iTRAQ® Service

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

Reduction/Alkylation & Trypsin Digestion

iTRAQ reagent labeling

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

Mix and clean up samples

Nano-HPLC

iTRAQ procedure: step 6 - peptide finger printing by MS

MS/MS

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
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 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.

Pricing

Services Description	Academic/Governmental Labs		Industrial/Commercial Labs
Services Description	Code	Price	Code	Price
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.