Biology Reference
In-Depth Information
wash buffer, and elute peptides in two steps by addition of
20
L elution buffer.
10. The peptides recovered from each SCX fraction are dried under
vacuum for further analyses by MS/MS.
μ
3.3 Sample Analysis
by MS/MS
Currently, different mass spectrometers and several types of pep-
tide fragmentation methods may be used to analyze iTRAQ-
labeled samples. Q-TOF mass spectrometers are the most widely
used instruments in which CID is the common fragmentation
used. As a valid alternative, the recently introduced LTQ Orbitrap
hybrid mass spectrometer able to perform higher energy collision-
induced dissociation (HCD) also allows iTRAQ analysis. Here, we
describe an iTRAQ analysis method using a parallel detection
method combining CID peptide fragmentation in a linear ion trap
(LIT) analyzer and HCD fragmentation in the HCD cell both in
the LTQ Orbitrap analyzer. Compared to other methods reported,
this method leads to higher protein identifi cation yields together
with improved quality of spectra and quantifi cation.
1. The protocol described is for use with an Agilent 1200 HPLC
system (chromatographic conditions and columns may vary
using alternative instrumentation). Fractions are resuspended
in 10
L injections are programmed to
ensure reproducible sample injection in this autosampler.
Tryptic peptides were pre-concentrated using Zorbax 300SB-
C18 cartridges (5 × 0.3 mm, 5
μ
L of 0.1 % FA and 8
μ
L/min
for 10 min in RPB-A followed by elution in a Zorbax 300SB
RP C
18
column (75
μ
m particle size) at 15
μ
m particle size)
(Agilent Technologies) using a 60-min linear gradient from 5
to 40 % RPB-B fl owing at 200 nL/min.
2. The eluent is sprayed directly into an LTQ Orbitrap mass spec-
trometer (Thermo Fisher Scientifi c) equipped with a Proxeon
electrospray ion source. The electrospray voltage is set to
1,800 V and the capillary voltage to 50 V at 190 °C.
3. The LTQ Orbitrap is operated in the parallel mode, allowing
for the accurate measurement of the precursor survey scan
(400-2,000
m
/
z
) in the Orbitrap selection, a 60,000 full-
width at half-maximum (FWHM) resolution at
m
/
z
400 con-
current with the acquisition of three CID data-dependent
MS/MS scans in the LIT, followed by three data-dependent
HCD MS/MS scans (100-2,000
m
/
z
) with 7,500 FWHM
resolution at
m
/
z
400. The LTQ Orbitrap XL mass spectrom-
eter includes an octopole acting as collision cell able to per-
form an alternative peptide fragmentation termed HCD [
27
].
The normalized collision energies used are 40 % for HCD and
35 % for CID. The maximum injection times for MS and MS/
MS are set to 50 and 500 ms, respectively. The precursor isolation
μ
m i.d. × 150 mm and 3.5
μ
