Biology Reference
In-Depth Information
4. Elution buffer B: 5 % pyrrolidine and 95 % Milli-Q water.
5. TiO
2
material (GL Sciences Inc., Tokyo, Japan).
2.4 LC-MS Analysis
1. Mobile phase buffer A: 0.5 % acetic acid in Milli-Q water.
2. Mobile phase buffer B: 0.5 % acetic acid, 89.5 % acetonitrile,
and 10 % Milli-Q water.
3. Sample loading buffer: 0.5 % TFA in Milli-Q water.
4. Mass spectrometer, e.g., 7-tesla LTQ-FT Ultra tandem mass
spectrometer (Thermo Fischer Scientifi c) equipped with a
nano electrospray ion source (Proxeon Biosystems).
5. LC system, e.g., Agilent 1100 Nanofl ow system.
6. Packing material for LC column: Reprosil-Pur C18-AQ, 3
μ
m
resin (Dr. Maisch GmbH).
7. Fused silica emitter.
3
Methods
In order to perform proteome analysis of pure virions it is impor-
tant to separate out host cell proteins that otherwise would inter-
fere and in the worst case would dominate in the detection. CsCl
gradient ultracentrifugation is an effi cient method for adenovirus
purifi cation as described by Pettersson and Sambrook [
22
]. Virus
is banded in a step gradient followed by equilibrium centrifuga-
tion. Purity of virus samples can be checked in a SDS-PAGE and
Coomassie staining. Before MS detection the proteins in the viri-
ons need to be extracted and digested into peptides. Ideally, viral
proteins should pass a minimal number of preparation steps before
MS detection, since each step causes losses of material. On the one
hand, in-solution digestion followed by a desalting step is the most
straight-forward procedure. On the other hand LC-MS analysis is
based on separation of digested peptides on a reversed-phase col-
umn and recording of mass spectra of eluted peptides. Eluting pep-
tides are competing with each other in order to be properly
detected by mass spectrometry. Therefore preliminary fraction-
ation is often needed and then it is favorable to initially separate the
proteins in a sample by 1D or 2D SDS-PAGE and then analyze
selected protein bands or spots with MS. Aspects on expected
detectability of Adenovirus structural proteins are discussed in
Note 2
and Table
1
.
Similarly, for PTM analysis, enrichment is highly valuable in
order to decrease the competition between modifi ed and non-
modifi ed peptides. Most of posttranslationally modifi ed peptides
(or original proteins) can be enriched by immunoaffi nity methods
using PTM specifi c antibodies attached to magnetic beads [
23
],
resin beads or columns [
24
]. This approach has been used for
