Biology Reference
In-Depth Information
worked best for Adenovirus type 2. The very basic proteins are
diffi cult to identify even when using basic Immobiline Dry
strips pH 7-11. Therefore we recommend pH 4-7 or pH 3-11
strips. The 7 cm strips have suffi cient capacity for the Adenovirus
proteome since they only consist of a dozen of proteins with a
maximum of ten isoforms.
7. For specifi c enrichment of tyrosine phosphorylation, a larger
amount of virus, i.e., around 1 mg is recommended to be incu-
bated with anti-tyrosine phosphorylated antibody as described
by Bergström Lind et al. [
23
].
8. As mentioned before, MS instrumentation is the most infl exi-
ble parameter due to the high cost of the equipment. We
describe the approach we have used with our LTQ-FTICR
mass spectrometer, but one can use alternative instruments.
Here we list the most important settings and parameters that
need to be taken into account during LC-MS experiments.
(a) Resolution. This is a very important parameter for obtain-
ing reliable protein and, especially, PTM identifi cations.
With a higher resolution a higher precision of mass deter-
mination is possible. Better confi dence is then achieved
when matching the theoretical mass of the proposed
sequence to the experimental value. Orbitrap and high-
resolution Q-TOF instruments can be alternatives to
LTQ-FTICR. The most crucial is to record the survey
scan with high resolution, while fragmentation spectra can
be less resolved [
23
].
(b) Acquisition rate. This parameter is very important for
highly complex samples, while viruses are normally com-
posed of a limited number of proteins, e.g., Ad2 consist of
about a dozen. Therefore it is not critical to detect a maxi-
mum number of peptides. More important is to record
high quality spectra for a limited set. Ion traps or conven-
tional Q-TOFs are very fast mass spectrometers with low
resolution and therefore less useful. If they are the only
available choice, the user must specify as high resolution as
possible sacrifi cing scanning speed.
(c) Fragmentation. Overall, CID provides very good results
for proteome profi ling and is recommended for most mass
spectrometers. Orbitrap instruments offer a special HCD
(Higher-energy collisional dissociation) fragmentation
possibility, which is more effective than CID. HCD was
also shown to be the best fragmentation technique for
identifi cation of phosphorylated peptides. Available on
many instruments, ETD (electron transfer dissociation) or
ECD (electron capture dissociation) fragmentation is very
useful for localization of labile PTMs, like glycosylations
