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
Reduction/Alkylation & Trypsin Digestion
iTRAQ reagent labeling
Mix and clean up samples
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 Mass (8.5KD to 800KD)
Protein pI (3.7 to 12)
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 Mass (8.5 to 622KD)
Protein pI (4.6 to 11.4)
*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|
|Rank||Ratios||MW (Da)||pI||Accession||Protein Name|
|1||0.92||120,027||7.5||MTP_MOUSE||Microsomal triglyceride transfer protein large subunit OS=Mus musculus GN=Mttp PE=2 SV=2|
|2||0.88||43,498||8.9||MDHM_MOUSE||Malate dehydrogenase, mitochondrial OS=Mus musculus GN=Mdh2 PE=1 SV=3|
|3||0.91||49,409||8.3||THIM_MOUSE||3-ketoacyl-CoA thiolase, mitochondrial OS=Mus musculus GN=Acaa2 PE=1 SV=3|
|4||0.83||69,450||9.2||ATPA_MOUSE||ATP synthase subunit alpha, mitochondrial OS=Mus musculus GN=Atp5a1 PE=1 SV=1|
|5||0.85||44,001||8.4||G3P_MOUSE||Glyceraldehyde-3-phosphate dehydrogenase OS=Mus musculus GN=Gapdh PE=1 SV=2|
|6||0.87||46,479||8.5||ALDOB_MOUSE||Fructose-bisphosphate aldolase B OS=Mus musculus GN=Aldob PE=1 SV=3|
|7||0.81||40,241||8.6||VDAC1_MOUSE||Voltage-dependent anion-selective channel protein 1 OS=Mus musculus GN=Vdac1 PE=1 SV=3|
|8||0.96||56,723||9.3||QCR2_MOUSE||Cytochrome b-c1 complex subunit 2, mitochondrial OS=Mus musculus GN=Uqcrc2 PE=1 SV=1|
|9||1.23||69,368||7.2||KPYM_MOUSE||Pyruvate kinase isozymes M1/M2 OS=Mus musculus GN=Pkm2 PE=1 SV=4|
|10||1.17||48,995||7.6||ANXA2_MOUSE||Annexin A2 OS=Mus musculus GN=Anxa2 PE=1 SV=2|
|11||1.18||40,911||9.1||LEG4_MOUSE||Galectin-4 OS=Mus musculus GN=Lgals4 PE=1 SV=2|
|12||1.85||66,905||7.9||AL1A1_MOUSE||Retinal dehydrogenase 1 OS=Mus musculus GN=Aldh1a1 PE=1 SV=5|
|13||0.84||195,543||6.5||CPSM_MOUSE||Carbamoyl-phosphate synthase [ammonia], mitochondrial OS=Mus musculus GN=Cps1 PE=1 SV=2|
|14||1.21||19,712||8.6||FABPL_MOUSE||Fatty acid-binding protein, liver OS=Mus musculus GN=Fabp1 PE=1 SV=2|
|15||1.17||57,299||8.0||PGK1_MOUSE||Phosphoglycerate kinase 1 OS=Mus musculus GN=Pgk1 PE=1 SV=4|
|16||0.97||17,990||8.5||PROF1_MOUSE||Profilin-1 OS=Mus musculus GN=Pfn1 PE=1 SV=2 PE=1 SV=2|
|17||1.02||102,141||8.1||ACON_MOUSE||Aconitate hydratase, mitochondrial OS=Mus musculus GN=Aco2 PE=1 SV=1|
|18||0.89||45,575||9.1||BDH_MOUSE||D-beta-hydroxybutyrate dehydrogenase, mitochondrial OS=Mus musculus GN=Bdh1 PE=1 SV=2|
|19||1.20||110.361||5.7||VILI_MOUSE||Villin-1 OS=Mus musculus GN=Vil1 PE=1 SV=3|
|20||1.34||87,074||8.6||ACOX1_MOUSE||Peroxisomal acyl-coenzyme A oxidase 1 OS=Mus musculus GN=Acox1 PE=1 SV=5|
|21||0.97||49,097||8.7||THIKA_MOUSE||3-ketoacyl-CoA thiolase A, peroxisomal OS=Mus musculus GN=Acaa1a PE=2 SV=1|
|22||0.95||64,684||9.1||EF1A1_MOUSE||Elongation factor 1-alpha 1 OS=Mus musculus GN=Eef1a1 PE=1 SV=3|
|23||0.53||30,707||8.6||CELA1_MOUSE||Chymotrypsin-like elastase family member 1 OS=Mus musculus GN=Cela1 PE=2 SV=1|
|24||1.01||41,646||8.9||DEOC_MOUSE||Putative deoxyribose-phosphate aldolase OS=Mus musculus GN=Dera PE=2 SV=1|
|25||0.93||43,872||8.8||HCDH_MOUSE||Hydroxyacyl-coenzyme A dehydrogenase, mitochondrial OS=Mus musculus GN=Hadh PE=1 SV=2|
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 Description||Academic/Governmental Labs||Industrial/Commercial Labs|
|iTRAQ® Experiment||350A||call for price||350N||call 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 SequestHT
- Data report (see sample report below)
Price does NOT cover sample preparation or the cost of iTRAQ kit.