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. 2014 Aug 13;13(9):3966–3978. doi: 10.1021/pr5002862

Figure 3.

Figure 3

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.