generally performed using the
C
18
-packed tips that trap the polypeptides while salts (and chao-
tropes) pass through. Trapped peptides are eluted by 50 % acetoni-
trile and can be further analyzed. This approach was used by us and
is described below in detail. Alternatively, online desalting can be
employed using trapping units in LC-MS system [
32
]. It is impor-
tant to note, that these considerations are for in-solution digestion
protocols, while in-gel based approaches do not require any addi-
tional cleanup. It is recommended to check the purity of samples
before LC-MS analysis as described in
Note 4
.
Further, the choice of mass spectrometer is crucial for the
result of analysis. Nevertheless, this is the most infl exible parameter
due to the very high cost of this instrumentation. In this protocol
we describe how sample preparation is performed to be compatible
with the electrospray ionization used in LC-MS as well as detailed
description of settings used for viral proteome analysis using the
linear ion trap Fourier transform (LTQ-FTICR) and instrument.
The strategies for proteome virus analysis of Adenovirus are sum-
marized in Fig.
1
.
1. Grow HeLa cells in MEM medium with spinner modifi cation
in a 37 °C incubator with 5 % CO
2
. Start the cells in a small
fl ask and transfer then all cells to a spinner fl ask. Use a mag-
netic stirrer to disperse the cells. Expand the culture by daily
counting the cells and addition of more medium. Keep the cell
density not higher than 1 × 10
6
cells/mL before dilution and
not below 0.3 × 10
6
cells/mL after dilution.
2. Infect the cells with adenovirus when the total cell culture vol-
ume is ~4 L. Spin down cells in 225 mL graduated conical
tubes. Spin at ~600 ×
g
, 4 °C for 15 min. Remove the medium
and save it for further use. Resuspend the infected cells in
400 mL medium for infection. Add adenovirus at a 5-10 FFU
(fl uorescent focus forming units) per cell. Incubate for 2-3 h
at 37 °C with stirring. Then, add back 3,600 mL of saved
medium for cell growth to a fi nal volume of 4 L. Allow the
virus to grow at 37 °C for 72 h before harvest.
3. Spin down infected cells in four 225 mL graduated conical
tubes at ~600 ×
g
, 4 °C for 5 min. Four cell pellets are obtained.
3.1 Virus Production
