The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Glaciers are considered to be the benchmark of airborne anthropogenic pollution [1,2]. Anthropogenic pollution may cause a darkening of the ice surface which increases solar energy absorption, and, therefore, snow and ice melting. The early stage of glacial melting is the formation of cryoconites— small cylindrical holes in the ablation zone of the glacier surface.
In addition to small rocks and inorganic particles, cryoconites also contain specific microbial communities consisting of algae, bacteria, fungi, and rotifers. Cryoconite holes as a microbiological habitat were found in glaciers around the world, including polar (Arctic and Antarctic) and temperate (alpine) ice .Microbial growth increases the deposition of dark organic matter, causing additional snowmelt . Thus, anthropogenic pollution might have an impact onthe deposition of dark organic matter through the effect on cryoconite communities.
The aim of our study is anunbiased functional comparison of the cryoconite community in two glaciers with different pollution levels.We did this bycomparing cryoconite metaproteomes in Novaya Zemlya (Mushketovaglacier with minimal anthropogenic pollution; 79 05’46’’ N, 101 51’25,35’’ E) and in Kabardino-Balkaria (Garabashihighly polluted glacier; N 43°18’18’’E 42°27’49’’). The samples were taken from the cryoconite holes by drilling form the depth 0-20 cm. The cryoconites consisted of skeletal fraction (up to 25 %) and fine earth (about 75 %) in both sampling sites. The рH was about 6.5-6.9, total organic carbon content was 0.05-0.25 %. Samples were frozen on-site and transported in frozen state to St.Petersburg. We obtained samples from five cryoconites in Kabardino-Balkaria and six in Novaya Zemlya.
The proteins were extracted bySDS solutionand digested by standard “in-solution” methodology. The samples were analyzed by label-free shotgun proteomics with nanoLC-MS/MS with ion mobility in PASEF DDA mode on TimsToF Pro (Bruker) instrument. Proteins were identified in the Peaks X Pro softwareagainst the UniProtKBdatabase.Only protein groups with at least two unique peptides and FDR < 1% were included into further data analysis in R. A more detailed description of the proteomics analysis is given in Supplementary materials 1.
We identified 475 protein groups (fig. 1; SM2).About a third of them were found in both glaciers. Such similarity despite significant geographic distance is in good accordance with theprevious observations: cryoconites from glaciers all over the world have similar invertebrate  and microbial  fauna.
Analysis of the phylum enrichment (fig. 1 b) revealed that most identified proteins were from the Cyanobacteria. Proteins from other phyla were much less represented (fig. 1 b). Contrary to this, insome Alpine glaciers the dominating component of cryoconite communitiesis heterotrophic Proteobacteria [7,8].This may be due to seasonal dynamics in the Alps, with autotrophic cyanobacteria dominating after snowmelt and heterotrophic bacteria becoming dominant towards the end of summer [9,10]. Protein groups from the mainly heterotrophic Actinobacteria, Proteobacteria, Bacteroidetesand Acidobacteria were also abundant (fig. 1 b). Interestingly, 24 Bacteroidetes protein groups were unique for the Caucasus. Seven of them are members of SusC/RagA family TonB-linked outer membrane protein. These outer membrane proteins form transporter complexes which import degradation products of proteins or carbohydrates. Acidobacterial protein groups were also identified mostly in the Caucasus (16 versus 3 protein groups; fig. 1 b).
In the Caucasus sample we found less Cyanobacterial protein groups than in the Novaya Zemlya sample (226/250 protein groups) and more Actino- /Proteobacterial proteins (40/35 and 13/16 protein groups).
Comparison of GO annotations of protein groups specific for the Caucasus and Novaya Zemlya cryoconites also revealed shifts from autotrophic bacteriadominating in Novaya Zemlya to heterotrophic organisms in the Caucasus (fig. 1 c, d). While in Novaya Zemlya the most enriched GO is associated with phycobilisomes, photosynthesis, protein-chromatophore linkage and thylakoid membrane; in the Caucasus the most enriched GO is associated with outer and cell membrane and may be regarded as transport proteins of heterotrophic bacteria.
In summary, we successfully performed metaproteomics analysis of cryoconitemicrobial communities by the shotgun proteomics with ion mobility. We compared metaproteomes of communities from cryoconites of the Caucasus (high anthropogenic pollution) and Novaya Zemlya (low anthropogenic pollution) glaciers and revealed slight shift from the dominance of phototrophic Cyanobacteria in Novaya Zemlya to heterotrophic bacteria in the Caucasus. This may be due to anthropogenic load. However, we cannot conclude this definitely due to differences in the weather conditions and temperature dynamics.Our observationsmay also be associated with seasonal shifts from autotrophic to heterotrophic communities.