Biotinylation of Peptides/Proteins Using Biocytin Hydrazide

Biocytin hydrazide is widely used to biotinylate the carbohydrate moieties of glycoproteins. In this study, however, biocytin hydrazide was found to be able to directly biotinylate peptides and proteins. This phenomenon may cause false identification of non‐glycopeptides/non‐glycoproteins as glycopeptides/glycoproteins. Here, we report a systematic investigation of the reaction of peptides/proteins with biocytin hydrazide. Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry is used to analyze the biotinylation reaction between peptides/proteins and biocytin hydrazide. Peptides/proteins were reacted with biocytin hydrazide in diverse solvent systems with different biocytin hydrazide concentrations for up to 96 h at temperatures ranging from 4 °C to 65 °C. Singly biotinylated or multiply biotinylated peptides/proteins are observed. The efficiency of the biotinylation reaction increases with higher temperature, higher biocytin hydrazide concentration, or longer reaction time. The influence of buffer pH on the biotinylation reaction of peptides/proteins is less pronounced. The biotinylation efficiency is optimum at neutral pH. Data suggests that the peptides are biotinylated as efficiently as proteins. The observation that peptides/proteins condense only with biocytin hydrazide, 2‐iminobiotin hydrazide, adipic dihydrazide and phenyl hydrazine but not with biocytin HCl and 2‐iminobiotin, indicates that the biotinylation reaction of peptides/proteins occurs with the hydrazide moiety but not with biotin moiety of the biotinylated reagent. The postsource decay data of biotinylated P₁₄R indicates that biocytin hydrazide condenses with the guanidino group of arginine's side chain of P₁₄R, indicating that besides N‐terminal and lysine residue of peptides/proteins, arginine residue is capable of reacting with biocytin hydrazide.