Identification of 18,956 Da MUP by gel electrophoresis, tandem mass spectrometry and peptide mass fingerprinting (PMF). (a) Urine pooled from five B6 males and five females was first resolved by non-denaturing (native) or SDS-PAGE electrophoresis (8 μg protein loaded). The male specific band indicated by the arrow was excised from the gel and digested with trypsin or endopeptidase LysC for peptide mass fingerprinting. (b) The peptide maps define peptides (trypsin: T1...T17, endopeptidase LysC: L1...L11) that would be obtained from the MUP of unmodified mass 18,956. Peptides that were identified by PMF (shown in (c)) or by MS/MS (shown in (d)) are shaded or highlighted with an asterisk. Overlaid narrow bands define peptides identified as part of a missed cleavage. (c) A representative MS/MS spectra of peptide ENIIDLTNVNR, m/z 1,300.67, [M+2H]2+ 650.7. This protein contains a putative glycosylation site at Asn66 (AFVENITVLENSLVFK77, peptide T5) and, after digestion with N-glycanase (NG, enzyme band indicated by an asterisk), shifted in electrophoretic mobility (a).