Simple, Rapid, and Selective Isolation of 2S Albumins from Allergenic Seeds and Nuts.

The 2S albumins belong to the group of seed storage proteins present in different seeds and nuts. Due to their pronounced allergenic potential, which is often associated with severe allergic reactions, this protein family is of special interest in the field of allergen research. Here we present a simple, rapid, and selective method for the purification of 2S albumins directly from allergenic seeds and nuts. We systematically optimized the parameters "buffer system", "extraction temperature", "buffer molarity", and "pH " and were able to achieve 2S albumin purities of about 99% without further purification and demonstrate transferability of this method to nine different allergenic food matrices. Compared to conventional isolation routines, significant reduction of hands-on time and required laboratory equipment is achieved, but nonetheless higher protein yields are obtained. The presented method allows for the rapid purification of different 2S albumins including the corresponding isoforms from natural material.

Characterization of mustard 2S albumin allergens by bottom-up, middle-down, and top-down proteomics: a consensus set of isoforms of Sin a 1.

The mustard allergen Sin a 1 belongs to the 2S-albumin family of seed-storage proteins. Because of its high abundance in mustard seeds and the potential to elicit severe allergenic reactions, Sin a 1 is considered to be a major allergen in mustard. Eight Sin a 1 isoforms have been identified using DNA cloning and sequencing, and we aim in this study to thoroughly investigate sequence heterogeneity using a novel combination of bottom-up, middle-down, and top-down proteomics. The characterization of purified Sin a 1 extract shows that sequence diversity is far more pronounced than previously assumed. We identified in total 24 sequence polymorphisms including 17 yet unpublished point mutations. Using middle-down and top-down approaches on the subunit and protein level of Sin a 1, we were able to detect eight consensus isoforms of Sin a 1(including four novel isoforms), which we detect in the majority of the four different mustard samples included in this study. In addition, we provide for the first time data on relative abundance of the main Sin a 1 isoforms and identify phytic acid as a potential ligand of Sin a 1. Together, these data can form the basis for a more detailed investigation of the effect of Sin a 1 polymorphic sites on allergenicity of isoforms.

Development and applications of simultaneous immunochemical staining and serial detection of overlapping proteins in blotting procedures.

Immunoblotting techniques are widely used for specific protein identifications and characterizations. The specific bindings of antibodies to epitopes in a protein sequence permits determination of antigens and gives detailed information about protein compositions and expression levels in complex suspensions. However the techniques are mostly restricted to one specific antibody determination. Overlaying proteins are detected using numerous repeated gel runs. For multiple but specific protein determinations on one immunoblot, here we describe the detection of several antigens by simultaneous incubation of antibodies originated from different species followed by sequential addition of secondary antibodies labelled with horseradish peroxidase (HRP) and binding to analogous primary antibodies. Particular signals were visualized step by step using a HRP chemiluminescence substrate while enzymatic HRP reactions were meanwhile inactivated irreversibly by hydrogen peroxide incubation. We demonstrate flexible applications of multiple antigen detections using the Western blotting technique with determination of the CNS protein markers neuron specific enolase, glial fibrillary acid protein and the physiological prion protein (PrP(C)) in brains and in meats as food contaminations and with glycotyping of PrP(C) using antibodies binding to different epitopes. We showed the use of the dot blotting technique with serial determination of different antigens in complex protein suspensions. The method is easy to handle and is flexible and applicable in the fields of diagnostics and public health for detection of overlaying proteins on one immunoblot.