- 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
Proteins
400 μg
Proteins
400 μg
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)