Biology Reference
In-Depth Information
width is 2 amu and the exclusion mass width is set to 10 ppm.
Monoisotopic precursor selection is allowed and singly charged
species are excluded. The minimum intensity threshold for
MS/MS is 1,000 counts for the LIT and 8,000 counts for the
Orbitrap. Target values for automatic gain control were set as
follows: 5 × 10
5
for the Orbitrap survey scans, 1 × 10
5
for the
Orbitrap MS/MS scans, 3 × 10
4
for LIT survey scans, and
1 × 10
4
for the LIT MS/MS scans.
To analyze the data, a number of different software platforms
may be used. The most extended used is for ProteinPilot software
(Life Technologies) which is used to analyze data from instruments
such as Q-TOF, all of them from Life Technologies company. The
use of the Spectrum Mill Proteomics Workbench (SMPW) (Agilent
Technologies) tool with a licensed module allowing the extraction
of data or raw fi les from Thermo instruments is described here
(
see
Note 12
).
3.4 Protein
Identifi cation
1. The search is jointly performed for all data fi les and SCX frac-
tions which results in a summed and averaged data in the fi nal
results output.
2. Prior to launching the search, the MS/MS spectra dataset is
processed with the extraction tool of SMPW to merge the
MS/MS spectra with the same precursor (
Δ
m
/
z
< 1.4 Da and
t
< 15 s) and fragmentation type.
3. A two-step search is performed. The reduced dataset is search
first against a protein amino acid database including the
following settings: trypsin, up to two missed cleavages, beta-
methylthiolation of Cys, incomplete iTRAQ labeling (from no
label to only Lys to only N-term to a complete label), Met-
oxidation, Asn- and Gln-deamidation, and a mass tolerance for
the LIT CID spectra of 2.5 Da for precursor and 0.7 Da for
product ions, mass tolerance for the Orbitrap HCD spectra of
25 ppm for precursor and 50 ppm for product ions, and
sequence tag length fi lter
chromatographic
Δ
4. In a second search, the previous
protein hits are researched using the following parameters:
phosphorylation at Ser, Thr, and Tyr, and N-term Glu-
pyroglutamic acid (
see
Notes 13
and
14
).
4. Peptide hits are validated in SMPW in the protein mode and
then in the peptide mode according to the manufacturer's rec-
ommended score settings. Briefl y, identities interpreted for
individual spectra are automatically designated as valid by
applying the following scoring threshold criteria to all spectra,
protein details mode: protein score >20, peptide details mode
(score, scored percent intensity, delta rank1 − rank2) in the fol-
lowing order: peptide charge +2 (>6, >60 %, >2), peptide
charge +1 (>6, >70 %, >2), peptide charge +3 (>8, >70 %, >2),
≥
