Microheterogeneity of the major grass group 6 allergen Phl p 6: Analysis by mass spectrometry.
The precise structural characterization of allergens is a basic requirement to improve diagnostics and to find therapeutic strategies against allergic disorders. Natural grass pollen allergens exhibit a wide variety of isoforms and it is still unknown whether this microheterogeneity is essential for the allergic reaction or has a functional effect on sensitization. Well-defined recombinant allergens are considered to replace natural allergens for clinical trials. For the major timothy grass pollen allergen Phl p 6 (approximately 12 kDa) and a recombinant rPhl p 6 we determined the structural microheterogeneity by two-dimensional electrophoresis (2-DE), high-resolution electrospray ionization-Fourier transform-mass spectrometry (ESI-FT-MS) of the intact molecules, and by tryptic peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Natural Phl p 6 is a mixture of mainly two isoforms that differ by two amino acids leading to a mass difference of 5 Da. For each of this two isoforms six variants were identified with modifications at the C- and/or N-terminus. The recombinant Phl p 6 comprises the same structure as one of the main isoforms indicating that it represents a major part of the natural Phl p 6.
Blume
Cornelia
C
Division of Biochemical and Molecular Allergology, Research Center Borstel, Germany.
Lindner
Buko
B
Becker
Wolf-Meinhard
WM
Petersen
Arnd
A
D000485:Allergens / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D015180:Electrophoresis, Gel, Two-Dimensional; D018740:Genetic Heterogeneity; D014644:Genetic Variation; D015151:Immunoblotting; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D010449:Peptide Mapping; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D006109:Poaceae / Q000235:genetics*; D011058:Pollen / Q000737:chemistry / Q000276:immunology; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D017386:Sequence Homology, Amino Acid; D016622:Silver Staining; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017550:Spectroscopy, Fourier Transform Infrared; D014357:Trypsin
D000485:Allergens; C095238:PHLPVI protein, Phleum pratense; D010446:Peptide Fragments; D010940:Plant Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D014357:Trypsin