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The iodoTMTsixplex reagents consist of six different isobaric isomers that can react with the sulfhydryl (-SH) group of protein via irreversible labeling step by forming an iodoacetyl-activated covalent bond (Figure 3C, and the chemical structures of iodoTMTsixplex reagents with the distribution of isotopes and their reaction with reduced Cys are shown in Figure 4C

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An advantage of on-column cleavage is the high level of purity obtained, but this comes at the expense of generally low yield due to less efficient protease cleavage and decreased control of the digestion conditions

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