Mass spectrometric analysis of electrophoretically separated allergens and proteases in grass pollen diffusates.
BACKGROUND
Pollens are important triggers for allergic asthma and seasonal rhinitis, and proteases released by major allergenic pollens can injure airway epithelial cells in vitro. Disruption of mucosal epithelial integrity by proteases released by inhaled pollens could promote allergic sensitisation.


METHODS
Pollen diffusates from Kentucky blue grass (Poa pratensis), rye grass (Lolium perenne) and Bermuda grass (Cynodon dactylon) were assessed for peptidase activity using a fluorogenic substrate, as well as by gelatin zymography. Following one- or two-dimensional gel electrophoresis, Coomassie-stained individual bands/spots were excised, subjected to tryptic digestion and analysed by mass spectrometry, either MALDI reflectron TOF or microcapillary liquid chromatography MS-MS. Database searches were used to identify allergens and other plant proteins in pollen diffusates.


RESULTS
All pollen diffusates tested exhibited peptidase activity. Gelatin zymography revealed high Mr proteolytic activity at approximately 95,000 in all diffusates and additional proteolytic bands in rye and Bermuda grass diffusates, which appeared to be serine proteases on the basis of inhibition studies. A proteolytic band at Mr approximately 35,000 in Bermuda grass diffusate, which corresponded to an intense band detected by Western blotting using a monoclonal antibody to the timothy grass (Phleum pratense) group 1 allergen Phl p 1, was identified by mass spectrometric analysis as the group 1 allergen Cyn d 1. Two-dimensional analysis similarly demonstrated proteolytic activity corresponding to protein spots identified as Cyn d 1.


CONCLUSIONS
One- and two-dimensional electrophoretic separation, combined with analysis by mass spectrometry, is useful for rapid determination of the identities of pollen proteins. A component of the proteolytic activity in Bermuda grass diffusate is likely to be related to the allergen Cyn d 1.
Raftery
Mark J
MJ
Department of Pathology, School of Medical Sciences, University of New South Wales, Sydney, Australia 2052. m.raftery@unsw.edu.au <m.raftery@unsw.edu.au>
Saldanha
Rohit G
RG
Geczy
Carolyn L
CL
Kumar
Rakesh K
RK
D052179:Antigens, Plant / Q000032:analysis*; D002853:Chromatography, Liquid / Q000379:methods; D031744:Cynodon; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D008129:Lolium; D013058:Mass Spectrometry / Q000379:methods; D010447:Peptide Hydrolases / Q000032:analysis*; D031785:Poa; D006109:Poaceae / Q000201:enzymology* / Q000276:immunology*; D011058:Pollen / Q000201:enzymology* / Q000276:immunology*; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D052179:Antigens, Plant; D010447:Peptide Hydrolases