Evolution of seed allergen quantification – From antibodies to mass spectrometry

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Abstract

Development of accurate, high-throughput approaches for protein allergen quantification is important for the seed industry as a means to monitor natural variability in expression and ensure introduced transgenes do not collaterally alter the expression of any known allergen. Analytical approaches for protein quantification have undergone a renaissance in recent years with the emergence of soft-ionization approaches and advanced mass spectrometers capable of achieving low attomolar sensitivity. These advances coupled with bioinformatic tools to mine mass spectral data are collectively referred to as proteomics, and allow for the large-scale study of proteins with high precision and quantitative accuracy. In this review, we discuss differential and quantitative proteomics workflows that proceed from discovery profiling to targeted, quantitative analysis of specific proteins using stable isotopically-labeled, synthetic peptide doping standards. These synthetic peptide standards, also referred to as AQUA peptides, are synthetic mimics to proteotypic peptides and allow for absolute quantification of proteins in complex biological mixtures. The approaches discussed herein are ideal for the analysis of prominently expressed proteins such as protein allergens from plant seed, as no gels or sample pre-fractionation is required. We discuss these new techniques in the context of traditional, antibody-based technologies for allergen detection and quantification.

Introduction

Incidences of food allergies have doubled in the last two decades and become a worldwide public health concern (Sicherer et al., 2003, van Hengel, 2007). Approximately 90% of adverse reactions are the result of ingestion of only eight foods (peanuts, soybean, milk, eggs, fish, crustacean, wheat, and tree nuts), three of which are seeds – soybean, peanuts, and tree nuts (World Health Organization, 2006). Together, tree nuts and peanuts cause approximately 80% of anaphylactic reactions and account for over 50% of children fatalities related to food allergies (Bock et al., 2001). Currently, avoidance of these seeds is the primary recommendation to prevent allergic reactions. For this reason, accurate and sensitive methods are necessary to identify and quantify seed allergens as a means to provide consumer information on seed and food-products from seed. New or advanced strategies and technologies (e.g. proteomics and mass spectrometry) provide the means to compare allergen concentrations among seed varieties to evaluate the contribution of both genetic and environmental factors. These technologies also have the potential to provide more precise information on allergen expression in genetically modified seeds and breeding lines before commercial release into the market.

Section snippets

Immunoassays for allergen discovery and detection

Historically seed allergens have been detected using antibody-based assays, or immunoassays, with or without protein separation techniques (Poms et al., 2004). In these assays, immunoglobulins are the primary source of allergen detection either through direct binding or competition. The “first responder” of the immune system, immunoglobulin E (IgE), has been used widely for this purpose (Williams, 2003). In this assay IgE is collected from allergic patient donors and used as a probe to discover

Proteomics tools for seed allergen identification and quantification

Proteomics has been a useful development towards the study of the allergen composition of seeds. The proteomics field has developed an advanced set of tools for specific, high-sensitivity identification and quantification of proteins. Analytical mass spectrometers produce molecular mass determinations and sequence information for peptides of interest that lead to the accurate, unambiguous identification of the original proteins (Thelen, 2009). Mass spectrometry, in combination with 2DGE and

Future perspectives

Methods for assessing the levels and types of allergens in seeds have benefited greatly from the impressive technological breakthroughs in the field of mass spectrometry spurred by the demands of the proteomics field. Advances in sensitivity have allowed for routine peptide measurements at mid to high attomole levels using triple quadrupole instrumentation, while advances in linear ion trap scan rates have doubled the number of peptides measured during an LC–MS/MS run. These attributes make

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

The authors would like to acknowledge Susan MacIntosh, Rod A. Herman, and Laura Privalle for their thorough review of this manuscript.

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    This research was supported by the National Science Foundation, Plant Genome Research Program Grant DBI-0332418.

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