title: The Application of Protein Mass Spectrometry to the Understanding of Behaviour in Mus Species
Creators: name: Lee, V
Publisher: name: University of Liverpool Repository
Year: 2016

description: The Application of Protein Mass Spectrometry to the Understanding of Behaviour in Mus Species The urine of mice contains a large amount of protein, approximately 99% of which are major urinary proteins (MUPs). These 18 – 19 kDa proteins have been shown to have a significant role in chemosignalling. MUPs bind, protect and slowly release the volatile components in scent marks that have been found to elicit various behavioural and physiological responses in mice, including aggression between male mice and the onset of puberty in female mice. MUPs themselves have also been found to be significant in chemical signalling, having roles in modulating identity signalling, attractiveness and aggressive responses. Whilst the MUPs of the house mouse have been comprehensively characterised, including their roles in social and reproductive behaviour, much less in known regarding the urinary proteins of other closely related Mus species and their roles in communication amongst conspecifics. In the first part of this thesis, protein expression in the urine of Mus spicilegus was investigated. Whilst M. spicilegus are genetically close to the Mus musculus subspecies group, their mating patterns and social behaviour are different to those of the house mouse, Mus musculus domesticus. Urinary MUPs play a significant part in the social and reproductive behaviours displayed by M. m. domesticus, yet little is known regarding the molecular causes of the unique social and reproductive behaviours displayed by M. spicilegus. Therefore, this part of the thesis determined whether M. spicilegus also invested in MUPs, and by inference, whether these could play a key role in their unusual behaviours. The protein content of the urine of male and female M. spicilegus was examined, and mass spectrometry was used to identify four MUPs (three of them male-specific) and characterise their primary structure in the absence of genomic data. Male mice expressed more MUP in their urine than female mice, with MUP expression patterns between different males varying quite significantly. In experiments relating to their sexual and social behaviour, male mice altered their MUP expression upon contact with females, and a link between male MUP output and aggressive behaviour amongst male mice was observed. As MUPs have roles in modulating a number of behaviours in the house mouse, including kin recognition, the second part of the thesis focuses on communal nursing in the house mouse, where kin recognition appears to play a role in the lactative investment in pups in a communal nest. Previous studies suggest communal nursing increases reproductive success, but that mutualistic cooperation is higher in nests where females are related. This study involved the metabolic labelling of adult female mice, who were communally nursing, using stable isotope labelled amino acids. The mass spectrometric analysis of MUPs expressed in adult female mice (mothers) urine, proteins in pup stomach contents and proteins in pup tissue samples enabled the confirmation of amino acid label incorporation into nursing mothers, and the determination of label incorporation into pups. Therefore, the investment received by pups from their mothers, in litters where their mothers are related and in litters where their mothers are unrelated was determined, assessing whether any discrimination in investment is evident in relation to relatedness of the female pairs in the nest. Stable isotope labelling strategies and mass spectrometry successfully enabled investment from communally nursing female mice to be tracked in their pups, determining that no females, related or unrelated, discriminated between their own pups and their female partners’ when investing. In most cases, however, one female appeared to invest significantly more in the entire communal litter than the other. Mass spectrometry has been the main tool for the accurate identification and characterisation of MUPs present in scent marks, but MUP quantification has proved more difficult due to the highly homologous nature of these proteins. Previous absolute quantification of MUPs has been based on QconCAT technology, but difficulties arose due to the high sequence similarity between MUP variants. It was therefore considered whether quantification of MUPs could take place at the intact protein level, since intact protein analysis by ESI-MS is already well established for the accurate identification of MUP isoforms. For ESI-MS analysis to be a suitable method for absolute quantification of MUPs, the responses of each individual MUP in ESI-MS analysis were determined using recombinant MUPs, assessing the relationship of MUP concentration and instrument response whilst considering how charge state distribution profiles and sample complexity affected MUP ionisation and instrument response. Despite the homology of MUPs, the differences in their ionisation efficiencies in ESI-MS analysis as part of an equimolar mixture compared to as a single protein means that whilst ESI-MS analysis of MUPs can be useful for the relative quantification of these proteins in urine samples, further preparatory experiments would be required to determine whether ESI-MS analysis of intact proteins could be suitable for absolute quantification.