Field-realistic exposure studies provide the most relevant assessment of the
effects of the intensity and diversity of urban and industrial contamination on
pollen structure and allergenicity. The significance of in-depth post-translational
modification (PTM) studies of pollen proteomes, when compared with studies on
other aspects of pollution and altered pollen allergenicity, has not yet been
determined; hence, little progress has been made within this field

Apostolović, Danijela

Burazer, Lidija M.

Mutić, Jelena

Prodić, Ivana

Smiljanić, Katarina

van Hage, Marianne

Ćirković-Veličković, Tanja

English

Ćirković Veličković, Tanja

Faculty of Chemistry Repository - Cherry

2     6 1st FoodEnTwin Workshop “Food and Environmental -Omics”, Belgrade, June 20-21, 2019      Invited lecture Highly improved method for in-depth post-translational modification profiling: example of Timothy grass (Phleum pratense) pollen proteomes from polluted and preserved environments Katarina Smiljanić1, Ivana Prodić2, Danijela Apostolović3, Jelena Mutić1,4, Marianne van Hage3, Lidija Burazer5, and Tanja Ćirković Veličković1,4,6,7  1University of Belgrade–Faculty of Chemistry, Centre of Excellence for Molecular Food Sciences, Belgrade, Serbia; 2Innovation Center Ltd, University of Belgrade–Faculty of Chemistry, Belgrade, Serbia; 3Karolinska Institute, Department of Medicine, Solna, Stockholm, Sweden; 4Ghent University Global Campus, Incheon, South Korea; 5Institute of Immunology, Virology and Sera Production, “Torlak Institut”, Belgrade, Serbia; 6Ghent University, Faculty of Bioscience Engineering, Ghent, Belgium; 7Serbian Academy of Sciences and Arts, Belgrade, Serbia  Field-realistic exposure studies provide the most relevant assessment of the effects of the intensity and diversity of urban and industrial contamination on pollen structure and allergenicity. The significance of in-depth post-translational modification (PTM) studies of pollen proteomes, when compared with studies on other aspects of pollution and altered pollen allergenicity, has not yet been determined; hence, little progress has been made within this field.  Therefore, we created a comprehensive approach for the comparison of pollen from polluted and environmentally preserved areas. To examine the effects of long-term, in vivo pollen exposure to multiple source pollutants, Phleum pratense (Timothy grass) pollen samples were collected along a regional road in Kruševac, central Serbia. This road experienced moderate traffic and was located near a chemical plant that produces fertilizers. Pollen samples from this location were compared with pollen samples collected from a rural, environmentally preserved area over two consecutive pollination seasons. We combined the quantitative comparison of proteome expression profiles from in-solution and 2D gels with unrestrictive in-depth quantitative PTM profiling using high resolution tandem mass spectrometry and the PEAKS 8.5 Suite platform. This was followed by Session 1: Omics and the environment 7 1st FoodEnTwin Workshop “Food and Environmental -Omics”, Belgrade, June 20-21, 2019   quantitative IgE enzyme-linked immunosorbent assays (ELISA) and one dimensional (1D) IgE immunoblots that were probed with the sera of grass pollen allergic patients and healthy control subjects from Serbia. In addition, elemental compositional analyses of Timothy grass pollen samples from both locations, and the surface grain structure and SPP releasing potential (including total protein and phenolic content), were assessed. An increased phenolic content and release of sub-pollen particles was found in pollen samples from the polluted area, including a significantly higher content of mercury, cadmium, and manganese. Antioxidative defense-related enzymes were significantly upregulated and seven oxidative PTMs were significantly increased (methionine, histidine, lysine, and proline oxidation; tyrosine glycosylation, lysine 4-hydroxy-2- nonenal adduct, and lysine carbamylation) in pollen exposed to the chemical plant and road traffic pollution sources. Oxidative modifications affected several Timothy pollen allergens; Phl p 6, in particular, exhibited several different oxidative modifications. The expression of Phl p 6, 12, and 13 allergens were downregulated in polluted pollen, and IgE binding to pollen extract was substantially lower in the 18 patients studied, as measured by quantitative ELISA.  Quantitative, unrestricted, and detailed PTM searches using an enrichment-free approach was used for the first time to map extensive modifications in the pollen allergome, which was shown to reflect the increased environmental oxidative stress, primarily caused by increased content of heavy metals in pollen. With some modifications, this PTM profiling approach is suitable for exploring the oxidative stress effects in any proteomic source in a quantitative in-depth manner, thus enabling further data-driven research.  Acknowledgements: This research work was funded the Ministry of Education and Science of the Republic of Serbia, GA No. OI172024, Ghent University Global Campus, Belgian Special Research Fund BOF StG No. 01N01718, Serbian Academy of Sciences and Arts Project F-26. The project leading to this application has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 810752.         8 1st FoodEnTwin Workshop “Food and Environmental -Omics”, Belgrade, June 20-21, 2019   CIP - Каталогизација у публикацији Народна библиотека Србије, Београд  577.1(048) 663/664(048) 502/504(048)  FOODENTWIN Workshop "Food and Environmental - Omics" (1 ; 2019 ; Beograd) Book of Abstracts / 1st FoodEnTwin Workshop "Food and Environmental - Omics", June 20-21, 2019 Belgrade, Serbia ; [editor in cheif Dragana Stanić-Vučinić]. - Belgrade : University, Faculty of Chemistry, 2019 (Belgrade : Službeni glasnik). - 49 str. ; 28 cm  Tiraž 50.  ISBN 978-86-7220-099-7  a) Биохемија -- Апстракти b) Храна -- Апстракти v) Животна средина -- Апстракти  COBISS.SR-ID 277360908 --   

Highly improved method for in-depth post-translational modification profiling: example of Timothy grass (Phleum pratense) pollen proteomes from polluted and preserved environments

Cherry - Repository of the Faculty of Chemistry;  University of Belgrade

2  
 
 
 
6 
1st FoodEnTwin Workshop “Food and Environmental -Omics”, Belgrade, June 20-21, 2019 
 
 
 
 
 
Invited lecture 
Highly improved method for in-depth post-translational modification 
profiling: example of Timothy grass (Phleum pratense) pollen proteomes 
from polluted and preserved environments 
Katarina Smiljanić1, Ivana Prodić2, Danijela Apostolović3, Jelena Mutić1,4, Marianne van 
Hage3, Lidija Burazer5, and Tanja Ćirković Veličković1,4,6,7 
 
1University of Belgrade–Faculty of Chemistry, Centre of Excellence for Molecular Food Sciences, 
Belgrade, Serbia; 
2Innovation Center Ltd, University of Belgrade–Faculty of Chemistry, Belgrade, Serbia; 
3Karolinska Institute, Department of Medicine, Solna, Stockholm, Sweden; 
4Ghent University Global Campus, Incheon, South Korea; 
5Institute of Immunology, Virology and Sera Production, “Torlak Institut”, Belgrade, Serbia; 
6Ghent University, Faculty of Bioscience Engineering, Ghent, Belgium; 
7Serbian Academy of Sciences and Arts, Belgrade, Serbia 
 
Field-realistic exposure studies provide the most relevant assessment of the 
effects of the intensity and diversity of urban and industrial contamination on 
pollen structure and allergenicity. The significance of in-depth post-translational 
modification (PTM) studies of pollen proteomes, when compared with studies on 
other aspects of pollution and altered pollen allergenicity, has not yet been 
determined; hence, little progress has been made within this field. 
 
Therefore, we created a comprehensive approach for the comparison of pollen 
from polluted and environmentally preserved areas. To examine the effects of 
long-term, in vivo pollen exposure to multiple source pollutants, Phleum pratense 
(Timothy grass) pollen samples were collected along a regional road in Kruševac, 
central Serbia. This road experienced moderate traffic and was located near a 
chemical plant that produces fertilizers. Pollen samples from this location were 
compared with pollen samples collected from a rural, environmentally preserved 
area over two consecutive pollination seasons. We combined the quantitative 
comparison of proteome expression profiles from in-solution and 2D gels with 
unrestrictive in-depth quantitative PTM profiling using high resolution tandem 
mass spectrometry and the PEAKS 8.5 Suite platform. This was followed by 
Session 1: Omics and the environment 
7 
1st FoodEnTwin Workshop “Food and Environmental -Omics”, Belgrade, June 20-21, 2019 
 
 
quantitative IgE enzyme-linked immunosorbent assays (ELISA) and one dimensional 
(1D) IgE immunoblots that were probed with the sera of grass pollen allergic 
patients and healthy control subjects from Serbia. In addition, elemental 
compositional analyses of Timothy grass pollen samples from both locations, and 
the surface grain structure and SPP releasing potential (including total protein and 
phenolic content), were assessed. 
An increased phenolic content and release of sub-pollen particles was found in 
pollen samples from the polluted area, including a significantly higher content of 
mercury, cadmium, and manganese. Antioxidative defense-related enzymes were 
significantly upregulated and seven oxidative PTMs were significantly increased 
(methionine, histidine, lysine, and proline oxidation; tyrosine glycosylation, lysine 
4-hydroxy-2- nonenal adduct, and lysine carbamylation) in pollen exposed to the 
chemical plant and road traffic pollution sources. Oxidative modifications affected 
several Timothy pollen allergens; Phl p 6, in particular, exhibited several different 
oxidative modifications. The expression of Phl p 6, 12, and 13 allergens were 
downregulated in polluted pollen, and IgE binding to pollen extract was 
substantially lower in the 18 patients studied, as measured by quantitative ELISA. 
 
Quantitative, unrestricted, and detailed PTM searches using an enrichment-free 
approach was used for the first time to map extensive modifications in the pollen 
allergome, which was shown to reflect the increased environmental oxidative 
stress, primarily caused by increased content of heavy metals in pollen. With some 
modifications, this PTM profiling approach is suitable for exploring the oxidative 
stress effects in any proteomic source in a quantitative in-depth manner, thus 
enabling further data-driven research. 
 
Acknowledgements: This research work was funded the Ministry of Education and Science of the Republic of 
Serbia, GA No. OI172024, Ghent University Global Campus, Belgian Special Research Fund BOF StG No. 
01N01718, Serbian Academy of Sciences and Arts Project F-26. The project leading to this application has 
received funding from the European Union's Horizon 2020 research and innovation programme under grant 
agreement No 810752. 
 
 
 
 
 
 
 
 
8 
1st FoodEnTwin Workshop “Food and Environmental -Omics”, Belgrade, June 20-21, 2019 
 
 
CIP - Каталогизација у 
публикацији Народна 
библиотека Србије, Београд 
 
577.1(048) 
663/664(048) 
502/504(048) 
 
FOODENTWIN Workshop "Food and Environmental - Omics" (1 ; 2019 
; Beograd) 
Book of Abstracts / 1st FoodEnTwin Workshop "Food and 
Environmental - Omics", June 20-21, 2019 Belgrade, Serbia ; [editor in 
cheif Dragana 
Stanić-Vučinić]. - Belgrade : University, Faculty of Chemistry, 2019 (Belgrade 
: Službeni glasnik). - 49 str. ; 28 cm 
 
Tiraž 50. 
 
ISBN 978-86-7220-099-7 
 
a) Биохемија -- Апстракти b) Храна -- Апстракти 
v) Животна средина -- Апстракти 
 
COBISS.SR-ID 277360908 
-- 
 
 


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Highly improved method for in-depth post-translational modification profiling: example of Timothy grass (Phleum pratense) pollen proteomes from polluted and preserved environments

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