Detectability of Biotin Tags by LC–MS/MS

The high affinity of biotin to streptavidin has made it one of the most widely used affinity tags in proteomics. Early methods used biotin for enrichment alone and mostly ignored the biotin-labeled peptide. Recent advances in labeling have led to an increase in biotinylation efficiency and shifted the interest to the detection of the site of biotinylation. This has increased the confidence in identification and provided additional structural information, yet it requires the efficient release of the biotinylated protein/peptide and the sensitive separation and detection of biotinylated peptides by LC-MS/MS. Despite its long use in affinity proteomics, the effect of biotinylation on the chromatographic, ionization, and fragmentation behavior and the ultimate detection of peptides is not well understood. To address this, we compare two commercially available biotin labels, EZ-Link Sulfo-NHS-Biotin and Sulfo-NHS-SS-Biotin, the latter containing a labile linker to efficiently release biotin to determine the effects of peptide modification on peptide detection. We describe an increase in the hydrophobicity and charge reduction with an increasing number of biotin labels attached. On the basis of our data, we recommend gradient optimization to account for more hydrophobic biotinylated peptides and include singly charged precursors to account for charge reduction by biotin.