A Practical Guide to Cancer Systems Biology

Quantitative Proteome 27

Sample A Sample B Sample C Sample D

Protein extraction

Large-scale experiment Small-scale experiment

iTRAQ labeling iTRAQ labeling

ZipTip desalting

LC-MS/MS analysis

SCX chromatography

LC-MS/MS analysis

Reduction, alkylation, gel-assisted digestion and gel extraction

ZipTip desalting

Tissue A Tissue B Tissue C Tissue D

Proteins 400 μg

Peptides Peptides Peptides Peptides

A 150 μg

B 150 μg

C 150 μg

D 150 μg

A 5 μg

B 5 μg

C 5 μg

D 5 μg

114 150 μg

115 150 μg

116 150 μg

117 150 μg

114 5 μg

115 5 μg

116 5 μg

117 5 μg

600 μg Combined labeled peptides

20 μg Combined labeled peptides

30-40 Fractions of labeled 0.5 μg Labeled peptides peptides

0.5 μg Labeled peptides/each fraction

Figure 2. Flowchart of sample preparation for iTRAQ quantitative proteomics. The
detailed description and protocols are shown below.

Acrylamide/bis-acrylamide (40%, v/v, 37.5:1)

Ammonium persulfate (APS)

N,N,N′,N′-Tetramethylethylenediamine (TEMED)

Acetonitrile (ACN)

Sequencing grade modified trypsin (Promega, cat. no. V5111)

Trifluoroacetic acid (TFA)

iTRAQ Reagents Multiplex Kit (SCIEX, cat. no. 4352135)