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ICAT
Isotope coded affinity tag (ICAT)-based
protein profiling
Because the mass
spectrometer used to provide this technology was
purchased under an NHLBI contract, priority access to ICAT-based
protein
profiling is given to members of the Yale/NHLBI Proteomics Center.
While both MALDI-MS based
peptide/protein disease biomarker discovery and DIGE analyses
comparatively profile the naturally occurring forms of peptides and
proteins, ICAT analysis profiles the relative amounts of cysteine-containing
peptides derived from tryptic digests of protein extracts. Recognizing
that only a single tryptic peptide is needed to quantify the expression
of the corresponding parent protein, the ICAT reagent was designed to
affinity isolate and quantify via the use of a stable isotope the
relative concentrations of cysteine-containing tryptic peptides
obtained from digests of control versus experimental samples. Hence,
the newest ICAT reagent from Applied Biosystems has a thiol-specific
reactive group adjacent to an alkyl linker, which contains either nine
[12C] or nine [13C] atoms - thus resulting in a
mass difference of 9 daltons between the control versus the
corresponding experimental version of the same tryptic peptide. A nice
feature of the ICAT approach is that the in vitro incorporation
of a stable isotope into one of the two samples being compared obviates
the need to analyze by mass spectrometry the control and experimental
samples separately. The alkyl linker in the ICAT reagent is connected
to a (cleavable) biotin group which allows rapid affinity isolation of cysteine-containing tryptic peptides. While a tryptic digest of a whole
cell human protein extract might produce 550,000 peptides, less than
100,000 of these might be expected to contain cysteine. Based on a
search of the Swiss Database, <5% of human proteins lack cysteine and
would be missed. As depicted in Fig. 3, following derivatization of the
control protein extract with the [12C]-ICAT reagent and of
the experimental protein extract with the [13C]-ICAT
reagent, the pooled samples are subjected to trypsin digestion followed
by cation exchange chromatography. Typically, a whole cell or tissue
protein extract would be divided into 36 cation exchange fractions with
each of them being subjected to avidin chromatography isolation of
cysteine-containing tryptic peptides followed by LC/MS/MS analysis to
identify ICAT peptide pairs and quantify the relative [12C]/[13C]
ratios. Fig. 3: Flow chart depicting ICAT/MS-based protein profiling.
The resulting ICAT data, which is
analogous to that obtained via the use of two different fluorescent
dyes in DNA microarray analysis of mRNA or DIGE analysis of protein
expression, provides the corresponding ratio for the level of
expression of the parent protein in the control versus experimental
sample. Currently, the largest number of proteins profiled by this
approach from a single sample were 491 proteins contained in microsomal
fractions of naïve and in vitro differentiated human myeloid
leukemia cells[i]. Based on reports on Applied Biosystems web pages, we
believe it may be possible to routinely identify and profile
approximately 300 proteins/sample that has been separated into 36
cation exchange HPLC fractions prior to LC/MS/MS.
| The Keck Laboratory's ICAT-based,
quantitative protein profiling technology will be carried out on a new
Applied Biosystems API QStar XL mass spectrometer. The QStar mass
spectrometer will be interfaced with a LC Packings Ultimate Capillary/Nanoflow
HPLC System which consists of a UltiMate™ Micro Pump and Detection
Module for accurate and reproducible micro- and nanoflow delivery, a
FAMOS™ Micro Autosampler for automated injections of small volume
samples with zero sample loss, and a Switchos™ Micro Column Switching
Module that allows for automated sample preparation and
mulitdimensional (e.g., 2D, 3D) LC. The Keck Laboratory expects to
follow the Applied Biosystems protocol for reduction and trypsin
digestion of 100 :g amounts of extracted protein which then will be
fractionated into 1, 12, or 36 pools by cation exchange HPLCi.
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Avidin chromatography will be used to
isolate Cys-containing tryptic peptides from each pool which then will
be individually subjected to LC-MS/MS at a flow rate of 300 nl/min with
a 75 micron x 15 cm LC Packings PepMap C-18 column equilibrated with
0.5% acetic acid, 5% acetonitrile and eluted with a 60 min acetonitrile
gradient.
ICAT derivatized peptide pairs that
differ by exactly 9 Da will be identified and quantified by the Applied
Biosystems ProICAT software. ProICAT can perform modified database
searches by extracting and using only data obtained on cysteine-containing
peptides, thus significantly reducing search and data analysis times.
ProICAT uses a 3-dimensional LC/MS reconstruct algorithm to locate and
accurately determine experimental:control (heavy:light) peak ratios in
complex proteomic samples. Applied Biosystems has been working closely
with Spotfire to link/export the ProICAT quantitation data directly
into DecisionSite which would then enable dynamic visualization and use
of analytic tools similar to those currently used for analysis of gene
expression data.
In order to perform high-throughput
proteomic profiling using ICAT technology, it is essential to automate
as many steps as possible. The standard ICAT procedure from Applied
Biosystems requires manual syringe-based purification steps on
both cation-exchange and avidin cartridges. To address these
challenges, the Keck Laboratory uses an Applied Biosytems Vision
workstation to automate both these steps. By using the Vision
workstation we will be able to automate both the cation exchange HPLC
and avidin cartridge chromatography steps. This greatly enhances our
ability to process samples, to maximize sample throughput on the QSTAR,
and to substantially reduce the possibility of errors associated with
manual syringe-based purification. The Vision workstation is a computer
controlled biocompatible PEEK™ based HPLC system that enables
unattended analysis and fraction collection for protein purification.
It is equipped with two positive displacement piston pumps, a robotic
sample handling system, eight column switching valve, and UV-Visible,
pH, and conductivity monitors. The robotic sample handling system acts
as both a fraction collector and autoampler, thus allowing automated
re-injection of cation-exchange fractions onto the avidin cartridge.
Quantification based on LC-MS peak
areas of stable isotope, internal standard analogs of an analyte has
been used extensively and thus ICAT technology rests on a very firm
foundation in this regard. The resulting peak area reports will be
analyzed by the Keck Biostatistics Resource using statistical criteria
that will be developed in the Yale/NHLBI Proteomics Center. Protein
identification will be based on database searches of the resulting
MS/MS spectra using Sequest and other algorithms. The interpreted
datasets will be returned to individual investigators via a
password-protected Web interface that launches an FTP file transfer and
that will be similar to the interface used for the
Yale Microarray Database.
Additional Information on ICAT:
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