Biology Reference
In-Depth Information
3. A 1.8 kV electrospray voltage was applied via a gold-electrode
liquid junction upstream of the C18 column.
4. Samples were injected onto the C18 column using a Surveyor
autosampler (Thermo, San Jose, CA).
5. Each sample was loaded onto the C18 column followed by an
initial wash step with buffer A (5 % (v/v) ACN, 0.1 % (v/v)
formic acid) for 10 min at 1
L/min.
6. Peptides were subsequently eluted from the C18 column with
0-50 % buffer B (95 % (v/v) ACN, 0.1 % (v/v) formic acid)
over a 30-min linear gradient at 500 nL/min followed by
50-95 % buffer B over 5 min at 500 nL/min and 5 min was
with 95 %B prior to column re-equilibration (
see
Note 11
).
7. The column eluate was directed into the nanospray ionization
source of the mass spectrometer.
8. Spectra were scanned over the range 400-1,500 amu.
Automated peak recognition, dynamic exclusion (90 s), and
tandem MS of the top six most intense precursor ions at 40 %
normalization collision energy were performed using Xcalibur
software (Thermo).
μ
1. The set of 16 data fi les from one experiment were acquired in
the proprietary .RAW format. These were fi rst converted to
.mzxmL format using the freeware Readw.exe program (
see
Note 12
).
2. The set of 16 .mzxmL data fi les from a given sample were then
placed into one directory and peptide-to-spectrum matching
was performed using the XTandem algorithm. We use the
Global Proteome Machine (GPM) software [
26
], which is freely
available and runs the XTandem Tornado version. Searching the
set of .mzxmL fi les stored in a directory enables the user to
choose for a single combined summary output fi le to be created
in addition to all 16 individual result fi les (
see
Note 13
).
3.4 Protein and
Peptide Identifi cation
3. The combined protein and peptide identifi cation output fi le
for all 16 gel slices was then exported to an Excel spreadsheet.
This contains six columns of data, with the headers Identifi er,
log(
I
), rI, log(
e
), pI, and Mr (kDa). Two other headers are
included, Description and Annotated Domains, but will only
have entries automatically provided if this information is avail-
able within the searched database. The Excel fi le is then
exported to comma-separated value format, which is then
compatible with input into the Scrappy software described
elsewhere [
27
].
An example of the output produced by this approach is pro-
vided in Table
1
. Included in this abridged table are the fi rst 20
proteins identifi ed from a rice leaf extract, sorted by the confi dence
