This page provides guideline for solution-based iTRAQ or TMT labeling approach in global proteome and phopshoproteome analyses.

Potential interference substances

It's important to avoid the following substances:

1. High concentration of thiols (for example, >30mM DTT and mercaptoethanol)
2. High amounts of detergents and denaturants. It's important to limit the concentration of detergent or denaturant.
   • SDS (<5%)
   • OG (octyl B-D-glucopyranoside) (0.5%)
   • NP®-40 (0.5%)
   • Triton® X-100 (0.5%)
   • Tween® 20 (0.5%)
   • CHAPS (0.5%)
   • Urea (<6M)
     Note: When using urea, always use a freshly-prepared solution. When a sample containing urea, don’t incubate your samples above 37 °C for >1 hour.
3. Active proteases
4. Reagents containing primary amines. Avoid using any reagents containing primary amines, for example those in:
   • Ammonium acetate
   • Ammonium bicarbonate
   • Ammonium citrate
   • Ammonium tartrate
   • AMPD [2-amino-2-methyl-1,3-propanediol]
   • Aminoguanidine bicarbonate salt
   • AMP [2-amino-2-methyl-1-propanol]
   • Ethanolamine
   • Gly-gly
   • Tris buffers

Alternative Buffer

Protein extraction tips for iTRAQ and TMT labeling project

1. Use your conventional protein extraction protocols optimized for your tissues, samples and biological systems (such as RIPA lysis buffer).
2. Replace any possible primary-amine containing buffer with the above alternative buffers. (If you have to do protein extraction buffer with primary amine, let us know. We will need to do acetone precipitation followed by wash to remove any primary amine reagents from your protein pellets for us to process labeling).
3. Add protease cocktails specifically for your tissues (such as for plant, for bacteria or for animals etc.) as the vendors’ (e.g. Sigma, Roche etc.) recommends to minimize the possible subsequent protein degradation. Add immediately phosphatase inhibitor cocktails as well while you do protein extraction if you plan to do phosphoproteomics simultaneously.
4. Keep extracted proteins’ concentration as high as you can (~2-3 ug/uL), which will make downstream denaturing and digestion steps a lot  easier.
5. Quantify the extracted protein concentrations by Bradford or BCA or 1D gel analysis. If you run an SDS gel for your extracted protein samples, send the gel image to us when you submit your samples.
6. If you are on Cornell Ithaca campus: please don’t freeze your extracted proteins. Submit your protein samples immediately after you get the protein quantitation done. Do not forget to contact Elizabeth Anderson to set up an appointment to submit unfrozen samples.
7. If you are not in Cornell Ithaca campus: freeze your samples immediately after you check protein concentration, and ship us your samples with dry ice on next day delivery service as described on our shipping instructions.

Initial steps we will do in our facility relevant to your samples

1. If your samples contain unknown concentration of detergents or unknown chemical components from protein extraction kits or elution kits for IP-pull down samples etc., we will either have to do acetone precipitation or use Strap workflow for your samples before we perform iTRAQ/TMT labeling.
2. As part of our standard protocol at the facility, in order to do accurate protein quantitation before we perform iTRAQ/TMT analysis, we will run SDS gels for all of your samples along with 4-5 lanes of our in-house made E coli lysate with variable amount of proteins used for generating a standard curve. Then we will scan the gel image and use ImageQuant TL software for determining the absolute amounts of proteins in your samples in each gel lane.  

Protein concentration and amount

• For simple samples (immunoprecipitate or affinity purified sample), use 5 μg of protein, at concentration of no less than 1µg/µL.
• For complex samples (samples containing more than 100 proteins, such as lysate): use up to 100 μg, at concentration of no less than 1µg/µL.
• Parallel phoshoproteome analysis require up to 300 μg of proteins for each sample  at concentration of no less than 2µg/µL.

If necessary, verify that you have sufficient protein by performing a quantitative protein assay (for example, bicinchoninic acid [BCA] assay). If you have less than 5 μg protein for your samples, and you want to do relative quantitation analysis for multiple samples, we suggest you use either dimethylation (DM) labeling approach or label-free quantitation (LFQ) analysis. The DM labeling approach will allow only heavy, medium and light reagents for three samples (3-plex) at the same time. In most cases, we will not conduct LC fractionation prior to nanoLC-MS/MS analysis by LFQ analysis.

RIPA buffer for mammalian cell and soft tissue lysis

Composition

• 50 mM Phosphate buffer saline (PBS) pH 7.4 with 150 mM NaCl
• 1% NP-40
• 0.25% sodium deoxycholate
• 1mM PMSF (add prior to use)
• 1% sodium dodecyl sulfate (SDS)
• 1x Roche complete mini protease inhibitor cocktail
• 1x Pierce phosphatase inhibitor cocktail

Making stock solution

• 5ml of NP-40
• 2.5g sodium deoxycholate in water (stored in room temperature in dark)
• 25ml 20% SDS solution
• 50ml 10XPBS
• 420ml milliQ water

Alternate stock solutions

• 100mM PMSF in isopropanol
• Roche complete mini protease inhibitor cocktail tablets (cat# 04 693 124 001)
• Pierce phosphatase inhibitor cocktail (100x) (cat# 78420)

Before the homogenization of the tissue, add following to a total volume of 10mL of the RIPA buffer:

• 100uL of PMSF (10mg/ml)
• 1x Roche anti protease cocktail
• 1x Pierce phosphatase inhibitor cocktail or [10ul orthovanadate (1000mM), 300uL aprotinin]