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Quantification of the Purinergic Receptor P2X3 using ICAT and ,,, Orthogonal 2D LC-MS/MS with an Ion Trap Mass Spectrometer

David Barnidge, Leo E. Bonilla, Gargi Choudhary

Neuromics, Inc., Minneapolis, MN
Thermo Finnigan, San Jose, CA

ICAT Analysis

The data presented here was acquired using the ProteomeX integrated proteomics workstation. Overview 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, is demonstrated. Introduction 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) represents 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- ICAT followed by proteolysis, affinity enrichment of the labeled peptides, and MS analysis.(2) 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. Goal In this report, we describe the analysis and quantification of the membrane receptor P2X3 using a technique that shows:
  1. The ease-of-use of the ProteomeX Workstation for multi-dimensional separation of complex mixtures.
  2. The ability of TurboSEQUEST software to identify ICAT-labeled proteins.
  3. The power of XPRESS to provide quantitative information for the proteins of interest.
Methods Preparation of Cell Membrane Fractions
Cell membranes used in this experiment were prepared from approximately 110 9 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-ICAT. 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: d8. A 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 ProteomeX. 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 NH4Cl 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).

Data Analysis
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:d8 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: d8 ratios, 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 (composite) . Results 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: d8 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 loop.

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: d8 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. Conclusions
  1. These experiments serve as a preliminary validation of the ProteomeX technology before attempting quantification of P2X3 in vitro and in vivo.
  2. 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.
  3. 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.

  1. Yates, J. R. et al. Nature Biotech. 17, 676-682 (1999)
  2. Aebersold, R. et al. Anal. Biochem. 297, 25-31 (2001)
  3. Aebersold, R. et al. Molecular Cellular Proteomics 1, 19-20 (2002)



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