Comparison of DiDBiT with conventional methods
to identify biotin-labeled
peptides from HEK cells labeled with NHS-biotin. (a) Starting with
equal amounts of material (6 mg of protein lysate) and using reverse-phase
separation coupled to MS analysis, we compared DiDBiT and 2 protein
enrichment methods (outputs of the fractions in 3A, 3Ai, 3Aii, and
3B described in Figure 1). (b–d) Venn
diagrams showing the overlap of modified proteins detected with the
DiDBiT strategy and unmodified proteins detected with protein elution
(b) on-bead digestion (c) and elution of bound peptides in the “on-bead
digestion release 2” fraction (d). The modified peptides detected
using DiDBiT are highly overlapping (88.2%) with the modified peptides
detected in the on-bead digestion release 2 fraction, however DiDBiT
detected 10× more biotin modified proteins (d). (e) Plot of the
number of peptides identified per protein for the three methods. Similar
coverage per protein was obtained with DiDBiT, on-bead digestion,
and protein elution.