title: Development of an isoform-specific tandem mass spectrometry assay for absolute quantitation of maize lipid transfer proteins.
abstract: Precise and accurate quantitation of maize grain allergens is important for seed and food industries. The major allergen in maize grain is Zea m 14, a lipid transfer protein (LTP). The B73 maize genome encodes for at least six LTPs sharing 15%-87% sequence identity to Zea m 14. Phylogenetic analysis of the maize LTP family revealed one gene that corresponds to Zea m 14 (denoted as LTPa) and two other genes sharing 43% (LTPc) and 74% (LTPb) identity with Zea m 14 that are putative homologues. Using stable isotope peptide mimics as internal standards for LTPs, we present a multiple reaction monitoring mass spectrometry approach for multiplexed, absolute quantitation of all three LTP proteins and alternative transcript models therein. To validate quantitative accuracy, a redundant peptide, simultaneously representing the two most abundant LTPs, was included. Analysis of 21 maize varieties revealed LTPa was most prominently expressed in maize grain, ranging from 9 to 32 μg LTP/mg protein. Proteins belonging to the LTPb and LTPc gene models were also expressed but at approximately 10- and 100-fold lower levels than LTPa, respectively. The quantitative results provided by the redundant peptide show around 95% agreement with the sum of the two unique peptides, thus providing support for the LTP gene models and validating the accuracy of this method. Though not all Zea m 14-related LTPs are abundant in grain, their high sequence homology and detectable expression in maize grain signify that LTPb and LTPc are putative allergens and should be accounted for in any quantitation strategy for maize LTP allergens.
authors.affiliation: University of Missouri, Department of Biochemistry, Christopher Bond Life Sciences Center , 1201 Rollins Avenue, Columbia, Missouri 65211, United States.
authors.forename: Severin E
authors.initials: SE
authors.lastname: Stevenson
authors.forename: Scott
authors.initials: S
authors.lastname: McClain
authors.forename: Jay J
authors.initials: JJ
authors.lastname: Thelen
mesh_terms: D000485:Allergens / Q000032:analysis; D000595:Amino Acid Sequence; D052179:Antigens, Plant / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D002352:Carrier Proteins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D020033:Protein Isoforms; D015203:Reproducibility of Results; D012639:Seeds / Q000737:chemistry; D016415:Sequence Alignment; D053719:Tandem Mass Spectrometry / Q000379:methods*; D003313:Zea mays / Q000737:chemistry*
chemical_list: D000485:Allergens; D052179:Antigens, Plant; D002352:Carrier Proteins; D010940:Plant Proteins; D020033:Protein Isoforms; C511920:lipid transfer proteins, plant
keywords: AQUA-MRM; LC-MS/MS; LTP; MRM; Zea mays; allergen; lipid transfer protein; maize; quantitation