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Protein Chemistry >
iTRAQ
Multiplexed Isobaric Tagging
Technology (iTRAQ™)
Overview
iTRAQ technology uses a chemical
tagging reagent which allows multiplexing of four samples and produces
identical MS/MS sequencing ions for all four 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 the four MS/MS reporter ions, which
range from 114 to 117 Da. The overall experiment involves:
- 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 four possible, different iTRAQ reagents.
- Pooling together of the (up to) 4
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
FAQ regarding iTRAQ
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) |
$5,080.00 |
$5,842.00 |
|
iTRAQ + 20
LC-MS/MS (3-plex) (20 fractions) |
$5,345.00 |
$6,146.75 |
|
iTRAQ + 20
LC-MS/MS (4-plex) (20 fractions) |
$5,620.00 |
$6,463.00 |
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) |
$90.00 |
$103.50 |
|
iTRAQ
labeling (2-plex) |
$480.00 |
$552.00 |
|
iTRAQ
labeling (3-plex) |
$655.00 |
$753.25 |
|
iTRAQ
labeling (4-plex) |
$840.00 |
$966.00 |
|
Cation-Exchange
Chromatography (20 fractions) |
$250.00 |
$287.50 |
|
LC-MS/MS,
Data Analysis, YPED, submission and Data Archive
(cost/fraction - 1-15 fractions) |
$278.00 |
$319.70 |
|
LC-MS/MS,
Data Analysis, YPED submission, and Data Archive
(all 20
fractions) |
$4,170.00 |
$4,795.50 |
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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