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.
- 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)