Quantification of Proteins by iTRAQ - LC-MS/MS in Proteomics: Methods and Applications - page 208

Lorganic
in the labelling reaction. Repeat for each label/sample.
13. Vortex to mix, pulse spin, and incubate on the bench for at
least 1 h ( see Note 2 ).
14. Samples may slowly form precipitates as addition of iTRAQ
reagent may result in decreased solubility. This is not a
problem, as this precipitate is solubilised before samples
are pooled prior to SCX chromatography. If precipitation
forms immediately on addition of the reagent, this could
indicate poor digestion and so samples should be assessed
by 1D SDS-PAGE for the presence of undigested protein
prior to further analysis.
15. Place samples in SpeedVac for 10-15 min to reduce the
volume to around 20-30

add this to the sample, resulting in a total of 70 μ

μ L, removing the ethanol.

20 ◦ C at this stage if required.
OPTIONAL - Remove 10% of each labelling reaction, clean
up on SCX cartridge/column as a single fraction, and analyse by
mass spectrometry to check labelling efficiency ( see Note 3 ).

16. Samples can be stored at −

1. To pool samples, add 100 L of SCX Buffer A (10 mM

KH 2 PO 4 , 20% ACN, pH 2.7) to each sample. This should
help solubilise any precipitate formed during the labelling
reaction (if precipitate is still present, add a further 50

μ 3.2.SampleAnalysis

by2D-Liquid
Chromatographyand
TandemMass
Spectrometry

μ

L).

Pool labelled peptides into a clean tube.
2. Wash out sample tubes with a further 100

L SCX Buffer
A and add to pooled material, ensuring all material is trans-
ferred.
3. Check pH of pooled peptide sample using pH paper.
Ensure pH<3.0 by adding small amounts of HCl, if neces-
sary. Spin in a microfuge at full speed for 5 min to remove
particulates or and remaining precipitate.
4. Transfer sample to a glass, round-bottomed sample vial and
make volume up to 1.6 mL with SCX Buffer A (SCX-A) for
loading onto SCX column.
5. Separate peptide sample by strong cation exchange chro-
matography. Gradient details and flow rate are sample and
LC System specific ( see Note 4 ). We use an ICS-3000 SP
pump (Dionex) coupled with a FAMOS autosampler and
UV detector. 1.6 mL sample is injected into a 2 mL loop,
followed by 200

μ

L SCX-A. Sample is then loaded from
the loop onto the column and washed for at least 30 min
with 100% SCX-A at a flow rate of 1 mL/min. UV signal
is monitored over this time and washing is not deemed to
be complete until the UV trace is flat.

μ