In the MS/MS spectrum shown in Figure the two-most intense fragment ions detected at m/z=1085.01, 2+ and m/z=1557.69, 1+ were not assigned automatically by pLink2. In addition, the poor sequence coverage of the peptide [ 70 A-K 87 ] make this assignment questionable. Knowing the lability of the hydrolyzed thiosuccinimide linker in gas phase (Figure ) the signals detected at m/z=1085.01, 2+ and m/z=1557.69, 1+ were manually assigned to the linker fragment ions ([ 70 A-K 87 ]+71) 2+ and (( 1 Z-K 16 )+98) 1+ , respectively. Also the third-most intense signal detected at m/z=779.34, 2+ was assigned to (( 1 Z-K 16 )+98) 2+ . All these linker fragment ions were also more intense than the signal detected at m/z=589.28, 1+ and assigned to the βy5, which is originated by the fragmentation of a very labile peptide bond at the N-terminal of proline . This result confirms that hydrolyzed thiosuccinimide linker is also cleavable in ESI-MS analysis when CID is used.