Application of Saturation Labeling in Lung Cancer Proteomics - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 259

Fig. 2. Determination of the required number of cells. Protein from different numbers of cells (( a ): 1,000 cells, ( b ): 4,000
cells, ( c ): 5,000) was labeled by saturation labeling and subsequently separated by 2DE. Using image analysis software
(DeCyder 6.5, GE Healthcare), 2,638 ( a ), 3,271 ( b ), and 3,725 ( c ) protein spots were detected. Therefore, 5,000 cells were
collected from each sample for subsequent experiments.

experiment, prepare six aliquots containing the optimal number
of cells (see Subheading 3.2 ) from a pool of 30,000 cells.

2. Label three of them with 2, 4, and 8 nmol Cy3 and another

three with 2, 4, and 8 nmol Cy5 (reduction of disulfi de bounds
is performed with 1, 2, and 4 nmol TCEP, respectively).

3. Mix samples with equal dye amounts and perform 2DE.

4. After gel scanning, analyze the overlay images in order to fi nd

the optimal ratio of protein and dye showing an accurate overlay
of Cy3 and Cy5 images in ImageQuant™ TL. As shown in Fig. 3 ,
applying too much dye results in horizontal shifts that could
occur due to nonspecifi c labeling of the amine groups on lysine
residues. This will result in an increased mass as well as a reduc-
tion of the protein's charge by 1. An insuffi cient amount of dye
causes vertical streaks due to inadequate protein labeling.

3.4. Reference
Proteome for Internal
Standardization and
Protein Identifi cation

1. 2DE analysis of 5,000 microdissected cells (~4 g) does not

deliver suffi cient sample material for protein identifi cation
using mass spectrometry. Therefore, a reference proteome
from a closely related source has to be defi ned allowing protein
identifi cation by protein spot matching. Additionally, this
reference proteome may be considered as an internal standard
(labeled with Cy3) which reduces consumption of microdis-
sected samples.

μ

2. A suitable reference proteome has to be identifi ed which not

only demonstrates a high correlation to microdissected cells
but also provides an adequate protein amount for subsequent