Abstract
Major urinary proteins (MUPs) are highly homologous proteoforms that function in binding, transporting and releasing pheromones in house mice. The main analytical challenge for studying variation in MUPs, even for state-of-the-art proteomics techniques, is their high degree of amino acid sequence homology. In this study we used unique peptides for proteoform-specific identification. We applied different search engines (ProteinPilot™vs. PEAKS®) and protein databases (MUP database vs. SwissProt + unreviewed MUPs), and found that proteoform identification is influenced by addressing background proteins (unregulated urinary proteins, non-MUPs) during the database search. High resolution Q-TOF mass spectrometry was used to identify and precisely quantify the regulation of MUP proteoforms in male mice that were reared in standard housing and then transferred to semi-natural enclosures (within-subject design). By using a designated MUP database we were able to distinguish 19 MUP proteoforms, with A2CEK6 (a Mup11 gene product) being the most abundant based on spectral intensities. We compared three different quantification strategies based on MS1- (from IDA and SWATH™ spectra) and MS2 (SWATH™) data, and the results of these methods were correlated. Furthermore, three data normalization methods were compared and we found that increased statistical significance of fold-changes can be achieved by normalization based on urinary protein concentrations. We show that male mice living in semi-natural enclosures significantly up-regulated some but not all MUPs (differential regulation), e.g., A2ANT6, a Mup6 gene product, was upregulated between 9-fold (MS1) and 13-fold (MS2) using the designated MUP database. Finally, we show that 85 ± 7% of total MS intensity can be attributed to MUP-derived peptides, which supports the assumption that MUPs are the primary proteins in mouse urine. Our results provide new tools for assessing qualitative and quantitative variation of MUPs and suggest that male mice regulate the expression of specific MUP proteoforms, depending upon social conditions.
Highlights
Paper proteoform specific Major urinary proteins (MUPs) expression and reflect inter and intra-individual MUP variation.The main technical challenge for measuring variation in MUPs is due to their remarkably high homology at genetic and protein levels
We show that male mice living in semi-natural enclosures significantly up-regulated some but not all MUPs, e.g., A2ANT6, a Mup[6] gene product, was upregulated between 9-fold (MS1) and 13-fold (MS2) using the designated MUP database
As already observed in the MS1 approach, additional MUPs could be quantified using the comprehensive database on MS2 level and here we show that A2ANT6 (Mup6), A2CEK6 (Mup13), A9C497 (Mup19), B8JI96 (Mup14), L7MUC7 (Mup7), P11589 (Mup2), and P11590 (Mup4) were significantly (p o 0.002) upregulated (Fig. 3(D))
Summary
The main technical challenge for measuring variation in MUPs is due to their remarkably high homology at genetic and protein levels (see Fig. 1). MUPs are encoded by at least 21 Mup loci that are closely linked within a large cluster containing central (497% homology) and peripheral (Mup 3, 4, 6, 20, and 21; 82–94% homology on c-DNA level) MUPs, which are easier to distinguish.[12,13,14] The analytical challenge is discriminating individual MUP family members (up to 34 different protein sequences are currently published in Uniprot; wild mice may have more). Conventional antibody-mediated methods and genetic analyses cannot distinguish individual MUPs, due to their homology on gene and protein levels.[2,15,16] MUPs represent a difficult and challenging analytical problem, even for state-of-the-art proteomic techniques
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