Chemical modification of proteins by air pollutants and metaproteomic analysis of atmospheric aerosol samples by HPLC‐MS analyses
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Abstract
Proteins are a major component of bioaerosols and can account for several percent of air particulate matter. They may influence the climate and public health depending on their properties, e.g., hygroscopicity, molecular composition and structure. The interaction with anthropogenic air pollutants can modify their physical, chemical and biological properties, thus altering their climate and health effects. In particular, chemical modifications of proteins (e.g., nitration and cross‐linking) induced by air pollutants, have been linked to an enhanced potency of allergenic proteins. The mechanisms and kinetics of the underlying chemical processes, however, are not yet well understood.
In this thesis, the reaction products, kinetics and mechanisms of atmospheric protein chemistry were studied, and the proteome of atmospheric aerosol samples was characterized using high performance liquid chromatography coupled with diode array detection and fluorescence detection (HPLC‐DAD and HPLC‐DAD‐FLD), and HPLC coupled to mass spectrometry (HPLC‐MS/MS). The focus areas of this thesis can be summarized as follows:
1.Development of a method to characterize proteins from atmospheric aerosol samples using a mass spectrometry‐based metaproteomic approach. Extraction solvents were optimized to overcome the interaction between proteins and glass fiber filters and achieve high protein recoveries. Size exclusion chromatography (SEC) was applied to remove matrix components. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) was applied for protein fractionation according to molecular size, followed by in‐gel digestion. The digested peptides were analyzed using a hybrid Quadrupole‐Orbitrap MS and database search functions. The developed method has been successfully applied for protein identification from filters samples collected in central Europe (Mainz, Germany). The presented method provides a tool for further studies of spatiotemporal variability of bioparticles and allergens in atmospheric aerosol samples.
2.Elucidation of the mechanisms and kinetics of protein nitration and oligomerization induced by ozone (O3) and nitrogen dioxide (NO2). Proteins were exposed to O3, and O3/NO2 mixtures in coated‐wall flow‐tube and bulk-solution experiments, using bovine serum albumin (BSA) as a model protein. An SEC‐HPLC‐DAD method was developed that enables the simultaneous detection of mono‐, di‐, tri‐, and higher protein oligomers as well as their individual nitration degrees (NDs). In the reaction of BSA with O3, the formation of protein dimers, trimers and higher oligomers was observed. The SDS‐PAGE and fluorescence analysis results revealed that the protein cross‐linking can be attributed to the formation of intermolecular dityrosine species. For the reactions of BSA with O3 and NO2, more tyrosine residues were found to react via the nitration pathways than via the oligomerization pathways. Depending on reaction conditions, oligomer mass fractions and NDs were in the range of 2.5‐25% and 0.5‐7%, respectively. The extent of protein nitration and oligomerization strongly depended on the phase state of proteins (i.e., amorphous solid, semi‐solid, liquid) and hence the diffusivity of oxidants and protein molecules, which change with relative humidity. The experimental results can be explained and described by a kinetic multi‐layer model of surface and bulk chemistry. The rates of both processes were sensitive to the increase of O3 concentrations but rather insensitive to the change in ambient NO2 concentrations.
3.Identification and quantification of free amino acids released upon oxidation of peptides and proteins by hydroxyl radicals. The oxidation products of proteins and peptides generated by hydroxyl radicals from Fenton reactions were analyzed using HPLC‐MS/MS and HPLC‐DAD‐FLD. Free amino acids were identified as products by HPLC‐MS/MS analysis. A site‐selective formation of free amino acids was also observed, which may be due to a reaction pathway involving nitrogen‐centered radicals. For protein oxidation reactions, the molar yields of glycine (Gly, ~32‐55% for BSA, ~10‐21% for ovalbumin (OVA)) were substantially higher than for the other identified amino acids (i.e., alanine, aspartic acid, and asparagine; ~5‐12% for BSA, ~4‐6% for OVA). Upon oxidation of tripeptides with Gly in C‐terminal, mid‐chain, or N‐terminal positions, Gly was preferentially released when it was located at the C‐terminal site.
The methods developed and reaction products, kinetics, and mechanisms studied in this thesis provide a basis for further investigations of atmospheric protein chemistry influenced by air pollutants. They shall help to understand the relations between air pollutant‐modified aeroallergens and their enhanced allergenicity.