Selected Reaction Monitoring Applied to Quantitative Proteomics - LC-MS/MS in Proteomics: Methods and Applications - page 157

3. Methods

This section describes typical instrument settings for both the
HPLC system and the mass spectrometer.
1) Analyses are typically carried out in the nano-flow mode
using 75-150

3.1. Instrument
Settings

m ID columns, at a flow rates rang-
ing from 0.3 to 1.0

μ

L/min. The nano-HPLC sys-
tem must be capable of delivering small flow rates reli-
ably, have minimal dead volumes, and be equipped with
an auto-sampler with a reproducible microliter injection
capability.
2) HPLC separations are performed on a reversed-phase col-
umn (C18 column; 3-5

μ

m particles) using a gradient,
for instance. 5% B to 45% B in 40 min. The gradient is
generated by mixing two mobile phases: water containing
0.1% formic acid (A) and acetonitrile containing 0.1% formic
acid (B).
3) LC-MS measurements are performed on a calibrated TSQ
Vantage triple quadrupole mass spectrometer ( see Note 2 )
operated in SRM mode using time-based SRM (t-SRM, see
below). For ionization, spray voltage and capillary tempera-
ture were set at 1,800 V and 200 ◦ C, respectively. The selec-
tivity for both Q1 and Q3 were set at 0.7 Da (FWHM).
Argon was used as collision gas, and the nominal gas pres-
sure was set at 1.2 mTorr. A cycle time of 2 s and window
size of 4-min are used for t-SRM experiments.
4) The instrument methods used to perform multiplexed SRM
experiments have to be carefully designed prior to actual
experiment to ensure success ( see Section 3.4 ).

μ

3.2.Sample
Preparation

As mentioned above, the sample preparation is a critical part of a
quantitative experiment. Protocols ensuring a high recovery and
the reduction of sample complexity are a prerequisite to achieve
low limits of detection and quantification.
1) The biological samples to be analyzed (e.g., cell lysates, bod-
ily fluids) are processed using established proteomic pro-
cedures to isolate the protein fraction. After a (optional)
crude fractionation, the protein samples are reduced, alky-
lated, and digested with trypsin prior to LC-MS analysis. It is
advisable to perform a sample cleanup using reversed-phase
cartridges to improve the analysis consistency by removal of
contaminants.
2) The digest is directly used for the LC-MS quantitative analy-
sis. In a typical experiment, up to a 1 μ g total protein digest

can be injected.