Quadrupole time-of-flight mass spectrometry: a method to study the actual expression of allergen isoforms identified by PCR cloning.
BACKGROUND
Over the past 2 decades, molecular biology has shown that most major allergens exist in multiple isoforms. Very little is known about the relevance of allergen isoforms at the level of expressed protein (ie, actual allergen exposure).


OBJECTIVE
The aim of this study was to evaluate the applicability of state-of-the-art quadrupole time-of-flight mass spectrometry (Q-TOF MSMS) to the identification and quantification of allergen isoforms at the protein level.


METHODS
Q-TOF MSMS is a mass spectrometric approach for sequencing peptides and proteins. In our study it was applied to recombinant (r)Mal d 1, rBet v 1a and rBet v 1d, and natural (n)Mal d 1 from fruits of Malus domestica, cultivar Granny Smith.


RESULTS
Q-TOF MSMS allowed sequencing of about 70% of all amino acids in Mal d 1 and about 60% of those in Bet v 1. Mixing experiments with rBet v 1 isoforms and with rMal d 1 and nMal d 1 revealed that the technique allows identification of isoforms in mixtures down to a level of at least 5%. Recombinant Mal d 1 was identified as a Mal d 1a representative, whereas Granny Smith apples were shown to contain Mal d 1b-like allergen isoforms. In this apple cultivar hitherto unreported modifications of Mal d 1b were identified. Q-TOF MSMS allowed semiquantitative measurement of allergen at the femtomole to picomole level.


CONCLUSIONS
Q-TOF MSMS is a powerful tool to find out whether an allergen isoform, detected at the cDNA level, is really expressed in quantities relevant for the allergic patient.
Plant Research International, Wageningen, Amsterdam, The Netherlands.
Johannes P F G
JP
Helsper
Luud J W J
LJ
Gilissen
Ronald
R
van Ree
Antoine H P
AH
America
Jan H G
JH
Cordewener
Dirk
D
Bosch
D000485:Allergens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D003001:Cloning, Molecular; D018744:DNA, Plant / Q000378:metabolism; D005512:Food Hypersensitivity; D027845:Malus / Q000276:immunology*; D013058:Mass Spectrometry / Q000379:methods; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA
D000485:Allergens; D052179:Antigens, Plant; D018744:DNA, Plant; C095551:MALD1 protein, Malus domestica; D010940:Plant Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; C061312:Bet v 1 allergen, Betula