I am trying to conjugate an antibody to another protein with S-HyNic and S-4FB. In Table 1, MSR of S-HyNic saturates to 8 or 9. MSR of S-4FB saturates to 26 or 28. What makes the difference between those two values? Does S-HyNic or S-4FB or conjugate-product have any charges that affect the conjugation efficiency?

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Different linkers have varying modification efficiencies. 4FB modifies much more efficiently than does HyNic. 4FB is very hydrophobic, and binds nonspecifically, initially, to protein hydrophobic regions, then is in close proximity to, and reacts with Lys residues very efficiently. HyNic does have a protonated group (pyridine), and does not crosslink to the biomolecule with the same efficiency as 4FB. The charge on the HyNic perhaps contributes to it’s lower modification efficiency, but does not affect its conjugation efficiency to 4FB.