An exploration of the protein component of scent marks from a range of mammalian species using proteomics approaches

Whilst semiochemistry is traditionally associated with volatile cues, the discovery of the major urinary proteins (MUPs) of the house mouse, and their demonstrable ability to convey information such as identity, kinship and mating availability, have identified proteins as an important tool for communication. Whilst protein-mediated scent signalling is understood relatively well in the house mouse and the Norway rat, little is known regarding the roles of proteins in chemical signalling beyond these species. The protein content of scent marks from three species, the closely related bank vole (Myodes glareolus) and field vole (Microtus agrestis), and the much more distantly related marsupial, the brushtail possum (Trichosurus vulpecula), were investigated. Overall protein content was initially assessed using polyacrylamide gel electrophoresis and electrospray ionisation-mass spectrometry of intact proteins. Prominent proteins of interest were separated by anion exchange chromatography and sequenced de novo using liquid chromatography allied to a tandem mass spectrometer. Total proteome analyses were performed by tryptic proteolytic cleavage followed by liquid chromatography-tandem mass spectrometry. Proteins were identified by cross-species matching and quantified using a label-free approach. Previous investigation into urinary protein expression in the bank vole, Myodes glareolus, identified three odorant-binding proteins (OBPs). The discovery of another OBP, glareosin, is described. Glareosin was determined as the single most abundant protein present in male bank vole urine during the breeding season, and the elucidation of its sequence and structure was published in 2017. Additional work continued to explore the total protein content, identifying additional OBP-like proteins at the peptide level in urine and scent marks of both sexes, including previously identified OBPs, although no corresponding intact masses were found. Behavioural research into the field vole (Microtus agrestis) has concentrated on its unusual dynamic population cycles, but olfactory communication remain largely unevaluated. Three abundant male-specific sequence variants, homologous to bank vole glareosin, were identified from male breeding season urine and sequenced de novo. Other proteins, including another OBP-like protein, a putatively glycosylated MUP-like protein, and a lipocalin-11-like protein were partially sequenced from urine and scent marks of both species. Global proteome analysis indicated further unidentified heterogeneity of OBP proteins at the peptide level, indicating a far more complex protein landscape within field vole scent marks when compared to the bank vole. In New Zealand, the status of the brushtail possum, Trichosurus vulpecula, as an invasive pest species has ignited a concerted effort to eradicate the species, despite a deficit in research into marsupial behaviour. Following proteome analyses, a glycosylated lipocalin was identified in the urine of both sexes, and sequenced de novo. Phylogenetic analysis with a range of mammalian lipocalins identified the novel sequence within a new clade of lipocalins unique to marsupials, suggesting that the marsupial lineage diverged prior to the establishment of described lipocalin classes in the placental mammals. The following investigations are three examples of the diversity of scent mark protein expression between both closely- and distantly-related species, and highlight the lack of understanding in this area. However, due to the evolutionary pressure placed on proteins with capacity to influence mate choice and sexual selection, a proteome approach reliant on identification by database matching is difficult, particularly for those species without a comprehensive genome, which will remain a rate-limiting factor until a wider range of genomes are available

Glareosin : A novel sexually dimorphic urinary lipocalin in the bank vole, Myodes glareolus

Electronic supplementary material is available online at https://dx.doi.org/10.6084/m9.figshare.c.3859369. Detailed methods are presented in the electronic supplementary material. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [32] partner repository with the dataset identifier PXD006645 and 10.6019/PXD006645 This work was funded in part by BBSRC (BB/J002631/1 and BB/M012557/1).Peer reviewedPublisher PD

Vulpeculin: a novel and abundant lipocalin in the urine of the common brushtail possum, Trichosurus vulpecula.

Lipocalins are a family of secreted proteins. They are capable of binding small lipophilic compounds and have been extensively studied for their role in chemosignalling in rodent urine. Urine of the common brushtail possum (Trichosurus vulpecula) contains a prominent glycoprotein of 20 kDa, expressed in both sexes. We have isolated this protein and determined its primary sequence by mass spectrometry, including the use of metabolic labelling to resolve the leucine/isoleucine isobaric ambiguity. The protein sequence was identified as a lipocalin, and phylogenetic analysis grouped the protein with other marsupial lipocalin sequences in a phylogenetic clade distinct from established cross-species lipocalin sub-families. The pattern of expression in possum urine and the similarity in sequence and structure to other lipocalins suggests this protein may have a role in brushtail possum chemosignalling

Supplementary Methods S1; Supplementary Figure S2; Supplementary Figure S3; Supplementary Figure S4; Supplementary Figure S5 from Glareosin: a novel sexually dimorphic urinary lipocalin in the bank vole, <i>Myodes glareolus</i>

Expanded Materials and Methods; Overall sequence coverage of glareosin; MS/MS spectra used for sequencing; Leu/Ile labelling for ambiguous peptide IYLRE; Alignment of glareosin with other lipocalin