FAQs for Proteome Mapping / iTRAQ

Q1) What is “Shotgun proteomics” or “MuDPIT” and proteome mapping?
Ans:These are alternative names for the same technique of identifying all the proteins in a system simultaneously using 1D- or 2D- LC MALDI and LC/MS/MS. 

 

Q2) Why should I use proteome mapping and not 2D-gels?
Ans: In LC-MALDI and LC/MS/MS all proteins are analysed and identified in one experiment, whereas in gels every spot must be cut out and identified individually. 

 

Q3) How many proteins can be identified?
Ans:
•    1D LC-MALDI is sufficient to obtain identities on 10s (to >100) of proteins. 
•    2D LC-MALDI is sufficient to obtain identities on 100s (to >1000) of proteins. 
NOTE: Complex samples such as plasma/serum require 2D LC. For simple tissues such as muscle 1D LC may be sufficient.  

 

Q4) Why should I use iTRAQ and not 2D-gels?
Ans: In 2D LC mass spectrometry with iTRAQ all proteins are analysed, identified and quantified in one experiment, whereas in gels every spot must be imaged, its intensity measured, and then the spot cut out and identified individually.

 

Q5) Why is 1D LC required before iTRAQ?
Ans: We requires a 1D LC experiment to understand the complexity of all unknown and new systems to ascertain sample viability.

 

Q6) I am working on an organism where the genome is not sequenced, can I use iTRAQ?
Ans: No, iTRAQ analysis cannot be performed on unknown genome.

 

Q7) How much sample is required?
Ans: Greater amounts of total protein will improve sensitivity towards low abundance proteins; lower quantities may provide sub-optimal results.

Product Service

Proteome mapping

iTRAQ

Amount protein required

  • 1D-LC: 10-20μg of pure protein
  • 2D-LC: 100-200μg of pure protein
  • Requires 25 μg total proteins per tag (for a 4 tag experiment).
  • In order to ensure sufficient protein is available after clean-up; ~250 μg protein per sample is required for labelling for 2 tags, ~150 μg per sample is adequate for 4 tags.

If additional clean-up is required, we therefore recommend that 200-500µg of sample is provided. The minimum specifications are because up to 80% of sample can be lost during sample clean-up.

# Note: We prefers samples to be delivered in lysis solution on dry ice.


Q8) What is the maximum volume of sample?
Ans: The sample volume must be less than 250µl.

 

Q9) What type of samples can be analysed? 
Ans: Cell lysates, secreted protein samples, but not radioactive samples.

# Note:  Secreted samples must be in serum/plasma free media.

Source

Protein extraction

Cell cultures

  • Lysis Buffer 1: 0.2% NP40, 40 Mm KCl, 10 mM Hepes, + protease inhibitors OR
  • Lysis Buffer 2: Prepare in PBS, 0.2% IGEPAL, 0.2% Triton X, 0.2% w/v CHAPS, 75 mM NaCl, 1 mM EDTA , protease inhibitors;
  • Centrifuge at 13,000g for 10 min at 4°C, retain supernatant and freeze at -80°C.

Fungus

 

  • Freeze dried, mechanically broken with mortar and pestle;
  • Solubilise proteins with 10mM Tris-Cl (pH 7.5);
  • Centrifuged at 20 000g for 15 min @ 4° C;
  • Supernatant treated with nucleases to remove nucleic acids;
  • Retain supernatant and freeze at -80°C.

Muscle tissue

  • Lysis buffer - 50 mM Tris (pH 7), 0.5 mM EDTA, 20% glycerol, + protease inhibitors;
  • Sonicate sample on ice twice for 10 s with a 5 s delay between bursts;
  • Centrifuge sample at 13000for 10 min at 4 °C;
  • Retain supernatant and freeze at -80oC.

Secreted Protein

Secreted proteins should be in serum/plasma free media for 24 hours prior to harvesting.

# Note: Lysis buffers used for 2D gel preps are unsuitable for iTRAQ as they contain substances which interfere with the process.

 

Q10) What if I used a buffer that may interfere with the process?
Ans: Acetone precipitation will be performed after extraction to remove all the salts.

Please indicate on order form exactly what solution/buffer used.

Substances that interfere with process

  • sucrose
  • urea