Integral membrane proteins are generally under-represented in routine proteomic analyses, mostly because of their relatively low abundance, hydrophobicity and lack of trypsin-cleavage sites. To increase the coverage of membrane proteomes, various strategies have been developed, targeting mostly the extra-membrane segments of membrane proteins. We focused our attention to the rather overlooked hydrophobic transmembrane segments. Such peptides can be isolated after carbonate stripping and protease "shaving" of membranes isolated by simple centrifugation procedure. The treated membranes with embedded hydrophobic peptides can then be solubilized in organic solvents, re-digested with CNBr, delipidated and subjected to LC-MS/MS analysis. We modified the original "hppK" method, and applied it for the analysis of human lymphoma cells. We identified 1224 proteins of which two-thirds were IMPs with 1-16 transmembrane segments. This method allowed us to identify 13 "missing proteins" - proteins with no previous evidence on protein level. BIOLOGICAL SIGNIFICANCE: Integral membrane proteins execute numerous essential functions and represent substantial part of eukaryotic proteomes. Our knowledge of their function and expression is, however, limited. Novel approaches extending our knowledge of membrane proteome are therefore highly desired. As we demonstrate here, a non-conventional method which targets rather overlooked hydrophobic transmembrane segments of integral membrane proteins has wide potential to provide the missing information on the membrane proteome. We show that it can deliver identification and potentially also quantification of hundreds of integral membrane proteins including the so called "missing proteins".
- MeSH
- chromatografie kapalinová metody MeSH
- hydrofobní a hydrofilní interakce MeSH
- lidé MeSH
- lymfom z plášťových buněk chemie MeSH
- membránové proteiny analýza MeSH
- nádorové proteiny analýza MeSH
- peptidy analýza MeSH
- proteom chemie MeSH
- proteomika metody MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- trypsin chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: The incidence of food allergy to wheat is increasing. Its diagnosis depends on the purity of major allergens and their inclusion in tests. Isolation and characterization of wheat allergens are therefore of utmost importance. OBJECTIVE: To purify and identify wheat flour allergens most frequently recognized by patients' IgE antibodies and to study their allergenicity. METHODS: Water/salt-soluble extracts from wheat flour were prepared and separated using a combination of ultrafiltration, isoelectric focusing and liquid chromatography. Purified proteins were analysed by immunoblotting using pooled sera from patients with atopic dermatitis who possessed IgE specific to wheat. Wheat proteins found to bind IgE were subsequently identified by matrix-assisted laser desorption/ionisation-time of flight mass spectrometry. The frequency and intensity of IgE binding of isolated proteins were tested using individual sera from patients and controls. RESULTS: We developed a procedure that allows isolation of wheat allergens from natural sources. Twenty-seven potential wheat allergens have been successfully identified; of these, the following seven are newly reported in food allergy: endogenous α-amylase/subtilisin inhibitor, trypsin/α-amylase inhibitor (AAI) CMX1/CMX3, thaumatin-like protein (TLP), xylanase inhibitor protein-1, β-glucosidase, class II chitinase and 26 kDa endochitinase. TLP and wheatwin were shown to activate patients' basophils to a similar extent as two well-known allergens, lipid transfer protein (Tri a 14) and AAI 0.19 (Tri a 28.0101). CONCLUSION AND CLINICAL RELEVANCE: Our new approach enables the isolation of water/salt-soluble wheat allergens in their native form in amounts sufficient both for biological testing (in vivo and in vitro) and for physicochemical characterization. Such studies will lead to a more detailed knowledge of allergenicity of wheat proteins and to improved accuracy of diagnostic tests.
- MeSH
- alergeny chemie imunologie izolace a purifikace MeSH
- alergie na pšenici diagnóza imunologie MeSH
- atopická dermatitida diagnóza imunologie MeSH
- bazofily imunologie MeSH
- dítě MeSH
- dospělí MeSH
- imunoglobulin E krev MeSH
- isoelektrická fokusace MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mouka analýza MeSH
- potravinová alergie diagnóza imunologie MeSH
- předškolní dítě MeSH
- pšenice chemie imunologie MeSH
- rostlinné proteiny chemie imunologie izolace a purifikace MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- ultrafiltrace MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- MeSH
- antigeny povrchové metabolismus MeSH
- buňky NK metabolismus MeSH
- disacharidy chemie metabolismus MeSH
- glykosylace MeSH
- konformace sacharidů MeSH
- krysa rodu rattus MeSH
- polysacharidy chemie metabolismus MeSH
- receptory imunologické metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- MeSH
- echokardiografie MeSH
- kardiomegalie diagnóza MeSH
- lidé MeSH
- Check Tag
- lidé MeSH
