Dear Sanjib,

It very much depends on what you want. The linker you talk about is often used to react the primary amino group at the N-terminus of the peptide as final step of a peptide synthesis. The alkyl spacer such as aminohexanoic acid (Ahx) is introduced to avoid the formation of fluorescein with the subsequent removal of the last amino acid, thanks to the linker the targeted peptide can now be released from the resin via non-acidic cleavage.

Steric hindrance is indeed sometimes the main reason for using Ahx, however not so much when you talk about side chain labeling to for example cysteine. The majority of commonly used dyes are large aromatic compounds and the incorporation of these bulky molecules could prevent interactions between the peptide and the dye. The inclusion of a flexible spacer, such as the six-carbon linker Ahx, can also attribute to increase the stability of the fluorescent label.

See for examples:

https://www.researchgate.net/publication/244236573_N-Terminus_FITC_labeling_of_peptides_on_solid_support_the_truth_behind_the_spacer

and

http://www.ontoresinc.com/Blog_WhyalinkerneededforN-terFITC-labeledpeptide_125.html

However FITC can also be linked readily to the thiol of a cysteine. For this purpose Fluorescein-5-Maleimide is used. Fluorescein-5-Maleimide is a sulfhydryl-reactive derivative of fluorescein that labels proteins and other molecules having free thiols (cysteine side chains).

See for example:

https://www.researchgate.net/profile/Marietta_Zille/publication/260253081_XTEN-Annexin_A5_XTEN_Allows_Complete_Expression_of_Long-Circulating_Protein-Based_Imaging_Probes_as_Recombinant_Alternative_to_PEGylation/links/00b7d5373861b3584f000000/XTEN-Annexin-A5-XTEN-Allows-Complete-Expression-of-Long-Circulating-Protein-Based-Imaging-Probes-as-Recombinant-Alternative-to-PEGylation.pdf

https://www.researchgate.net/profile/Xue-Yuan_Bai/publication/51390221_Membrane_topology_structure_of_human_high-affinity_sodium-dependent_dicarboxylate_transporter/links/0fcfd501814d06bd98000000.pdf

So again it depends on what you want. There is no method essentially better than the other.

In my view: The maleimide approach is likely to be more specific for cysteine labeling. Obviously the maleimide method requires the presence of cysteines or the introduction (by for example site directed mutagenesis of specific cysteines). While the linker approach might eliminate possible hindrance in peptide or protein functioning. But for the cysteine labeling this hindrance plays no role.

Hope this clarifies the matter somewhat for you.

Thanks Rob Keller for valuable information. However, if I synthesis FITC-Ahx-Cys-sequence and then my drug has maleimide for binding with SH of Cys, do you think FITC group will make the access of SH group difficult for Maleimide of drug because of steric hindrance or it will be just fine while conjugation.

My answer was related to the question whether you better can use either the FITC-Ahx-Cys or FITC-maleimide-Cys approach.

If you want to use both on the same Cys... I guess it is hard to predict. Both approaches use different points to attach itself to cysteine. I think it could work, but I understand (now) your fear that the FITC-Ahx-Cys might hinder the availabilty of the -SH group.

I am afraid that you simply have to try it. If you reach a good protein (or peptide) and label ratio then it seems to be working. Interesting enough to see what will be the result!

PS. I do not fully understand why you want this, but I guess you have your reasons for it.

Thanks Rob Keller for your discussion.