This assignment could be also questionable despite of a more efficient fragmentation method such as HCD was used . In this MS/MS spectrum, the most intense fragment ion (m/z=1557.69, 1+) was also assigned manually to ( 1 Z-K 16 )+98) 1+ . The intense signal detected at m/z=1085.01, 2+ and assigned to ([ 70 A-K 87 ]+71) 2+ in Figure decreased considerably the intensity in Figure , suggesting that it was further fragmented by HCD. When this possibility was considered the sequence coverage of ([ 70 A-K 87 ]+71) 2+ was improved by matching new eleven *yn ions (from *y14 to *y4) containing a β-alanine modified lysine (conjugation site at Lys 85 ) with the molecular mass increased by 71Da (Figure ). Also four *bn fragment ions (from *b6 to *b9) of the peptide ( 1 Z-K 16 )+98) 1+ were assigned manually after considering the backbone fragment ions derived from this linearized peptide containing a cysteine residue alkylated by a succinic anhydride molecule (Figure ).