Urinary Proteome Profiling Using 2D-DIGE and LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 295

(16) Stock ammonium bicarbonate solution: 10 mM ammo-
nium bicarbonate in 100 ml of Milli-Q 18 water, store

at 4 ◦ C.
(17) DTT solution: 10 mM DTT in 10 mM ammonium bicar-
bonate stock solution, store at 4 ◦ C.
(18) IAM solution: 10 mM IAM in 10 mM ammonium bicar-
bonate, store at 4 ◦ C.
(19) Stock solution of modified porcine trypsin: 40 μ l trypsin

re-suspension buffer to 20 g of trypsin provided in vial.

This is of low pH and allows suspension to be stored with-
out autolysis occurring. Further dilution and activation
can be achieved with the addition of 10 mM ammonium
bicarbonate solution. The diluted trypsin can be stored at
−

μ

20 ◦ C.
(20) HPLC Buffer A: 0.1% formic acid in 100% LC-MS grade
H 2 0.
(21) HPLC Buffer B: 5% LC-MS grade H 2 0, 0.1 % formic acid,
made up to 100% with LC-MS grade acetonitrile.

3. Methods

The standardisation of collection and processing procedures of
samples is a vital first step in expression profiling ( 20 ) . During
collection, EDTA-free protease inhibitors should be used to pre-
vent sample degradation, as they do not interfere with Cy dye
labelling. Collection and processing methods are sample driven
and a variety of cell types (whole tissue, cell lines) and body fluids
(saliva, cerebrospinal fluid, plasma and urine) have already been
subject to proteomic analysis ( 21 - 26 ) ; these references can be
used to obtain optimal processing conditions of particular sample
types.

3.1.Sample
Collection
andPreparation

2D-DIGE experiments require careful design to ensure that sta-
tistically meaningful data can be obtained. The use of an inter-
nal standard permits robust analysis and more precise quanti-
tation. Labelling a pooled internal standard (sample composed
of equal aliquots of each sample used in the experiment) with
Cy2 dye, while Cy3 and Cy5 are used to label test and reference
samples is the most popular experimental design. Statistical anal-
ysis can be further improved by increasing the number of gels
per experiment, performing both technical and biological repli-
cates (minimum of three per sample group). Differential expres-
sion is then calculated as the average fold change (the average

3.2.Experimental
Design