Data-independent acquisition (DIA) approaches provide comprehensive records of all detectable precursor and fragment ions. Here we introduce midiaPASEF, a novel DIA scan mode using mobility-specific micro-encoding of overlapping quadrupole windows to optimally cover the ion population in the ion mobility-mass to charge plane. Using overlapping ion mobility-encoded quadrupole windows, midiaPASEF maximizes information content in DIA acquisitions which enables the determination of the precursor m/z of each fragment ion with a precision of less than 2 Th. The MIDIAlyzer pipeline integrates algorithms for multidimensional peak detection and for machine-learning-based classification of precursor-fragment relationships. MIDIAlyzer enables fully automated processing and multidimensional deconvolution of midia-PASEF files and exports highly specific DDA-like MSMS spectra which are suitable for de novo sequencing and can be searched directly with established tools including PEAKS, FragPipe and Mascot. midiaPASEF acquisition identifies over 40 unique peptides per second and provides powerful library-free DIA analyses including phosphopeptidome and immunopeptidome samples.

Abstract. Archaea are ubiquitous in terrestrial and marine environments and play an important role in biogeochemical cycles. Although air acts as the primary medium for their dispersal among different habitats, their diversity and abundance is not well characterized. The main reason for this lack of insight is that archaea are difficult to culture, seem to be low in number in the atmosphere, and have so far been difficult to detect even with molecular genetic approaches. However, to better understand the transport, residence time, and living conditions of microorganisms in the atmosphere as well as their effects on the atmosphere and vice versa, it is essential to study all groups of bioaerosols. Here we present an in-depth analysis of airborne archaea based on Illumina sequencing of 16S rRNA genes from atmospheric coarse and fine particulate matter samples and show seasonal dynamics and discuss anthropogenic influences on the diversity, composition, and abundance of airborne archaea.The relative proportions of archaea to bacteria, the differences of the community composition in fine and coarse particulate matter, and the high abundance in coarse matter of one typical soil related family, the Nitrososphaeraceae, point to local phyllosphere and soil habitats as primary emission sources of airborne archaea.We found comparable seasonal dynamics for the dominating Euryarchaeota classes and Crenarchaeota orders peaking in summer and fall. In contrast, the omnipresent Cenarchaeales and the Thermoplasmata occur only throughout summer and fall. We also gained novel insights into archaeal composition in fine particulate matter (< 3 µm), with Cenarchaeaceae, Nitrososphaeraceae, Methanosarcinales, Thermoplasmata, and the genus Nitrosopumilus as the dominating taxa.The seasonal dynamics of methanogenic Euryarchaeota point to anthropogenic activities, such as fertilization of agricultural fields with biogas substrates or manure, as sources of airborne archaea. This study gains a deeper insight into the abundance and composition of archaea in the atmosphere, especially within the fine particle mode, which adds to a better understanding of the overall atmospheric microbiome.

Oomycetes, also named Peronosporomycetes, are one of the most important and widespread groups of plant pathogens, leading to significant losses in the global agricultural productivity. They have been studied extensively in ground water, soil, and host plants, but their atmospheric transport vector is not well characterized. In this study, the occurrence of airborne Oomycetes was investigated by Sanger sequencing and quantitative PCR of coarse and fine aerosol particle samples (57 filter pairs) collected over a 1-year period (2006–2007) and full seasonal cycle in Mainz, Germany. In coarse particulate matter, we found 55 different hypothetical species (OTUs), of which 54 were plant pathogens and 29 belonged to the genus Peronospora (downy mildews). In fine particulate matter (<3 μm), only one species of Hyaloperonospora was found in one sample. Principal coordinate analysis of the species composition revealed three community clusters with a dependence on ambient temperature. The abundance of Oomycetes rRNA genes was low in winter and enhanced during spring, summer, and fall, with a dominance of Phytophthora, reaching a maximum concentration of ∼1.6 × 106 rRNA genes per cubic meter of sampled air in summer. The presence and high concentration of rRNA genes in air suggests that atmospheric transport, which can lead to secondary infection, may be more important than currently estimated. This illustrates the need for more current and detailed datasets, as potential seasonal shifts due to changing meteorological conditions may influence the composition of airborne Oomycetes. An insight into the dynamics of airborne plant pathogens and their major drivers should be useful for improved forecasting and management of related plant diseases.