Protocol:
1.
To each sample containing 100ug of sample (almost dried completely) add 20uL Dissolution Buffer (Dissolution Buffer =0.5 M triethylammoniumbicarbonate at pH 8.5) i.e., a concentration of 5 mg/mL = 5 ug/ul
2.
Add 1uL of the Denaturant (2% SDS) and vortex
3.
Add 1uL of Reducing Reagent tris-(2-carboxyethyl) phosphine [TCEP] to each sample to make 5 mM TCEP concentration.
4.
Vortex, spin
5.
Incubate the tubes at 60°C for 1hr
6.
Spin the sample
7.
Add 1uL of freshly prepared 84mM solution of iodoacetamide.
8.
Vortex, spin
9.
Incubate the tubes in the dark at room temperature for 30 minutes (wrap tubes in foil)
10.
Reconstitute a vial of Promega sequencing grade trypsin w/ 21uL of reconstitution buffer.
11.
Vortex, spin
12.
Prepare a 1mg/ml solution of trypsin and add 10ul to each sample
13.
Vortex, spin
14.
Incubate samples at 38°C overnight (12-16hrs)
15.
Spin samples
*In order to maximize labeling efficiency, the volume of the sample digest must be less than 50uL. If the volume of the sample digest is greater than 50uL, dry the sample in a centrifugal vacuum concentrator, then reconstitute with 30uL Dissolution Buffer.
16.
Bring each vial of iTRAQ Reagent that you need to room temperature
17.
Add 70uL of ethanol to each iTRAQ Reagent vial
18.
Vortex each vial for 1 minute, then spin
19.
Transfer the contents of one iTRAQ Reagent vial to one sample tube
20.
Vortex, Spin
21.
Incubate the tubes at room temperature for 1hr
22.
After 1 hour, add 100uL of Milli-Q water to each tube to quench the iTRAQ reaction. Incubate at room temperature for 30 minutes.
23.
Combine the contents of all iTRAQ Reagent-labeled sample tubes into one tube
24.
Vortex, Spin
25.
Dry the tube containing all the iTRAQ mixes.
26.
Add 100uL of water to the tube.
27.
Vortex to mix, spin
28.
Dry sample completely
29.
Repeat steps 26-28 two more times (a total of 3 times)
30.
Add 500uL of Cation Exchange Buffer-Load to the tube (We use 12 mM Ammonium Formate in 25% acetonitrile at pH 2.5-3.0, or alternately 10 mM potassium phosphate (KH2PO4) in 25% acetonitrile at pH 2.5-3.0).
31.
Vortex to mix.
32.
Check the pH. If the pH is not between 2.5 and 3.3 adjust by adding concentrated HCl (<1uL at a time), or formic acid if using the alternate Cation Exchange Buffer-Load from above
2D-LC separations:
After iTRAQ labeling is complete, or for LC-MALDI MudPit experiments, 2D-LC separation of the labeled tryptic peptides is carried out as follow: the samples are dried down and resuspended in SCX loading buffer.
SCX Separations are performed on a passivated Waters 600E HPLC system, using a 4.6 X 250 mm PolySULFOETHYL Aspartamide column (PolyLC, Columbia, MD) at a flow rate of 1 ml/min. Buffer A contains 10 mM ammonium formate, pH 3.6, in 20% acetonitrile/80% water. Buffer B contains 666 mM ammonium formate, pH 3.6, in 20% acetonitrile/80% water.
The gradient is Buffer A at 100% (0- 30 minutes following sample injection), 0%à35% Buffer B (30-48 min), 35%à100% Buffer B (48-49 min) 100% Buffer B (49-56 min), then at 56 min switched back to 100% A to re-equilibrate for the next injection. The first 28 ml of eluant (containing all flow-through fractions) are combined into one fraction, then 14 additional 2-ml fractions are collected. All 15 of these SCX fractions are dried down completely to reduce volume and to remove the volatile ammonium formate salts, then resuspended in 15 µl of 2% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid and filtered prior to reverse phase C18 nanoflow-LC separation.
For 2nd dimension separation by reverse phase nanoflow LC, each SCX fraction is autoinjected onto a Chromolith CapRod column (150 X 0.1 mm, Merck) using a 5 µl injector loop on a Tempo LC MALDI Spotting system (ABI-MDS/Sciex). Buffer C is 2% acetonitrile, 0.1% trifluoroacetic acid, and Buffer D is 98% acetonitrile, 0.1% trifluoroacetic acid.
The elution gradient is 100% C (0-4 min), 0à10% D (4-10 min), 10%à25% D (10-30 min), 25%à40% D (30-35 min), 40%à80% D (35-38 min), 80% D (38-42 min), 80%à0% D (42-43 min), 0% D (43-50 min).
