A spacer is not always absolutely required, but it is often helpful for fluorescent peptide modifications. Large dyes such as FITC or biotin can influence peptide conformation and accessibility, and a spacer such as Ahx can help separate the label from the peptide sequence.
A spacer can reduce direct interaction between the label and the peptide, helping preserve conformation and biological activity.
N-terminal modification is often easier in SPPS because it is introduced at the final stage of synthesis.
For bulky dyes, a flexible spacer such as Ahx is commonly recommended. Without a spacer, fluorescent labels may interact more directly with nearby residues such as cysteine or lysine, or influence peptide behavior more strongly.