Separation of urinary proteins in B6 mouse urine by anion exchange chromatography. Urinary proteins from (A) adult male and (B) adult female B6 mice were separated by strong anion-exchange chromatography, monitored by ultraviolet (UV) absorbance (central trace, blue or pink line). Four specific fractions corresponding to peak UV absorbance were collected from male urine, together with the corresponding fractions from female urine (I - IV). The protein masses in each of these four fractions, along with unfractionated MUPs, were analysed by electrospray ionisation mass spectrometry (outer traces), confirming that each mass corresponded to a known MUP mass in B6 mice . Male fractions I and IV each contained a single male-specific MUP with a mass that was not detectable in matching female fractions. Resolution of each fraction on SDS-PAGE confirmed the absence of MUPs in female fractions I and IV, while male fraction IV contained the male-specific high mobility 18893Da MUP, darcin.