A Practical Guide to Cancer Systems Biology

44 A Practical Guide to Cancer Systems Biology

The reagent amount used in the following steps are designed for 100μL
samples:

III. Add 4μLofCH 2 O (light) or^13 CD 2 O (heavy) to different groups of

samples.

IV. Add 4μLofNaBH 3 CN to each group of sample as a catalyst for the

labeling reaction.

V. Agitate for 1 minute to completely mix the reagents and samples.

VI. Incubation by rotating at room temperature for 60 minutes.

VII. Buffer preparation:

i. 1% ammonia

ii. 10% formic acid

VIII. Spin down and put the samples on ice.
IX. Add 16μL of 1% ammonium to consume the remaining labeling
reagents.
X. Agitatesamplesfor1minuteandthenspindownthesamples.
XI. Add 20μL of 10% formic acid to further stop the labeling reaction.
XII. Agitatesamplesfor1minuteandthenspindownthesamples.
XIII. Transfer the differentially labeled samples to a new 1.5 mL tube.
XIV. Agitate for 1 minute to completely mix the samples.
XV. Desalting using EmporeTMSDB-XC 6 mL cartridge.

5. Preparation and preservation of TiO 2

Materials:

• Titansphere TiO 2 (#5020-75010, GL science)

Procedure:

I. The newly purchased titansphere TiO 2 should be heat-treated at 500◦C
for 4 hours for complete dehydration and decomposition of polymers.
The color of TiO 2 would change from pale yellow to white after heating
treatment.
II. Store TiO 2 in a desiccator.

6. Preparation of StageTips and tubes^11

Materials:

• Empore C8 disc (#22214, 3M)