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Protein Chemistry >
iTRAQ
4 and 8-plex iTRAQ Protein Profiling
Technology
Overview
iTRAQ Technology (Multiplexed Isobaric
Tagging Technology) uses a chemical tagging reagent which allows
multiplexing of two to eight protein samples and produces identical
MS/MS sequencing ions for all eight versions of the same derivatized
tryptic peptide. This greatly facilitates peptide identification due to
the resulting higher intensities of the parent and fragment ions.
Quantitation is achieved by comparison of the peak areas and resultant
peak ratios for either four MS/MS reporter ions, which range from 114
to 117 Da or eight MS/MS reporter ions, which range from 113-119 and
121 Da.
- Hydrolysis/amino acid analysis of
an aliquot of each sample – to accurately determine the protein
concentration of each sample so that they can be mixed together in
the same relative amounts. The optimum sample amount is approximately
50-100 μg per sample.
- Trypsin digestion of an equal total
amount of each sample.
- Labeling of each sample with one of
the eight possible, different iTRAQ reagents.
- Pooling together of the (up to) 8
samples
- Cation exchange fractionation –
with the number of fractions collected being a function of the
complexity of the sample. Whole cell/tissue extracts typically would
be divided into 20 cation exchange fractions.
- Reverse-phase LC/MS/MS on each of
the cation exchange fractions.
- Database searching, quantitation,
and merging together of the data from all LC/MS/MS experiments and
posting/archiving the resulting data summary on the Yale Protein
Expression Database (YPED).
Email iTRAQ Questions to Keck Staff
Sample Submission Form
Pricing
Table 1 below shows the cost for a
complete iTRAQ experiment which includes amino acid analysis, iTRAQ
labeling, cation-exchange chromatography, LS-MS/MS, Data Analysis, YPED,
submission and Data Archive.
Table 2 shows ala carte pricing for
each step in the iTRAQ experiment.
Table 1. Yale University Keck
Biotechnology Pricing for complete iTRAQ analysis
|
Service |
Yale |
Non-Yale |
|
iTRAQ + 20
LC-MS/MS
(2-plex) (20 fractions) |
$3705 |
$4260 |
|
iTRAQ + 20
LC-MS/MS
(4-plex) (20 fractions) |
$4065 |
$4675 |
|
iTRAQ + 20
LC-MS/MS
(8-plex) (20 fractions) |
$4665 |
$5360 |
Table 2. Yale University Keck
Biotechnology Pricing for ala carte iTRAQ analysis
|
Service |
Yale
|
Non-Yale |
|
50ug of
control and 1-3 test protein mixtures |
|
Amino Acid
Analysis (cost/sample) |
$90 |
$103 |
|
iTRAQ
labeling (2-plex) |
$410 |
$472 |
|
iTRAQ
labeling (4-plex) |
$590 |
$679 |
|
iTRAQ
labeling (8-plex) |
$829 |
$954 |
|
Cation-Exchange
Chromatography (20 fractions) |
$250 |
$288 |
|
LC-MS/MS,
Data Analysis, YPED, submission and Data Archive
(cost/fraction - 1-15 fractions) |
$200 |
$230 |
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LC-MS/MS,
Data Analysis, YPED submission, and Data Archive
(all 20
fractions) |
$2865 |
$3295 |
Sample Preparation
Sample should be dissolved in 0.5 M
triethylammonium bicarbonate at pH 8.5 to a concentration of 5 mg/mL.
Samples can be denatured with SDS at a level of 0.1% SDS.
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