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iTRAQ® (isobaric tags for relative and absolute quantitation)*

 

 

 

iTRAQ® is a liquid-based proteomics technique. It labels the Lysine residues and N terminus of all peptides with isotopes of identical masses, thus allows accurate quantitative comparisons between 2 (2-plex) to 8 (8-plex) samples. This approach can provide both protein identification and protein ratios in one experiment. At Applied Biomics, we have made the following improvements that have significantly increased the sensitivity of detecting low abundant proteins and the total number of proteins (see an example here):
1) Optimized protein extraction on samples such as serum, clinical samples with interfering substances, protein complex and whole cell lysate
2) Optimized a proprietary fractionation protocol specifically targeting the low-abundant basic proteins (pI > 8.0), such as membrane proteins (see example)
3) Extended nanoLC gradient coupled with optimized MSMS parameters, totaling a 12 hour instrument time

We have optimized protein extraction for iTRAQ experiment on the following sample types:
1) Whole cell lysate from cultured cells, bacteria and other organisms
2) Subcellular organelles such as Mitochondria, Lysosome and Exosome etc
3) Protein complex such as IP or column purified complex with very low protein amount (<2ug)
4) Clinical samples with interfering substances such as high salt in urine
5) Serum samples with high abundant proteins and a wide dynamic range

Protein samples
Reduction/Alkylation & Trypsin Digestion
iTRAQ reagent labeling
Mix and clean up samples
NanoLC-MS/MS
MS
MS/MS
Reporter ion

 

 

Example-1: Characteristics Of Proteins Detected In A Single iTRAQ Experiment

Protein Ratios (0.14 to 3.7)
Protein Ratios
Protein Mass (8.5 to 800KD)Protein Mass

Protein pI (3.7 to 12)Protein pl

 

Example-2: Characteristics Of Proteins Detected In An iTRAQ Experiment Using High pI Fractionation

Protein Ratios (0.46 to 3.1)
Protein Ratio
Protein Mass (8.5 to 622KD)Protein Mass

Protein pI (4.6 to 11.4)
Protein PL

* 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

 

* iTRAQ is the trademark of AB SCIEX