Tandem mass spectrum of a precursor ion with m/z 656.8 and charge +4 (the charge was determined from mass difference between its isotopic peaks). The precursor ion is labeled with an asterisk. The spectrum was partially interpreted by considering precise mass differences between the adjacent fragment ions. Doubly charged fragment series rendered the sequence PGDTASLTWF which was further extended toward the N-terminus using very low abundant ions in the m/z range > 1100 (not shown), but the sequence of the two N-terminal amino acid residues remained ambiguous (VL or LV). It was possible to determine the C-terminal amino acid (K) and a short sequence stretch (TVLP) spaced from the C-terminal lysine by two or three unknown amino acid residues (X). Bridging between the sequence stretches PGDTASLTWF and TVLP could have been achieved by one of the three isobaric combination of amino acid residues, and the order of amino acid residues remained unknown. Hence the peptide might contain the sequence WF-QGET-VL, or WF-QEGT-VL, or WF-QADT-VL, or WF-QDAT-VL..., etc.