Many proteins involved in signal-transduction pathways are concentrated in membrane microdomains enriched in lipids with distinct physical properties. Since these microdomains are insoluble in non-ionic detergents in cold, proteins associated with them could be efficiently purified by techniques such as sucrose-density gradient centrifugation. The complexity of the resulting protein mixture requires powerful MS technique for its analysis. We have found that successful identification of biologically relevant proteins is critically dependent on the enrichment of the starting material (plasma membranes), and on the extraction procedure. Applying these conditions in combination with microHPLC-ESI (electrospray ionization)-MS/MS, we have identified proteins involved in signalling, cytoskeletal association and cellular adhesion in Jurkat cells that are not stimulated by any antibody incubation.

The role of plasma membrane microdomains

Recent biochemical, pharmacological and imaging data indicate that plasma membranes of most cells contain distinct structures called GEM (glycolipid-enriched membranes) or lipid rafts. These components of the plasma membrane concentrate sphingolipids, cholesterol and intracellular proteins involved in signalling, cytoskeletal adhesion and other processes. Owing to their high lipid content, they separate in low-density fractions during sucrose-density gradient centrifugation [1]. In T-cells and T-cell lines, such as Jurkat cells used in the present study, signal-transduction molecules (src-kinases Lck and Fyn, Ras, G-proteins and others) concentrate in lipid rafts upon cellular activation, and initiate polymerization of cytoskeletal proteins leading to the formation of an immune synapse [2]. Since the rapid development of MS techniques makes them suitable for analysis of complex protein mixtures, we applied the methodology for analysis of lipid rafts in unstimulated Jurkat cells. Compared with the previous work [3], we have avoided anti-CD3 stimulation, applied a more rigorous plasma membrane purification scheme and tested several extraction conditions [4].

Optimization of plasma membrane purification and extractions

A two-step centrifugation technique has been employed. First step is the isolation of plasma membranes after lysis in hypotonic buffer and gentle cell stripping through the needle. The fact that lipid rafts are rich in lipids and cholesterol is used in the second preparative step: solubilization of the plasma membrane under four different extraction conditions was followed by ultracentrifugal flotation in sucrose-density gradient [4]. Formation of an opalescent ring after an overnight ultracentrifugation and immunostaining with monoclonal antibodies against typical GEM proteins such as Lck (Figure 1A) provide a visible control of successful separation of lipid rafts. Subsequently, proteins were identified by microHPLC-MS/MS (Figure 1B).

Scheme of methods used for identification of proteins associated with lipid rafts

Figure 1
Scheme of methods used for identification of proteins associated with lipid rafts

(A) Western-blot and immunostaining analyses with monoclonal antibody against Lck. (B) MS analysis showing MS/MS spectrum of a tryptic peptide of Lck (1) and flotillin (2).

Figure 1
Scheme of methods used for identification of proteins associated with lipid rafts

(A) Western-blot and immunostaining analyses with monoclonal antibody against Lck. (B) MS analysis showing MS/MS spectrum of a tryptic peptide of Lck (1) and flotillin (2).

How to obtain and interpret large data sets?

The mixture of proteins in each fraction after ultracentrifugation was precipitated, digested and injected into the microcapillary column, which was connected online to ion trap mass spectrometer (LCQ™Deca; ThermoFinnigan, Palm Beach, FL, U.S.A.). The peptides were eluted with a linear acetonitrile gradient. Owing to the high separation capacity of the microcapillary column (Magic C18, 5 Å particles), the MS/MS spectra of the bulk peptides were obtained. The collected MS/MS spectra were searched using SEQUEST™ software against the database of human proteins [4].

Comparison of MS with other techniques

Compared with other techniques used for studying GEM proteins (immunostaining methods, light and confocal microscopy [5]), MS allows us to identify large numbers of proteins from a single experiment in an unbiased way. The representative list of proteins identified in GEM of Jurkat cells is shown in Table 1. The results obtained under four extraction conditions were comparable, except for the mild detergent Brij 58 that preserves weakly associated proteins resulting in GEM floating at lower densities.

Table 1
Partial list of proteins identified in lipid rafts by different extraction conditions

Numbers refer to fractions collected from the sucrose-density gradients as shown in Figure 1. Proteins identified with high confidence are marked x.

Proteins 1% Brij 58 1% Nonidet P40 1% Triton X−100 Na2CO3 
 
Actin          
ATPase            
Calmodulin                
CD9                
CD98              
Flotillin               
Galectin-9              
G-proteins           
Lamin               
LAR kinase               
CD45               
Lck         
Myosin               
Ras proteins              
Tubulin            
Proteins 1% Brij 58 1% Nonidet P40 1% Triton X−100 Na2CO3 
 
Actin          
ATPase            
Calmodulin                
CD9                
CD98              
Flotillin               
Galectin-9              
G-proteins           
Lamin               
LAR kinase               
CD45               
Lck         
Myosin               
Ras proteins              
Tubulin            

The number of proteins identified here using unstimulated Jurkat cells were smaller than that previously reported for CD3-activated cells [3]. Nevertheless, several molecules of interest in biology of Jurkat cells could be found. In addition to the well-documented resident GEM proteins (flotillin, calmodulin/NAP-22), we could detect additional proteins involved in cellular signalling, cytoskeletal association, cellular adhesion and cell-surface receptors. Signalling proteins found to be associated with GEM include Lck, G-proteins, LAR kinase regulating Lck and Fyn and Ras proteins. Most of these proteins are known to be associated with GEM [6]. CD45 has a dynamic association with GEM that may depend on the cellular activation status [7]. Cytoskeletal proteins such as actin, tubulin and myosin form a scaffold for protein kinases, and participate in the formation of immunological synapse upon activation [2]. Cell adhesion molecule CD9 is a regular component of GEM [8], whereas galectin-9, described as a novel eosinophil attractant, is bound as a peripheral membrane protein [9]. MHC class I was also found in GEM which may be caused by peripheral cis association with specific receptors localized to the lipid rafts [10]. S-100 (calgranulin B) is the intracellular calcium-binding protein and marker of T-cell lymphoproliferative disorders [11] shown here for the first time to be a GEM component.

Conclusion

The fast development of MS makes it suitable for analysis of complex mixtures of proteins including those co-purified with lipid rafts. In our study of unstimulated Jurkat cells, we identified in GEM not only the typical proteins such as Lck or flotillin, but also other signalling, cytoskeletal, and adhesion proteins. Rigorous purification of the starting material and efficient extraction and digestion of all proteins are the most important factors for a successful identification.

Structure Related to Function: Molecules and Cells: A Focus Topic at BioScience2004, held at SECC Glasgow, U.K., 18–22 July 2004. Edited by D. Alessi (Dundee, U.K.), T. Cass (Imperial College London, U.K.), T. Corfield (Bristol, U.K.), M. Cousin (Edinburgh, U.K.), A. Entwistle (Ludwig Institute for Cancer Research, London, U.K.), I. Fearnley (Cambridge, U.K.), P. Haris (De Montfort, Leicester, U.K.), J. Mayer (Nottingham, U.K.) and M. Tuite (Canterbury, U.K.).

Abbreviations

     
  • GEM

    glycolipid-enriched membrane

We thank Professor Václav Hořejší for the gift of monoclonal antibodies and Zora Nováková for cultivation of the cell line. This work was supported by Ministry of Education (MSM 113100001), and by the Institutional Research Concept AV0Z 5020903.

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