A Practical Guide to Cancer Systems Biology

26 A Practical Guide to Cancer Systems Biology

Combined

Sample

A

Sample

C

Sample

D

Isobaric tag

145 Da

Reporter group

114-117 Da

Balance group

31-28 Da

Peptide

reactive group

iTRAQ structure

Sample

B

Balance

LC-MS/MS

iTRAQ reporter ions

Reporter Peptide

PepƟde

fragment ions

QuanƟtaƟon IdenƟficaƟon

Intensity

m/z

114

115

116

117

Figure 1. Structure and workflow of iTRAQ.

protocol of iTRAQ method prior to beginning the experiment. The possible
interference of those substances is reaction with iTRAQ reagent, inactivation
of trypsin digestion and interference with protein reduction.

2. Protocol of sample preparation for iTRAQ
   quantitative proteomics

Materials

Reagents

• Tris-base


• 6M HCl


• 10% Sodium dodecyl sulfate (SDS)


• Glycerol


• Protease inhibitor


• Liquid nitrogen


• Pierce BCA Protein Assay Kit (Pierce cat. no. 23225)


• Triethylammonium bicarbonate buffer (TEAB) (Sigma cat. no. T7408)


• Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (Sigma cat. no.
  C4706)


• S-Methyl methanethiosulfonate (MMTS) (Sigma cat. no. 208795)