66
buffer A with a fl ow rate of 0.1 mL/min with a tee splitter to
reduce the fl ow rate to 300–400 nL/min.- Resuspend peptide samples from in-gel digestion in 10 μL of
buffer A. - Pressure-load sample onto column equilibrated in step 4.
- Connect column to instrument and ensure that it is actively
fl owing. - Create a method in Xcalibur with the following parameters:
Mass spectrometer: scanning in data-dependent scanning
mode with one full MS scan (400–1,800 m / z ) followed by
seven MS^2 scans of the n th most abundant ion.
HPLC: Create a 125 min gradient with the following
buffer concentrations:
0–10 min: 100 % A
10–20 min: 100–85 % A
20–80 min: 85–55 % A
80–90 min: 55–0 % A
100–105 min: 0–100 % A
105–125 min: 100 % A- Run control samples in chronological order.
- Prepare new column for experimental samples and run in
chronological order.
The instructions below are intended to be used on the Microsoft
Windows operating system. However, DTASelect and Perl are
both platform independent and can be run successfully on most
operating systems (e.g., OS X and Linux) although some details
will differ slightly.- Raw mass spectrometry data must be searched with SEQUEST
and fi ltered using DTASelect. Detailed instructions for using
these programs are beyond the scope of this protocol, but
details can be found in the literature [ 3 ]. A separate folder
should be created for the DTASelect output fi les for each gel
band and for each sample (Fig. 2 ). - DTASelect should be run with the following parameters: “y -1
–Stn RKD –Stc RKD –p 1.” These parameters specify one
peptide per protein (-p 1) and fully tryptic peptides or half
tryptic ending on an arginine, lysine, or aspartic acid (indica-
tive of caspase cleavage) (y -1 –Stn RKD –Stc RKD). These
parameters are not required but are helpful for the identifi ca-
tion of candidate sites of caspase cleavage. - Download the PROTOMAP software from http://www.
scripps.edu/chemphys/cravatt/protomap
3.4 Data Analysis
and Generation of
Peptographs
Melissa M. Dix et al.http://www.ebook3000.com