Biology Reference
In-Depth Information
source (ProxeonBiosystems) for viral proteome LC-MS analysis.
For users of other instruments,
Note 8
describes the main param-
eters of a mass spectrometer that need to be taken into account.
1. Set a 40-min linear gradient from 2 % to 45 % of buffer B at
200 nL/min on Agilent 1100 Nanofl ow system equipped with
homemade 15-cm fused silica emitter packed with Reprosil-
Pur C18-AQ 3
m resin (Dr. Maisch GmbH), and coupled
on-line to the mass spectrometer.
2. Create a program for operating the mass spectrometer in posi-
tive ion mode that automatically switches between a high reso-
lution (resolving power 50,000) survey mass spectrum in the
FTMS cell and consecutive low resolution CID (collisionally
induced dissociation) spectra of the 5 most abundant ions in
the ion trap. Set normalized collision energy to 30 %. Set
dynamic exclusion for 30 s for 5 peptides.
3. Dilute samples in water-TFA (1:0.005 v/v), load them into
LC-MS system and acquire the data.
4. Convert the acquired data (.RAW-fi les) to Mascot generic for-
mat (MGF) fi les using appropriate software. Submit acquired
MGF fi les to Mascot search engine (version 2.1.3, Matrix
Science) by searching the Uniprot-Swissprot database with
following settings:
(a) Set “Viruses” taxonomy.
(b) Set the enzyme specifi city (i.e., trypsin) and number of
allowed missed cleavages (we used two in our
experiments).
(c) Set mass deviation for precursor and fragment ions. For
our runs with LTQ-FTICR they were set to 0.02 Da and
0.7 Da correspondingly.
(d) Set “Carbamidomethylation” as a fi xed modifi cation.
(e) Set variable modifi cations to deamidation (N, Q) and
oxidation (M). Then set up to for extra modifi cations if
PTM identifi cation is required, for instance, extra phos-
phorylation (S,T), phosphorylation (Y), methylation
(C-terminus), and methylation (D, E) can be set (
see
also
Note 8
).
(f) Choose instrument setting (i.e., “ESI-FTICR”)
5. The search results will present the identifi ed proteins, peptides,
and suggested PTMs. The identifi cations have a certain proba-
bility specifi ed in the result fi le, and newly discovered PTMs
must be validated. We recommend manual interpretation of
MS/MS spectra [
34
] for verifi cation. Examples of verifi ed PTM
spectra are presented in Fig.
2
. As a reference database contain-
ing already discovered viral PTMs we recommend online
virPTM database [
35
] (
http://virptm.hms.harvard.edu/
)
.
μ
