, Leo E. Bonilla
Neuromics, Inc., Minneapolis, MN
Thermo Finnigan, San Jose, CA
The data presented here was acquired using the
ProteomeX integrated proteomics workstation.
The differential quantification of the purinergic
receptor P2X3 in membrane preparations of kidney cells is demonstrated using
a fully integrated 2D LC-MS/MS system to detect and quantify ICAT-labeled
tryptic peptides. Optimization of ICAT procedures, by removal of the need
for off-line ion exchange (IEX) and affinity clean-up after ICAT derivatization,
The systematic identification and quantification
of the constitutive proteins in a complex system (e.g., organelle, cell,
tissue) is the central task of proteomics. Orthogonal two-dimensional chromatography
(2D-LC) coupled with tandem mass spectrometry (MSn
a powerful technology platform for the global study of proteomes.(1)
Stable-isotope labeling, such as isotope-coded affinity tags (ICAT), allows
the implementation of MS-based methods for quantitative protein profiling.
The ICAT procedure relies on the selective alkylation of the free thiol
groups of cysteines with either a d0
- or d8
followed by proteolysis, affinity enrichment of the labeled peptides, and
Quantitative ICAT labeling of membrane proteins
remains an imp
ortant analytical challenge. As expected, cysteine thiols
that occur in highly hydrophobic environments, such as transmembrane protein
domains, are less accessible for labeling. In instances like this, the success
of the analysis hinges on the ability to efficiently label free thiols (i.e.,
on cysteines localized in extra-cellular loops), and to resolve and measure
the labeled peptides in the midst of a very complex matrix.
We are reporting on the quantification of the membrane receptor P2X3 expressed
in HEK cells using ICAT and 2D LC-MS/MS. The P2X family of purino-receptors
constitutes a class of ligand-gated ion channels where ATP is the ligand.
P2X3 occurs in small diameter sensory neurons and is implicated in the modulation
of chronic pain.
In this report, we describe the analysis and
quantification of the membrane receptor P2X3 using a technique that shows:
- The ease-of-use of the ProteomeX Workstation for multi-dimensional
separation of complex mixtures.
- The ability of TurboSEQUEST software to identify ICAT-labeled
- The power of XPRESS to provide quantitative information for
the proteins of interest.
Preparation of Cell Membrane Fractions
Cell membranes used in this experiment were prepared from approximately
HEK cells, grown to ca. 90% confluency. Cells were harvested
in cold PBS, pelleted, re-suspended to 2 mL in PBS and frozen at 80C.
The cell suspension was thawed, diluted to 5 mL in cold PBS, and sonicated
for 30 seconds followed by ultracentrifugation at 100,000 xg
for 1 hou
r. The supernatant was removed and the pellet resuspended in 1
M NaCl PBS by sonication and then pelleted again at 100,000 xg
for 1 hour.
The resulting cell membranes were solubilized by sonication in 500 L of
buffer containing 6 M urea and 0.1% SDS in 50 mM TRIS pH 8.0.
Figure 1. Fluidic path configuration of the
orthogonal 2D LC-MS/MS system used for analysis of complex protein mixtures.
ICAT Labeling and Proteolysis The cell membrane
suspension was reduced with TCEP (5 mM) for 30 minutes at room temperature.
The sample was diluted to 3 mL to reduce the urea concentration and then
split into two fractions prior to alkylation with ICAT. Fraction A (2 mL)
was labeled with d0
-ICAT and Fraction B (1 mL) with d8
Fractions A and B were alkylated with ICAT reagent for 1 hour in the dark
at room temperature. After alkylation, both fractions were recombined to
yield a theoretical ratio of 1: 0.5 for d0
total of 90 g of trypsin was added to the mixture followed by overnight
incubation at 37C.
After digestion, the samples were acidified with 40 L of formic acid and
5 L of TFA to a pH < 2. A cloudy precipitate was formed which was removed
by centrifugation. The supernatant was removed and examined by RP-HPLC to
verify the presence of peptides prior to analysis by 2D LC-MS/MS.
Figure 2. Graphical user interface for programming
of the 2D LC gradients (SCX and RP) and MS acquisition parameters using
Orthogonal 2D LC- MS/MS
Orthogonal 2D LC-MS/MS was performed
using a ProteomeX Workstation (Thermo
Finnigan, San Jose, CA). The system was fitted with a strong cation exchange
column (SCX, 320 m ID 100 mm, DEV SCX, Thermo Hypersil- Keystone) and
two C18 reversed phase columns (RP, 180 m x 100 mm, BioBasic C18, 5 m,
Thermo Hypersil-Keystone), as shown in Figure 1. The salt steps used were
0, 25, 50, 100, 150, 200, 250 and 500 mM NH4
Cl synchronized with
seven 90-min RP gradients. RP solvents were 0.1% HCOOH in either water (A)
or acetonitrile (B). Data Dependent mass spectral acquisition (MS/MS)
was used for the most intense ion in the range of 300-1800 m/z (full-scan)
with the following Dynamic Exclusion settings: repeat count, 1; repeat
duration, 0.5 min; exclusion duration, 3.0 min. SCX and RP gradient programming,
as well as MS settings, which can be set using the Multidimensional Protein
ID method of the ProteomeX Workstation (Figure 2).
Database searching was performed using the TurboSEQUEST program in the BioWorks
3.0 software suite. The database used was a subset (human) of the NR database
available on 02/20/02 (ftp:// ftp. ncbi. nih. gov/blast/db/). No enzyme
was specified for the search. Calculation of the d0
ratios was performed automatically using the XPRESS feature of BioWorks
3.0. Only MS/MS spectra with Xcorr > 2.0 were considered for positive identification
of a gene product. For the compilation of d0
only those MS/MS spectra tagged by XPRESS, that also showed at least two
of the ICAT diagnostic ions, were considered.
Figure 3. Western blot of P2X3 expressed
in HEK cells.
Figure 4. Multiple File Consensus Report
To monitor the effectiveness of ICAT derivatization
of P2X3, Western blot analysis was done using sample preparations before
and after ICAT labeling (Figure 3). The results shown in Figure 3 confirmed
the accessibility of free thiols for labeling by ICAT. This result was also
consistent with previous reports on the effects of labeling on the migration
behavior in SDS-PAGE.
Using a new feature in BioWorks 3.0 called the Multiple File Consensus Report,
a composite of the results output was created, shown in Figure 4. This composite
shows the 4357 gene products identified in a single 2D LC-MS/MS experiment.
Only highly significant MS/MS matches (Xcorr>2.0) were considered for positive
identification. P2X3 was ranked as #537 on the list.
Figure 5 shows the MS/MS spectrum (bottom left) and calculated d0
ratio (bottom right) of the ICAT-labeled peptide TGGVLGIKIGWVC*IPK
using XPRESS. Note that in addition to the expected yand b-ions labeled
with blue and red in the spectrum, the spectrum also shows two diagnostic
ICAT ions at m/z 288 and 485.
There were a total of seven Cys-containing fragments in P2X3 (Table 1).
At least two of them (T1, T10) were inaccessible for labeling due to their
location inside trans-membrane regions. In fact, the only labeled peptides
detected in our experiments came from four Cys residues in the large extracellular
Three other non-Cys containing peptides were also identified with high confidence
between residues 218-243 and 358-367.
Figure 5. MS
/MS spectrum and calculated d0:
ratio of P2X3 peptide.
Table 1. P2X3 ICATlabeled peptides
and their corresponding d0:d8 ratios.
Figure 6 shows a hydropathy plot for the P2X3
receptor protein. The y-axis plots the hydrophilicity/hydrophobicity of
the protein for each of its amino acid residues along the x-axis. Each of
the Cys-containing peptides listed in Table 1 are plotted along with their
locations within the cell. Most of the labeled peptides are located within
the extra-cellular loop, while two are found in trans-membrane regions.
Figure 6. Hydropathy plot of P2X3 showing
the location of Cys-containing peptides.
- These experiments serve as a preliminary validation of the ProteomeX
technology before attempting quantification of P2X3 in vitro and in
- Off-line IEX and affinity clean-up of samples prior to LC/ MS analysis
was not required. Non-specific sample losses were minimized. Both ICAT-
labeled and unlabeled peptides were seen.
- In the future, we plan to quantify the levels of P2X3 in systems
treated with RiboTAG (Neuromics Inc., Minneapolis, MN) antisense
oligonucleotides in gene function studies.
- Yates, J. R. et al. Nature Biotech. 17, 676-682
- Aebersold, R. et al. Anal. Biochem. 297, 25-31 (2001)
- Aebersold, R. et al. Molecular Cellular Proteomics 1, 19-20 (2002)
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