Figure 1.

Schematic of DiDBiT and conventional strategies for sample preparation and analysis of purified biotinylated proteins. Conventional methods, schematized on the left in steps 1A–3A, involve incubating a complex mixture of proteins with NeutrAvidin beads (step 1A), washing the beads to remove unlabeled proteins (step 2A), elution of labeled proteins and protease digestion of eluted proteins (step 3A), or direct protease digestion of proteins bound to beads (step 3Ai). Note that labeled proteins may be a minority within the sample and that nonspecific or indirect binding to the beads may further decrease the representation of the labeled proteins. Coelution and codigestion of both labeled and unlabeled proteins often produces a mixture in which tagged peptides are too dilute for direct detection of tags (A and 3Ai; see outcome in Figure 3a). DiDBiT is schematized on the right in steps 1B–3B, showing the improvement in enrichment (step 1B), recovery (step 2B), and analysis (step 3B) of biotinylated peptides. Complete protease digestion of the input material allows the incubation of highly concentrated peptide mixtures with NeutrAvidin beads due the higher solubility of peptide mixtures in aqueous buffer (PBS) compared to their input protein extracts (step 1B). Washes to remove nonspecific bound peptides can be done in 5–10% acetonitrile in PBS (step 2B). Peptides bound to NeutrAvidin are efficiently eluted by boiling in TFA/FA/acetonitrile. This sample preparation protocol significantly increases labeled/nonlabeled peptide ratio (step 3B; see output results in Figure 3b). Biotinylated peptides were consistently detected using DiDBiT. This peptide elution strategy can also be used after the conventional on-bead digestion to release the bound peptides from the resin after trypsinization (A3ii or “on-bead release 2”) (see output results in Figure 3a).