iTRAQ is a liquid-based proteomics analysis. It labels the Lysine residues and N terminus of all peptides with isotopes of identical masses, thus allowing quantitative comparisons between 2 (2-plex) to 8 (8-plex) samples. This approach can provide both protein identification and protein ratios in one experiment. We have optimized our iTRAQ protocol to significantly increase the sensitivity of detecting low abundant proteins and the total number of proteins (see application gallery):

  • Optimized sample preparation on samples such as serum, clinical samples with interfering substances, protein complex and whole cell lysate
  • Optimized a fractionation protocol specifically targeting the low-abundant basic proteins (pI > 8.0), such as membrane proteins, or a certain protein mass/pI range of your choice
  • Extended nanoLC gradient coupled with optimized MSMS parameters, totaling a 12 hour instrument time

We have optimized protein extraction for iTRAQ analysis on the following sample types:

  • Whole cell lysate from cultured cells, bacteria and other organisms
  • Subcellular organelles such as Mitochondria, Lysosome and Exosome etc
  • Protein complex such as IP or column purified complex with very low protein amount (<2ug)
  • Clinical samples with interfering substances such as high salt in urine
  • Serum samples with high abundant proteins and a wide dynamic range

iTRAQ experimental procedure

iTRAQ procedure: step 1 - protein extraction and buffer exchange

Protein samples

iTRAQ procedure: step 2 - Reduction/Alkylation & Trypsin Digestion

Reduction/Alkylation & Trypsin Digestion

iTRAQ procedure: step 3 - iTRAQ reagent labeling

iTRAQ reagent labeling

iTRAQ procedure: step 4 - mix and clean up labeled peptides

Mix and clean up samples

iTRAQ procedure: step 5 - peptide separation by Nano-HPLC


iTRAQ procedure: step 6 - peptide finger printing by MS


iTRAQ procedure: step 7 - peptide fragmentation by MSMS


iTRAQ procedure: step 8 - quantitation by reporter ion

Reporter ion

Application Gallery

Example 1:

The following example analyzed the protein characteristics from a standard iTRAQ experiment on cell lysate following Applied Biomics’ protocol. Among all detected proteins, molecular weight ranges from 8.5-800KD and pI ranges from 3.7-12.

Characteristics of proteins detected in a standard iTRAQ experiment

Protein Ratios (0.14 to 3.7)

Protein ratio distributions in a standard iTRAQ experiment
log (Protein Ratio)

Protein Mass (8.5KD to 800KD)

Protein mass distributions in a standard iTRAQ experiment
Molecular Weight (KD)

Protein pI (3.7 to 12)

Protein pI distributions in a standard iTRAQ experiment
Protein PI

Example 2:

The following example analyzed the protein characteristics from a modified iTRAQ experiment with upstream protein fractionation by pI focusing on basic proteins. The data shows that about 50% of identified proteins are basic (pH >=8.0).

Characteristics of proteins detected in an iTRAQ experiment with high pI fractionation

Protein Ratios (0.46 to 3.1)

Protein ratio distributions in an iTRAQ experiment with high pI fractionation
log (Protein Ratio)

Protein Mass (8.5 to 622KD)

Protein mass distributions in an iTRAQ experiment with high pI fractionation
Molecular Weight (KD)

Protein pI (4.6 to 11.4)

Protein pI distributions in an iTRAQ experiment with high pI fractionation
Protein PI

*257 out of 545 proteins (~50%) of identified proteins are basic (pH >=8.0)

Top 25 (Out Of 545) Hits Obtained In High pI Fractionation iTRAQ Experiment
RankRatiosMW (Da)pIAccessionProtein Name
10.92120,0277.5MTP_MOUSEMicrosomal triglyceride transfer protein large subunit OS=Mus musculus GN=Mttp PE=2 SV=2
20.8843,4988.9MDHM_MOUSEMalate dehydrogenase, mitochondrial OS=Mus musculus GN=Mdh2 PE=1 SV=3
30.9149,4098.3THIM_MOUSE3-ketoacyl-CoA thiolase, mitochondrial OS=Mus musculus GN=Acaa2 PE=1 SV=3
40.8369,4509.2ATPA_MOUSEATP synthase subunit alpha, mitochondrial OS=Mus musculus GN=Atp5a1 PE=1 SV=1
50.8544,0018.4G3P_MOUSEGlyceraldehyde-3-phosphate dehydrogenase OS=Mus musculus GN=Gapdh PE=1 SV=2
60.8746,4798.5ALDOB_MOUSEFructose-bisphosphate aldolase B OS=Mus musculus GN=Aldob PE=1 SV=3
70.8140,2418.6VDAC1_MOUSEVoltage-dependent anion-selective channel protein 1 OS=Mus musculus GN=Vdac1 PE=1 SV=3
80.9656,7239.3QCR2_MOUSECytochrome b-c1 complex subunit 2, mitochondrial OS=Mus musculus GN=Uqcrc2 PE=1 SV=1
91.2369,3687.2KPYM_MOUSEPyruvate kinase isozymes M1/M2 OS=Mus musculus GN=Pkm2 PE=1 SV=4
101.1748,9957.6ANXA2_MOUSEAnnexin A2 OS=Mus musculus GN=Anxa2 PE=1 SV=2
111.1840,9119.1LEG4_MOUSEGalectin-4 OS=Mus musculus GN=Lgals4 PE=1 SV=2
121.8566,9057.9AL1A1_MOUSERetinal dehydrogenase 1 OS=Mus musculus GN=Aldh1a1 PE=1 SV=5
130.84195,5436.5CPSM_MOUSECarbamoyl-phosphate synthase [ammonia], mitochondrial OS=Mus musculus GN=Cps1 PE=1 SV=2
141.2119,7128.6FABPL_MOUSEFatty acid-binding protein, liver OS=Mus musculus GN=Fabp1 PE=1 SV=2
151.1757,2998.0PGK1_MOUSEPhosphoglycerate kinase 1 OS=Mus musculus GN=Pgk1 PE=1 SV=4
160.9717,9908.5PROF1_MOUSEProfilin-1 OS=Mus musculus GN=Pfn1 PE=1 SV=2 PE=1 SV=2
171.02102,1418.1ACON_MOUSEAconitate hydratase, mitochondrial OS=Mus musculus GN=Aco2 PE=1 SV=1
180.8945,5759.1BDH_MOUSED-beta-hydroxybutyrate dehydrogenase, mitochondrial OS=Mus musculus GN=Bdh1 PE=1 SV=2
191.20110.3615.7VILI_MOUSEVillin-1 OS=Mus musculus GN=Vil1 PE=1 SV=3
201.3487,0748.6ACOX1_MOUSEPeroxisomal acyl-coenzyme A oxidase 1 OS=Mus musculus GN=Acox1 PE=1 SV=5
210.9749,0978.7THIKA_MOUSE3-ketoacyl-CoA thiolase A, peroxisomal OS=Mus musculus GN=Acaa1a PE=2 SV=1
220.9564,6849.1EF1A1_MOUSEElongation factor 1-alpha 1 OS=Mus musculus GN=Eef1a1 PE=1 SV=3
230.5330,7078.6CELA1_MOUSEChymotrypsin-like elastase family member 1 OS=Mus musculus GN=Cela1 PE=2 SV=1
241.0141,6468.9DEOC_MOUSEPutative deoxyribose-phosphate aldolase OS=Mus musculus GN=Dera PE=2 SV=1
250.9343,8728.8HCDH_MOUSEHydroxyacyl-coenzyme A dehydrogenase, mitochondrial OS=Mus musculus GN=Hadh PE=1 SV=2

Sample Info

Sample type: We accept in-solution, in-gel samples or IP beads for this service.

Protein amount: 5-200 µg of total protein from each sample.

Protein concentration: 5-20 mg/ml is preferred for in-solution samples..

Buffer: Please feel free to submit samples in whichever buffer that you would normally use.  Please inform our scientists about buffer composition. Additional charge may apply on samples that require extensive amount of the extra work such as protein eluting, concentrating, buffer exchange or serum abundant protein depletion.

Biohazardous Materials: We do NOT accept samples containing any biohazardous materials. If your samples contain any biohazardous materials, please make sure to deactivate them completely using the appropriate procedure and declare them in the order form. In addition, we do NOT accept any samples containing Level 4 biohazardous materials even if they are deactivated. Please view here for the rank of biohazardous materials.

Sample type: Please inform our scientists about your sample type. We will send you additional tips for each sample type.


Services DescriptionAcademic/Governmental LabsIndustrial/Commercial Labs
iTRAQ® Experiment350Acall for price350Ncall for price

Turnaround time is 7-9 days. Price covers the following:

  • Reduction and alkylation
  • Trypsin digestion
  • iTRAQ labeling followed by sample cleaning up
  • Nano-LC coupled with auto-spotting
  • MS and MS/MS
  • Standard database search against one species in NCBI or SwissProt database using MASCOT
  • Data report (see sample report below)

Price does NOT cover sample preparation or the cost of iTRAQ kit.