26Major urinary proteins (MUP) are the major component of the urinary protein fraction in house mice (Mus 27 spp.) and rats (Rattus spp.). The structure, polymorphism and functions of these lipocalins have been well 28 described in the western European house mouse (Mus musculus domesticus), clarifying their role in 29 semiochemical communication. The complexity of these roles in the mouse raises the question of similar 30 functions in other rodents, including the Norway rat, Rattus norvegicus. Norway rats express MUPs in urine 31 but information about specific MUP isoform sequences and functions is limited. In this study, we present a 32 detailed molecular characterization of the MUP proteoforms expressed in the urine of two laboratory 33 strains, Wistar Han and Brown Norway, and wild caught animals, using a combination of manual gene 34 annotation, intact protein mass spectrometry and bottom-up mass spectrometry-based proteomic 35 approaches. Detailed sequencing of the proteins reveals a less complex pattern of primary sequence 36 polymorphism than the mouse. However, unlike the mouse, rat MUPs exhibit added complexity in the form 37 of post-translational modifications including phosphorylation and exoproteolytic trimming of specific 38 isoforms. The possibility that urinary MUPs may have different roles in rat chemical communication than 39 those they play in the house mouse is also discussed. 40 41 42Physiological production of substantial protein in the urine is well known in both rats and house mice [1, 2]. 43 The protein fraction is dominated by 18-19 kDa, eight stranded beta-barrel lipocalins known as major 44 urinary proteins (MUPs, also named as α2u-globulins when first identified in rats [2, 3]). Urinary MUPs are a 45 heterogeneous mixture of multiple isoforms that are very similar in mass and isoelectric point [4][5][6]. The 46 functions of MUPs have largely been studied in the western European house mouse (Mus musculus 47 domesticus) where they play critical roles in olfactory communication. First, they act as carriers for low 48 molecular weight pheromones and other constituents, delaying their release from urinary scent marks [7][8][9]. 49 MUP polymorphism also provides an identity signal for individual and kin recognition [10][11][12][13][14] and may play a 50 role in species recognition [6]. Finally, MUPs act as pheromones in their own right [14][15][16][17]. In particular, 51 darcin (MUP20, MGI nomenclature; http://www.informatics.jax.org/) has a number of unique properties, 52 including a highly specialized role as a male sex pheromone that also induces competition between males. 53 This protein binds most strongly the abundant volatile male pheromone (S)-2-(sec-butyl)-4,5-54 dihydrothiazole in mouse urine [16][17][18][19]. The pheromonal properties of darcin are retained in the 55 recombinant protein showing that it acts as a pheromone in the absence of bound ligands [16, 17]. 56 57The structure and functions of MUPs in the house mouse are well established and serve to emphasi...