Biology Reference
In-Depth Information
were allowed. The searches can be done using public databases
such as NCBInr or SwissProt (
see
Note 33
). A protein is
considered identifi ed when the minimal following criteria are
fulfi lled: a score of 65, four matched peptides, and sequence
coverage of 25 %.
2.
MS/MS
. Protein extracted from extracellular media can be ana-
lyzed directly by mass spectrometry without a previous protein
gel-based separation. In this case, a protein in-liquid digestion
is performed [
41
]. Alternatively, in order to decrease sample
complexity a peptide separation by strong cation/anion
exchange chromatography (SCX/SAX) can be performed pre-
viously to MS/MS analysis which is known as MuDPIT
approach [
42
].
The tryptic fragments are analyzed by LC-MS/MS using
an Agilent 1100 series nano-HPLC system lined on an XCT
plus ion trap mass spectrometer (Agilent technologies)
equipped with a nano-ESI source. Sample concentration and
desalting are performed on a Zorbax 300SB-C18 trap column
at 3
L/min of RPB-A while peptide separation was achieved
on a Zorbax 300SB-C18 analytical column using a 30-min linear
gradient of 5-35 % RPB-B at a constant fl ow rate of 0.3
μ
L/min.
MS and MS/MS spectra are acquired in the standard enhanced
mode (26,000
m/z
per second) and the ultra-scan mode
(8,100
m/z
per second), respectively. Mass spectrometer set-
tings for MS/MS analyses included an ionization potential of
1.8 kV and an ICC smart target (number of ions in the trap
before scan out) of 400,000 or 150 ms of accumulation. MS/MS
analyses are performed using automated switching with a pref-
erence for doubly charged ions and a threshold of 10
5
counts
and 1.3 V fragmentation amplitude. Each MS/MS spectral
dataset (ca. 1,200 spectra/run) is processed to determine
monoisotopic masses and charge states, to merge MS/MS
spectra with the same precursor (
μ
Δ
m/z
< 1.4 Da and chro-
matographic
t
< 15 s) and to select high-quality spectra with
the extraction tool of the Spectrum Mill Proteomics Workbench
(SMPW). A two-step search is performed. The reduced dataset
is searched against a suitable protein database, e.g., NCBInr, in
the identity mode with the MS/MS Search tool of the SMPW
using the following parameters: trypsin, up to two missed
cleavages, fi xed modifi cation
S
-carbamidomethyl, variable
modifi cations Met-oxidation, Asn- and Gln-deamidation, and
a mass tolerance of 2.5 Da for the precursor and 0.7 Da for
product ions. A sequence tag length minimum of three and
four minimum peaks detected is selected and calculate reversed
database scores. Peptide hits are validated fi rst in the peptide
mode and then in the protein mode according to the manu-
facturer's recommended score settings. Validated fi les are
summarized in the protein mode to assemble peptides into
Δ
