Application of Saturation Labeling in Lung Cancer Proteomics - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 265

3.8. Micropreparation
of Signifi cantly
Regulated Protein
Spots

1. After the differential analysis, protein spots are identifi ed using

mass spectrometry (MALDI MS, ESI MS) subsequent to
tryptic in-gel digestion. For this analysis, 400 g of the internal

standard (reference proteome) must be labeled and applied to
2DE. A preparative label kit with 400 nmol Cy3 is available.

μ

2. For preparative gels label 400 g of tissue lysate with 320 nmol

of Cy3 (160 nmol TCEP) (see Note 11).

μ

3. Directly after gel scanning, isolate the protein spots of interest

manually (see Note 12). Assign the positions of the spots using
a print-out of the gel placed underneath the glass plate.

4. Put the isolated protein spots into sample glass containers and

store them at −80°C.

5. For protein identifi cation, different MS techniques can be

applied subsequent to enzymatic in-gel digestion. Matrix-
assisted laser desorption/ionization MS (MALDI MS) allows
fast and sensitive identifi cation performing peptide mass fi nger-
printing (PMF) ( 8 ). In cases where more sequence information

is necessary (e.g., post-translational modifi cation), liquid chro-
matography-coupled electrospray ionization (LC-ESI) with its
high performance for peptide fragment/sequence analysis using
tandem MS (MS/MS) is preferable ( 4, 9 ) .

3.9. Validation
of Differentially
Regulated Proteins

As 2D gels separate protein isoforms, protein spots found to be
differentially expressed between the groups can represent protein
isoforms. To validate the identifi ed differentially regulated proteins
(also in respect of false positives), a validation with alternative
methods is mandatory (see Fig. 7 ). Typical methods are western
blotting, immunohistochemistry (providing also information about
the spatial distribution in tumor tissues), and ELISA as antibody-
based methods or selected reaction motoring (SRM) as MS-based
method. Sample material not used in the initial analysis should be
used for these experiments.

Fig. 7. Validation of a differential regulated protein using western blot analysis. Western blot analysis confi rms the higher
abundance of Annexin A2 in tumor cells of both grade 2 (G2) and grade 3 (G3) tumors compared to bronchial epithelium
cells (BE). Some additional bands can also be detected which might represent splice variants or proteolytic products of
Annexin A2.