Mass spectrometry and tandem MS/MS

iTRAQ Sample Preparation Protocol

iTRAQ Sample Prep Protocol

Mass Spec & Proteomics Facility

Penn State College of Medicine

Click Here to Download iTRAQ/MudPit Sample Submission Form

Prior to labeling with iTRAQ reagents it is necessary to ensure that your samples contain equal amounts of total protein. Many groups quantitate their protein from each sample (e.g., by BCA or BioRad/Bradford assay) and then run a gel and silver stain the gel to verify that each sample appears to have the same amount of stained material - a normalization procedure in our Protein Pilot analysis software can compensate for some differences in initial protein amount, but it is obviously best to start with equivalent amounts of protein in each sample. Also be sure that your sample does not contain, nor was prepared with a solution containing any interfering substance (see end of protocol for the list of interfering substances)

We also suggest doing steps 1-15 below with slightly more than 100ug of protein. By digesting more than 100ug of protein you will be able to run a gel and silver stain it after step 15 to make certain that your sample was digested well (note that a gel cannot tell you if your sample is completely digested, but can show you if it is NOT well digested). When digesting more than 100ug of protein, adjust the final volume of solution used at step 15 so that the volume used after step 15 contains 100ug of protein (less than 100 ug of protein can be used, as long as the same amount of total protein is labeled from each sample; however, you will get proportionally fewer IDs with less input protein, so 100 ug is the ideal amount to use for each sample).

Products we suggest using:

iTRAQ Multiplex (4-plex) Kit, Applied Biosystems # 4352135
iTRAQ Multiplex(8-plex) Kit, Applied Biosystems # 4390811 (single kit), # 4390812 (5 kits)
(NOTE: We order the kits in bulk to get a discount, so the least expensive way to get the kits is to get them through us, $250 for a 4Plex and $500 for an 8plex. You can order a Multiplex Buffer Kit (ABI #4381664), but note that we do not recommend using all of the reagents in that kit in our optimized labeling protocols below (e.g., we recommend using iodoacetamide instead of MMTS for an alkylating agent), and all of the reagents in that kit are common laboratory reagents whose concentrations are listed in the protocol below, so it is not necessary to purchase that buffer kit)

TCEP (tris-(2-carboxyethyl) phosphine) reducing agent, Pierce #20490 (www.piercenet.com/Products/Browse.cfm?fldID=02051012) or Sigma #C4706 (powder) or #646547 (solution) (www2.sigmaaldrich.com/catalog/search/ProductDetail/SIGMA/C4706)

Iodoacetamide, Sigma, # A3221-10vL

Sequencing Grade Modified Trypsin, Promega # V511

Protocol:

1. To each sample containing 100ug of sample (almost dried completely) add 20uL Dissolution Buffer (Dissolution Buffer = 0.5 M triethylammoniumbicarbonate (TEAB) at pH 8.5, e.g., Sigma product 17902, T7408, or 90360 diluted to 0.5 M with water), i.e., a protein 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 (note that this is NOT the MMTS included in iTRAQ kits)

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 Resuspension buffer (50 mM acetic acid, supplied with the Promega trypsin).

11. Vortex, spin

12. Prepare a 1mg/ml solution of trypsin and add 10ul to each sample

13. Vortex, spin

14. Incubate samples at 48°C overnight (12-16hrs)

15. Spin samples

*In order to maximize labeling efficiency, the volume of the sample digest must be less than 50uL (33ul MAX for 8Plex reagents). If the volume of the sample digest is greater than 50uL (33 ul), 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. (for 4Plex reagents, add 70uL of ethanol to each iTRAQ Reagent vial being used)
(for 8Plex reagents, add 50 ul of isopropanol to each iTRAQ Reagent vial being used - the percentage of isopropanol in the reaction tube below (step 19) must be AT LEAST 60% v/v)

18. Vortex each vial for 1 minute, then spin. Check the pH, if it is not at least 7.8-8.5 then add up to 5ul of Dissolution Buffer to get the pH at or above 7.8. You must simultaneously add isopropanol as needed to keep the FINAL concentration of isopropanol in step 19 below AT LEAST 60% v/v)

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 (4Plex) 2 hours (8Plex)

22. After 1 hour (4Plex) or 2 hours (8Plex), 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 combined 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.

33. Fill out a iTRAQ Sample Submission Sheet, email the filled out sheet to bas12@psu.edu (or submit the sheet directly to the LabLink/Proteus LIMS system at proteus.hmc.psu.edu) and take the sample to the core facility (C1734) for analysis. The 2D separations and Mass Spec analyses will take 6-8 days to complete once your samples get to the head of the queue.

Table 2-1 Substances that may interfere with the iTRAQ Reagents Protocol. *from iTRAQ Reagents Protocol, Applied Biosystems

Potential Interfering Substance	Potential Interference	Potential Interfering Substance Potential Interference When to Perform Acetone Precipitation
Thiols (for example, DTT and mercaptoethanol)	Interfere with cysteine blocking	Before beginning the protocol
High amounts of detergents and denaturants* (see next table)	Inactivate Trypsin	If the substance is needed to solubilize your sample, after reducing the protein and blocking the cysteine.
active proteases	Inactivate Trypsin	“
Primary amines (for example those in: · Ammonium acetate · Ammonium bicarbonate · Ammonium citrate · AMPD · Aminoguanidine bicarbonate salt · AMP · Ethanolamine, gly-gly, tris)		Before trypsin digest