As allergy sufferers know all too well, proteins from environmental allergens such as grass pollen and dust mites trigger immune system responses. This can lead to a variety of respiratory symptoms: itchy eyes, runny nose, coughing, sneezing and wheezing are among the most common effects. Previous research studying allergen proteins has implicated glycans as important in promoting the uptake of allergens and targeting carbohydrate lectin receptors on antigen presenting cells (APCs).1
To more fully understand the post-translational modifications (PTMs) involved in immune response, Halim et al. recently staged a detailed PTM characterization of seven main allergens used in allergy tests and vaccines.2 Utilizing a combined top-down and bottom-up mass spectrometry strategy, Halim and colleagues characterized Phl p 1, Phl p 5, Der p 1, Der p 2, Der f 1, Der f 2 and Bet v 1. These allergens originated from grass pollen, house dust mite (HDM) pollen and birch trees, respectively.
The research team purified samples of each allergen, separated proteins using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and digested the proteins with trypsin. For the bottom-up strategy, the team purified digested proteins and analyzed them using an ultra-high-performance EASY-nLC 1000 liquid chromatograph (Thermo Scientific) interfaced via a Nanospray Flex ion source (Thermo Scientific) to an Orbitrap Velos Pro hybrid ion trap-Orbitrap mass spectrometer (Thermo Scientific).
The scientists performed acquisition MS1 precursor scans at m/z 350–1,700 in the Orbitrap Velos Pro instrument using a nominal resolution of 30,000 followed by HCD-MS2 fragmentation of the five most abundant multiply-charged precursor ions.The team chose a minimum signal threshold of 50,000 ions to trigger HCD-MS2 and ETD-MS2 fragmentation; they acquired MS2 scans (m/z 100–2,000) in the Orbitrap Velos Pro mass analyzer using a resolution setting of 15,000.
For the top-down strategy, the team analyzed each allergen (1 pmol/l in 50% methanol and 0.1% formic acid, v/v) on the Orbitrap Velos Pro mass spectrometer by direct infusion via a TriVersa NanoMate ESI-Chip interface (Advion Biosystems).
This work revealed more complex glycan structures than previously thought. In the major grass pollen group 1 and 5 allergens, the team increased the number of Phl p 1’s previously published number of structural variants from three to eight glycoforms. In Phl p 5, they confirmed the presence of zero to seven Hyps and up to seven Pen residues on a defined N-terminal region.
Looking at the HDM allergens, the researchers demonstrated that the natural Der p 1 is N-glycosylated with one or two HexNAc residues at Asn150, while the combination of bottom-up and top-down proteomics revealed hexose modifications at multiple sites in both Der p 2 and Der f 2.The team confirmed previous work indicating no major PTMs are involved in birch pollen.
Collectively, Halim and colleagues report that these findings implicate important roles for carbohydrates in allergen recognition and response by the immune system.
1. Royer, P., et al. (2010) “The mannose receptor mediates the uptake of diverse native allergens by dendritic cells and determines allergen-induced T cell polarization through modulation of IDO activity,” Journal of Immunology, 185 (pp. 1522–31).
2. Halim, A., et al. (2015, January) “Glycoproteomic analysis of seven major allergenic proteins reveals novel post-translational modifications,” Molecular and Cellular Proteomics, 14(1) (pp. 191–204), doi: 10.1074/mcp.M114.042614.
Post Author: Emily Humphreys. Emily has previous research experience in eye development, infectious diseases, and aging. Emily has been a regular contributor to Accelerating Science since 2012.